ASSAY FOR SARS-CoV-2 INFECTION OF VULNERABLE HUMAN CELLS

Abstract

Provided herein are methods and compositions useful for identifying compounds that can inhibit SARS-CoV-2 infection or the effects thereof, especially in cardiomyocytes (CMs), which are highly infectible by SARS-CoV-2 corona viruses.

Description

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file, “3730176US1.txt”, created on Feb. 14, 2024 and having a size of 446,507 bytes. The contents of the text file are incorporated by reference herein in their entirety.

BACKGROUND

The World Health Organization has declared Covid-19 a global pandemic. A highly infectious coronavirus, officially called SARS-CoV-2, causes the Covid-19 disease. Even with the most effective containment strategies, the spread of the Covid-19 respiratory disease has only been slowed. The available vaccines are likely best way to prevent people from getting sick, but some refuse to be vaccinated and some vaccinated people can still suffer from Covid-19 infection. Compositions and methods to facilitate recovery from Covid-19 infection are needed.

SUMMARY

Provided are methods and compositions useful for identifying compounds that can inhibit SARS-CoV-2 infection or the effects thereof. As illustrated herein, cardiomyocytes (CMs), are highly infectible by corona viruses, including SARS-CoV-2. Even low multiplicities of infection (MOI) of SARS-CoV-2 (e.g., about 1 virion particle per 1000 cells) can infect cardiomyocytes and support SARS-CoV-2 viral replication.

COVID-19 causes severe heart failure, but specific pathological consequences in cardiomyocytes have yet to be identified. Here the inventors describe the consequences of COVID-19 infection on cardiomyocytes, and upon the functioning of the heart. As demonstrated herein, human cardiomyocytes exposed to the virus exhibit significant myofibrillar disruption and a distinct patterns of sarcomeric fragmentation. Many cardiomyocytes exposed to coronavirus lack nuclear DNA by common detection methods, such as Hoechst or hematoxylin staining. In contrast, SARS-CoV-2 does not appear to infect induced pluripotent stem cells (iPSCs), endothelial cells, or cardiac fibroblasts. The adverse morphologic features of virally infected cardiomyocytes are distinct and potentially unique compared to other genetic or environmental stresses that are known to induce cardiomyopathy phenotypes.

Human iPSC-derived cardiac cells were used as described herein for infection with SARS-CoV-2 to reveal robust transcriptomic and morphological signatures in cardiomyocytes, which allowed identification of clear markers of viral damage in human autopsy specimens. Cardiomyocytes display a distinct pattern of sarcomere fragmentation, with specific cleavage of thick filaments, and COVID-19 autopsy samples displayed similar sarcomeric disruption. Numerous iPSC-cardiomyocytes lacked nuclear DNA. Surprisingly, enucleated cardiomyocytes were prevalent in the hearts of COVID-19 patients. These striking cytopathic features are useful for identifying new therapies for COVID-19-related heart failure.

Methods and assay mixtures are described herein that involve use of human cells, for example, cardiomyocytes or cells generated from human induced pluripotent stem cells (iPS) for identifying compounds useful for treatment of SARS-CoV-2. Screening of viral infection and cytopathic effects of such infection in cardiomyocytes can be performed in multi-well plate formats that are compatible with high-throughput screening platforms.

In some cases, cardiomyocytes derived from induced pluripotent stem cells of different genotypes are used in the assays, allowing identification of compounds for treatment of SARS-CoV-2 in patients with different genetically induced cardiac conditions.

The screening assay described herein provides multiple distinct visual indications of cytopathic effects induced by coronavirus that can be used to identify different cellular responses to coronavirus infection and to test whether compounds are useful therapeutics to attenuate adverse consequences of SARS-CoV-2 viral infection. The methods are highly sensitive and can provide information on multiple parameters useful for evaluating cytopathic effects of SARS-CoV-2 viral infection. Thus, in addition to serving as a frontline screening platform for prophylactic and therapeutic effects of the virus on cardiac cells, the methods also serve as a sensitive assay for distinct cytopathic effects that could adversely impact other human cells and tissues that are vulnerable to coronavirus infection and inflammatory responses.

The therapeutic target can, for example, be the titin protein at the M-line in relation to infection. Titin is involved in sarcomere assembly and function through its elastic adaptor and signaling domains. Titin's M-line region contains a unique kinase domain that may regulate sarcomere assembly via its substrate titin cap (T-cap). Studies indicate that the titin M-line region is required to form a continuous titin filament and to provide mechanical stability.

DESCRIPTION OF THE FIGURES

FIG. 1A-1H illustrate that SARS-CoV-2 induces cytopathic effects in iPS-derived cardiac cell types, and productively infects cardiomyocytes. FIG. 1A graphically illustrates quantification of SARS-CoV-2 viral RNA by RT-qPCR quantification of the viral nucleocapsid (N5) gene in cell cultures exposed to SARS-CoV-2. Error bars: SEM. **: p-val<0.01, one-way ANOVE with Tukey's multiple comparisons. FIG. 1B graphically illustrates the toxicity of SARS-CoV-2 to cardiac cell types, as quantified by nuclear retention. Y-axis depicts the % of nuclei counted (relative to mock). Nuclei were counted automatically at 10× magnification (10 images/condition). Vehicle treatment (mock; left bars), heat inactivated SARS-CoV-2 (MOI=0.1; middle bars), and SARS-CoV-2 (MOI=0.006; right bars) nucleic counts are shown. FIG. 1C shows a representative image of a SARS-CoV-2 infected cardiomyocyte, as observed by transmission electron microscopy (TEM) of osmium tetroxide/potassium ferricyanide stained cells. Cells were exposed to SARS-CoV-2 virus for 48 hours at an MOI of 0.006 before fixation. This view shows the nucleus to the right, in addition to remnant ER-Golgi, with a closed membrane of viral particles. This image is a less magnified view of the images shown in FIG. 1D-1E; the line in the lower right corresponds to 0.5 μm. FIG. 1D shows an expanded view of the inset shown in FIG. 1C, further illustrating that SARS-CoV-2 virions were present and showing that a double membrane cannot be discerned by transmission electron microscopy. The line in the lower right corresponds to 0.4 μm. FIG. 1E shows an expanded view of the center of FIG. 1D, further illustrating that SARS-CoV-2 virions were present and showing the 500-750 nm diameter membrane and the 50-60 nm diameter viral particles within. The line in the lower right corresponds to 100 nm. FIG. 1F graphically illustrates ACE2 transcript levels in CMs compared to undifferentiated iPS cells as quantified by RT-qPCR quantification. **: p-value<0.01. FIG. 1G graphically illustrates the SARS-CoV-2 viral N5-fold change in infected iPSC and CM cells. Infection of iPSCs yielded no detectable levels of viral N5. FIG. 1H graphically illustrates the SARS-CoV-2 fold change as detected by viral N5 fold changes relative to the N5 levels in IPSCs.

FIG. 2A-2C illustrate pharmacological modulation of SARS-CoV-2 infection and host innate immune responses in CMs. FIG. 2A graphically illustrates viral Nucleocapsid (N5) levels of CM samples exposed to SARS-CoV-2 for 48 h (MOI=0.006) after 2 h pretreatment with the indicated reagents to block viral entry. RT-qPCR was used to quantify N5 levels. The agents used included a vehicle control (DMSO), an ACE2 blocking antibody (‘ACE2ab’), a PIKfyve inhibitor Apilimod, an autolysosome acidification blocker bafilomycin, a cathepsin-L inhibitor Z-Phe-Tyr(tBu)-diazomethylketone (Z-FY-DK), a serine protease inhibitor aprotinin, a cathepsin-B inhibitor CA-074, and a TMPRSS2 inhibitor camostat mesylate. Dots represent separate replicates. *: p-val<0.05, **: p-val<0.01. N>=3 for all conditions. One-way ANOVA with Tukey's multiple comparisons. FIG. 2B graphically illustrates SARS-CoV-2 RNA (N5) levels in CMs pretreated with different viral infection blocking agents as detected by RT-qPCR quantification of N5 levels. CMs were pretreated with either vehicle control (DMSO), ACE2 blocking antibody (‘ACE2ab’) or a cathepsin-B and -L blocker (E64D) for 2 hours before infection with SARS-CoV-2 (MOI=0.006). The graph depicts fold changes relative to a vehicle control (DMSO). Duplicates were analyzed for significance by one-way ANOVA with Tukey's multiple comparisons. ***: p-value<0.001. FIG. 2C graphically illustrates levels of factors that prime the cell's innate immune response in CM samples exposed to SARS-CoV-2 for 48 h (MOI=0.006) after 2 h pretreatment with the indicated reagents to block viral entry. Dots represent separate replicates. *: p-val<0.05, **: p-val<0.01. N>=3 for all conditions. One-way ANOVA with Tukey's multiple comparisons.

FIG. 3A-3J illustrate the transcriptional effects of SARS-CoV-2 exposure to cardiac cells. FIG. 3A graphically illustrates the percentage of total viral reads that map to the SARS-CoV-2 viral genome in multiple cell types. iPSCs, ECs or CFs were exposed at an MOI of 0.006, and CMs were exposed to the virus at three different MOIs: 0.001 (‘Low’), 0.01 (‘Mid’) and 0.1 (‘High’). **: p-val<0.01; ***: p-val<0.001. FIG. 3B graphically illustrates principal component analysis of transcriptomic samples. Dot shapes and colors represent the different cell types and whether they were exposed to SARS-CoV-2 virus and, in the case of CMs, the different MOIs used. FIG. 3C shows a loading plot for genes marking cardiomyocyte state (forward-slashed hatching ///), SARSCoV-2 infection related factors (no shading), and immune response (reverse-slashing \\\). FIG. 3D is a bar graph comparing genes involved in sarcomeric structure and myosin contractility between the high infection and mock infection CM groups. FIG. 3E graphically illustrates single cell transcript levels of ACE2 in iPS-derived cardiac cells. Each dot represents normalized transcript levels in a single cell. FIG. 3F graphically illustrates single cell transcript levels of FURIN in iPS-derived cardiac cells. Each dot represents normalized transcript levels in a single cell. FIG. 3G graphically illustrates single cell transcript levels of cathepsin-L (CTSL) in iPS-derived cardiac cells. Each dot represents normalized transcript levels in a single cell. FIG. 3H graphically illustrates single cell transcript levels of cathepsin-B (CTSB) in iPS-derived cardiac cells. Each dot represents normalized transcript levels in a single cell. FIG. 3I graphically illustrates single cell transcript levels of PIKfyve in iPS-derived cardiac cells. Each dot represents normalized transcript levels in a single cell. FIG. 3J graphically illustrates single cell transcript levels of DPP4 in iPS-derived cardiac cells. Each dot represents normalized transcript levels in a single cell.

FIG. 4A-4F illustrate cytopathological features induced by SARS-CoV-2 infection in CMs. FIG. 4A shows representative immunofluorescence images of myofibrillar fragmentation in CMs at different timepoints after exposure to SARS-CoV-2. White arrows indicate fragments consisting of two bands of cTnT positive staining. Scale bars: 50 μm. FIG. 4B graphically illustrates numbers of cells presenting myofibrillar fragmentation at 24 h and 48 h post-exposure to SARS-CoV-2 (defined as at least one event of a cTnT doublet unaligned and dissociated from other myofibrils). The number of cells was normalized to total number of nuclei in the images counted. Each dot represents a separate infection sample. Each replicate is the additive count of nine randomly acquired fields of view. ***: p-val<0.001. FIG. 4C shows representative images of immunostained cells infected with SARS-CoV-2, illustrating that cells staining positively for viral dsRNA are adjacent to other cells with different degrees of myofibrillar fragmentation. White squares indicate zoomed in areas, with labels corresponding to insets. White arrows point at examples of cTnT doublets (myofibrillar fragments). FIG. 4D shows representative images of stained CMs displaying myofibrillar fragmentation. White arrows indicate cTnT-ACTN2-cTnT staining positive fragments. Scale bars: 50 μm, inset view: 15 μm. FIG. 4E shows TEM images of sarcomeres in mock (‘healthy’) and SARS-CoV-2 infected (MOI=0.006) CM cultured cells (top). Darker gray arrows denote the sarcomeric z-disks; lighter gray arrows indicates M-line locations. Healthy sarcomeres display clear I and A-bands, but fragmented SARS-CoV-2 exposed sarcomeres only possess thin filaments. The image at the upper right is an expanded image of sarcomeric z-disks (arrows). The two images at the bottom are representative TEM image of a healthy nucleus (left), and a nucleus of a cell infected with SARS-CoV-2 (right). FIG. 4F shows an image of a cultured CM that was immunofluorescently stained after incubation with live SARS-CoV-2. The view to the right is an expanded view of the inset shown at the left, indicating that cells that have lost nuclear material.

FIG. 5A-5G illustrate pathological features of autopsy myocardial tissue from SARS-CoV-2 infected patients. FIG. 5A shows images of healthy neonatal left ventricle tissue stained with Hematoxylin and Eosin (H&E) to facilitate identification of the nucleus and other cellular structures. FIG. 5B shows images of H&E stained myocardial tissue from a COVID-19 patient with diagnosed myocarditis. Black boxes indicate the regions shown directly below that are at higher magnification. Arrows indicate cardiomyocytes showing a loss of nuclear material. FIG. 5C graphically illustrates the numbers of nuclei per field of view of intact myocardium and disrupted myocardium from SARS-CoV-2 patients. Statistical significance was determined by fitting to a Poisson generalized linear model, p-val<0.02. FIG. 5D shows representative H&E staining images of myocardial tissue from COVID-19 patients without diagnosed myocarditis. Darker gray arrows denote putative nuclear locations with loss of nuclear material. Lighter grey arrows indicate sarcomeric condensation. Black arrows indicate breakage at the intercalated disks between cardiomyocytes. FIG. 5E shows representative images from the myocardial tissue of a COVID-19 myocarditis patient immunohistochemical stained for troponin (cTnt, green in the original), collagen IV (grey in the original), and DAPI (blue in the original). Autofluorescence was also used to facilitate visualization of the images. Cardiomyocytes show diffuse and disorganized troponin staining with occasional cells in the blood vessel staining positively for troponin. White boxes indicate the regions shown directly below that are at higher magnification. White arrows indicate cardiac troponin T material in the cytoplasm of a mononuclear cell within a blood vessel. FIG. 5F shows images of a region of the heart from a COVID-19 patient denoting the transition from healthy to sick myocardium. White boxes indicate the regions shown to the right that are at higher magnification. The disrupted myocardium region is characterized by extensive breaks in α-actinin 2 (ACTN2) staining. FIG. 5G shows immunohistochemically stained images illustrating that viral nucleocapsid protein (magenta in the original; e.g., lower right-center) and α-actinin 2 (green in the original; striated tissue throughout) yielded no recognizable signal aside from occasional, unidentified puncta.

DETAILED DESCRIPTION

As illustrated herein, cardiomyocytes (CMs) can easily be infected by corona viruses, including SARS-CoV-2. Methods are described herein for identifying compounds that can inhibit or prevent such infection.

Such methods can include (a) contacting cardiomyocytes with one or more test agents either before, during or after the cardiomyocytes have been contacted (infected) with corona viruses, for example SARS-CoV-2; and (b) observing whether the cardiomyocytes are enucleated, observing whether the cardiomyocytes have cleaved cardiac myofibrils, observing whether the cardiomyocytes have cleavages in their titin proteins. The assays can also include measuring the number or reproduction rate of the corona viruses compared to a control. The measurements can be performed at one or more time points after the cardiomyocytes are contacted with the one or more test agents. The control can be untreated cardiomyocytes, meaning cardiomyocytes that were not contacted with a test agent. In some cases, the control can be cardiomyocytes contacted with a compound or biological known to inhibit or prevent corona virus infection.

The cardiomyocytes can be obtained from a variety of sources, for example, from existing cardiomyocyte cell lines, from healthy subjects, and/or from patients with cardiac conditions or cardiac diseases. In some cases, the cardiomyocytes can be obtained from induced pluripotent stem cells (iPSCs), which can be generated from cells obtained from healthy subjects or from patients with cardiac conditions or cardiac diseases. For example, cardiomyocytes can be obtained from induced pluripotent stem cells (iPSCs) generated from cells with genetic mutations, including genetic mutations that adversely affect heart function, that adversely affect immune function, or a combination thereof. The cardiomyocytes can, in another example, be obtained from induced pluripotent stem cells (iPSCs) that have mutations in one or more of their immune-related genes, for example, in their innate immune genes. Such mutations can make an individual more vulnerable to COVID-19 infection.

Test Agents

A variety of test agents (e.g., compounds and/or biological agents) can be tested to identify useful agent that reduce SARS-CoV-2 virally induced myofibrillar disruption, sarcomeric fragmentation, nuclear staining, enucleation, cardiac troponin solute levels, or a combination thereof in cardiomyocytes compared to a control assay of cardiomyocytes in the presence of SARS-CoV-2 virus without the test compound(s)/biological agents. For example, the test agents can be one or more small molecules, antibodies, nucleic acids, carbohydrates, proteins, peptides, or a combination thereof. Any such test agents can be tested and/or evaluated in the assays.

Cells for Test Assays

A population of cardiomyocytes for testing can be derived from essentially any source and can be heterogeneous or homogeneous. In certain embodiments, the cells to be tested as described herein are adult cells, including adult cardiomyocytes from essentially any accessible source. In other embodiments, the cells used are cardiomyocytes generated from induced pluripotent stem cells (iPSCs). The cells used to generate the iPSCs can be adult cells, adult stem cells, progenitor cells, or somatic cells obtained from healthy subjects or from patients with cardiac conditions or cardiac diseases. In still other embodiments, the cells used to generate iPSCs include any type of cell from a newborn, including, but not limited to newborn cord blood, newborn stem cells, progenitor cells, and tissue-derived cells (e.g., somatic cells). Accordingly, a starting population of cells that is used to generate iPSCs, can be essentially any live somatic cell type.

The cardiomyocytes can be autologous or allogeneic cells (relative to a subject to be treated or who may receive the cells).

In some cases, cardiomyocytes from healthy subjects are used in the test assays. In other cases, cardiomyocytes from subjects with cardiac conditions are used in the test assays. Cardiomyocyte cell lines can be used in the test assays. Alternatively, the cardiomyocytes can be isolated from a healthy subject, a subject with a cardiac condition, or the cardiomyocytes can be generated from induced pluripotent stem cells (iPSCs) from either healthy subjects or subjects with a cardiac condition. For example, cardiomyocytes can be obtained from induced pluripotent stem cells (iPSCs) generated from cells with genetic mutations, including genetic mutations that adversely affect heart function, that adversely affect immune function, or a combination thereof. The cardiomyocytes can, in another example, be obtained from induced pluripotent stem cells (iPSCs) that have mutations in one or more of their immune-related genes, for example, in their innate immune genes. Such mutations can make an individual more vulnerable to COVID-19 infection.

Cardiomyocytes can be generated from induced pluripotent stem cells (iPSCs) by any convenient method. For example, the cardiomyocytes can be generated from iPSCs using the methods described in WO 2015/038704, which is incorporated herein by reference in its entirety.

Cardiomyocytes from subjects with a variety of cardiac diseases and conditions can be used in the assays described herein. For example, the cardiomyocytes can be from any subject with any cardiac pathology or cardiac dysfunction.

The terms “cardiac pathology” or “cardiac dysfunction” are used interchangeably and refer to any impairment in the heart's pumping function. This includes, for example, impairments in contractility, impairments in ability to relax (sometimes referred to as diastolic dysfunction), abnormal or improper functioning of the heart's valves, diseases of the heart muscle (sometimes referred to as cardiomyopathies), diseases such as angina pectoris, myocardial ischemia and/or infarction characterized by inadequate blood supply to the heart muscle, infiltrative diseases such as amyloidosis and hemochromatosis, global or regional hypertrophy (such as may occur in some kinds of cardiomyopathy or systemic hypertension), and abnormal communications between chambers of the heart.

As used herein, the term “cardiomyopathy” refers to any disease or dysfunction of the myocardium (heart muscle) in which the heart is abnormally enlarged, thickened and/or stiffened. As a result, the heart muscle's ability to pump blood is usually weakened. The etiology of the disease or disorder may be, for example, inflammatory, metabolic, toxic, infiltrative, fibroplastic, hematological, genetic, or unknown in origin. There are two general types of cardiomyopathies: ischemic (resulting from a lack of oxygen) and non-ischemic.

Ischemic cardiomyopathy is a chronic disorder caused by coronary artery disease (a disease in which there is atherosclerotic narrowing or occlusion of the coronary arteries on the surface of the heart). Coronary artery disease often leads to episodes of cardiac ischemia, in which the heart muscle is not supplied with enough oxygen-rich blood.

Non-ischemic cardiomyopathy is generally classified into three groups based primarily on clinical and pathological characteristics: dilated cardiomyopathy, hypertrophic cardiomyopathy and restrictive and infiltrative cardiomyopathy.

In another embodiment, the cardiac pathology is a genetic disease such as Duchenne muscular dystrophy and Emery Dreiffuss dilated cardiomyopathy.

For example, the cardiac pathology can be selected from the group consisting of congestive heart failure, myocardial infarction, cardiac ischemia, myocarditis and arrhythmia.

Titin

Cardiac muscle is striated, like skeletal muscle, with actin and myosin arranged in sarcomeres to enable contractile function. The actin and myosin filaments have a specific and constant length of about a few micrometers. The filaments are organized into repeated subunits along the length of the myofibril. These subunits are called sarcomeres. Muscle cells are largely filled with myofibrils running parallel to each other along the long axis of the cell. The sarcomeric subunits of one myofibril are in nearly perfect alignment with those of the myofibrils next to it. This alignment provides optical properties so that cells to appear striped or striated.

Titin constitutes the third myofilament of cardiac muscle, with a single giant polypeptide spanning from Z-disk to the M-band region of the sarcomere. Titin has two general regions, an N-terminal I-band and a C-terminal A-band. An approximate 1.0 MDa region in the I-band is extensible and consists of tandemly arranged immunoglobulin (Ig)-like domains that make up proximal (near Z-disk) and distal (near A-I junction) segments, interspersed by the PEVK sequence (rich in proline, glutamate, valine, and lysine residues) and an N2B element.

The C-terminal titin region of about 2 MDa includes the A-band and is inextensible. This C-terminal region is composed of regular arrays of Ig and fibronectin type 3 (Fn3) modules forming so-called super-repeats. The A-band is thought to act as a protein-ruler and possesses kinase activity. An N-terminal Z-disc region and a C-terminal M-band region bind to the Z-line and M-line of the sarcomere, respectively, so that a single titin molecule spans half the length of a sarcomere. Titin also contains binding sites for muscle associated proteins and serves as an adhesion template for assembly of contractile machinery in muscle cells. The M-band is encoded by TTN exons 359-364.

Considerable variability exists in the I-band, the M-line, and the Z-disc regions of titin. Variability in the I-band region contributes to the differences in elasticity of different titin isoforms and, therefore, to the differences in elasticity of different muscle types. Mutations in this gene are associated with familial hypertrophic cardiomyopathy. Autoantibodies to titin are produced in patients with the autoimmune disease scleroderma.

The titin protein is encoded by the TTN gene, which is located on human chromosome 2, at NC_000002.12 (178525989..178807423, complement; see website at ncbi.nlm.nih.gov/gene?LinkName=protein_gene&from_uid=291045223). Alternative splicing of the TTN gene results in multiple transcript variants.

One example of a human titin protein sequence has UniProt accession number A0A0A0MRA3-1; this titin protein sequence is shown below as SEQ ID NO:1.

10         20         30         40         50
MTTQAPTFTQ PLQSVVVLEG STATFEAHIS GFPVPEVSWF RDGQVISTST
60         70         80         90        100
LPGVQISFSD GRAKLTIPAV TKANSGRYSL KATNGSGQAT STAELLVKAE
110        120        130        140        150
TAPPNFVQRL QSMTVRQGSQ VRLQVRVTGI PTPVVKFYRD GAEIQSSLDF
160        170        180        190        200
QISQEGDLYS LLIAEAYPED SGTYSVNATN SVGRATSTAE LLVQGEEEVP
210        220        230        240        250
AKKTKTIVST AQISESRQTR IEKKIEAHED ARSIATVEMV IDGAAGQQLP
260        270        280        290        300
HKTPPRIPPK PKSRSPTPPS IAAKAQLARQ QSPSPIRHSP SPVRHVRAPT
310        320        330        340        350
PSPVRSVSPA ARISTSPIRS VRSPLLMRKT QASTVATGPE VPPPWKQEGY
360        370        380        390        400
VASSSEAEMR ETTLTTSTQI RTEERWEGRY GVQEQVTISG AAGAAASVSA
410        420        430        440        450
SASYAAEAVA TGAKEVKQDA DKSAAVATVV AAVDMARVRE PVISAVEQTA
460        470        480        490        500
QRTTTTAVHI QPAQEQVRKE AEKTAVTKVV VAADKAKEQE LKSRTKEVIT
510        520        530        540        550
TKQEQMHVTH EQIRKETEKT FVPKVVISAA KAKEQETRIS EEITKKQKQV
560        570        580        590        600
TQEAIMKETR KTVVPKVIVA TPKVKEQDLV SRGREGITTK REQVQITQEK
610        620        630        640        650
MRKEAEKTAL STIAVATAKA KEQETILRTR ETMATRQEQI QVTHGKVDVG
660        670        680        690        700
KKAEAVATVV AAVDQARVRE PREPGHLEES YAQQTTLEYG YKERISAAKV
710        720        730        740        750
AEPPQRPASE PHVVPKAVKP RVIQAPSETH IKTTDQKGMH ISSQIKKTTD
760        770        780        790        800
LTTERLVHVD KRPRTASPHF TVSKISVPKT EHGYEASIAG SAIATLQKEL
810        820        830        840        850
SATSSAQKIT KSVKAPTVKP SETRVRAEPT PLPQFPFADT PDTYKSEAGV
860        870        880        890        900
EVKKEVGVSI TGTTVREERF EVLHGREAKV TETARVPAPV EIPVTPPTLV
910        920        930        940        950
SGLKNVTVIE GESVTLECHI SGYPSPTVTW YREDYQIESS IDFQITFQSG
960        970        980        990       1000
IARLMIREAF AEDSGRFTCS AVNEAGTVST SCYLAVQVSE EFEKETTAVT
1010       1020       1030       1040       1050
EKFTTEEKRF VESRDVVMTD TSLTEEQAGP GEPAAPYFIT KPVVQKLVEG
1060       1070       1080       1090       1100
GSVVFGCQVG GNPKPHVYWK KSGVPLTTGY RYKVSYNKQT GECKLVISMT
1110       1120       1130       1140       1150
FADDAGEYTI VVRNKHGETS ASASLLEEAD YELLMKSQQE MLYQTQVTAF
1160       1170       1180       1190       1200
VQEPKVGETA PGFVYSEYEK EYEKEQALIR KKMAKDTVVV RTYVEDQEFH
1210       1220       1230       1240       1250
ISSFEERLIK EIEYRIIKTT LEELLEEDGE EKMAVDISES EAVESGEDSR
1260       1270       1280       1290       1300
IKNYRILEGM GVTFHCKMSG YPLPKIAWYK DGKRIKHGER YQMDFLQDGR
1310       1320       1330       1340       1350
ASLRIPVVLP EDEGIYTAFA SNIKGNAICS GKLYVEPAAP LGAPTYIPTL
1360       1370       1380       1390       1400
EPVSRIRSLS PRSVSRSPIR MSPARMSPAR MSPARMSPAR MSPGRRLEET
1410       1420       1430       1440       1450
DESQLERLYK PVFVLKPVSF KCLEGQTARF DLKVVGRPMP ETFWFHDGQQ
1460       1470       1480       1490       1500
IVNDYTHKVV IKEDGTQSLI IVPATPSDSG EWTVVAQNRA GRSSISVILT
1510       1520       1530       1540       1550
VEAVEHQVKP MFVEKLKNVN IKEGSRLEMK VRATGNPNPD IVWLKNSDII
1560       1570       1580       1590       1600
VPHKYPKIRI EGTKGEAALK IDSTVSQDSA WYTATAINKA GRDTTRCKVN
1610       1620       1630       1640       1650
VEVEFAEPEP ERKLIIPRGT YRAKEIAAPE LEPLHLRYGQ EQWEEGDLYD
1660       1670       1680       1690       1700
KEKQQKPFFK KKLTSLRLKR FGPAHFECRL TPIGDPTMVV EWLHDGKPLE
1710       1720       1730       1740       1750
AANRLRMINE FGYCSLDYGV AYSRDSGIIT CRATNKYGTD HTSATLIVKD
1760       1770       1780       1790       1800
EKSLVEESQL PEGRKGLQRI EELERMAHEG ALTGVTTDQK EKQKPDIVLY
1810       1820       1830       1840       1850
PEPVRVLEGE TARFRCRVTG YPQPKVNWYL NGQLIRKSKR FRVRYDGIHY
1860       1870       1880       1890       1900
LDIVDCKSYD TGEVKVTAEN PEGVIEHKVK LEIQQREDFR SVLRRAPEPR
1910       1920       1930       1940       1950
PEFHVHEPGK LQFEVQKVDR PVDTTETKEV VKLKRAERIT HEKVPEESEE
1960       1970       1980       1990       2000
LRSKFKRRTE EGYYEAITAV ELKSRKKDES YEELLRKTKD ELLHWTKELT
2010       2020       2030       2040       2050
EEEKKALAEE GKITIPTFKP DKIELSPSME APKIFERIQS QTVGQGSDAH
2060       2070       2080       2090       2100
FRVRVVGKPD PECEWYKNGV KIERSDRIYW YWPEDNVCEL VIRDVTAEDS
2110       2120       2130       2140       2150
ASIMVKAINI AGETSSHAFL LVQAKQLITF TQELQDVVAK EKDTMATFEC
2160       2170       2180       2190       2200
ETSEPFVKVK WYKDGMEVHE GDKYRMHSDR KVHFLSILTI DTSDAEDYSC
2210       2220       2230       2240       2250
VLVEDENVKT TAKLIVEGAV VEFVKELQDI EVPESYSGEL ECIVSPENIE
2260       2270       2280       2290       2300
GKWYHNDVEL KSNGKYTITS RRGRQNLTVK DVTKEDQGEY SFVIDGKKTT
2310       2320       2330       2340       2350
CKLKMKPRPI AILQGLSDQK VCEGDIVQLE VKVSLESVEG VWMKDGQEVQ
2360       2370       2380       2390       2400
PSDRVHIVID KQSHMLLIED MTKEDAGNYS FTIPALGLST SGRVSVYSVD
2410       2420       2430       2440       2450
VITPLKDVNV IEGTKAVLEC KVSVPDVTSV KWYLNDEQIK PDDRVQAIVK
2460       2470       2480       2490       2500
GTKQRLVINR THASDEGPYK LIVGRVETNC NLSVEKIKII RGLRDLTCTE
2510       2520       2530       2540       2550
TQNVVFEVEL SHSGIDVLWN FKDKEIKPSS KYKIEAHGKI YKLTVLNMMK
2560       2570       2580       2590       2600
DDEGKYTFYA GENMTSGKLT VAGGAISKPL TDQTVAESQE AVFECEVANP
2610       2620       2630       2640       2650
DSKGEWLRDG KHLPLTNNIR SESDGHKRRL IIAATKLDDI GEYTYKVATS
2660       2670       2680       2690       2700
KTSAKLKVEA VKIKKTLKNL TVTETQDAVF TVELTHPNVK GVQWIKNGVV
2710       2720       2730       2740       2750
LESNEKYAIS VKGTIYSLRI KNCAIVDESV YGFRLGRLGA SARLHVETVK
2760       2770       2780       2790       2800
IIKKPKDVTA LENATVAFEV SVSHDTVPVK WFHKSVEIKP SDKHRLVSER
2810       2820       2830       2840       2850
KVHKLMLQNI SPSDAGEYTA VVGQLECKAK LFVETLHITK TMKNIEVPET
2860       2870       2880       2890       2900
KTASFECEVS HFNVPSMWLK NGVEIEMSEK FKIVVQGKLH QLIIMNTSTE
2910       2920       2930       2940       2950
DSAEYTFVCG NDQVSATLTV TPIMITSMLK DINAEEKDTI TFEVTVNYEG
2960       2970       2980       2990       3000
ISYKWLKNGV EIKSTDKCQM RTKKLTHSLN IRNVHFGDAA DYTFVAGKAT
3010       3020       3030       3040       3050
STATLYVEAR HIEFRKHIKD IKVLEKKRAM FECEVSEPDI TVQWMKDDQE
3060       3070       3080       3090       3100
LQITDRIKIQ KEKYVHRLLI PSTRMSDAGK YTVVAGGNVS TAKLFVEGRD
3110       3120       3130       3140       3150
VRIRSIKKEV QVIEKQRAVV EFEVNEDDVD AHWYKDGIEI NFQVQERHKY
3160       3170       3180       3190       3200
VVERRIHRMF ISETRQSDAG EYTFVAGRNR SSVTLYVNAP EPPQVLQELQ
3210       3220       3230       3240       3250
PVTVQSGKPA RFCAVISGRP QPKISWYKEE QLLSTGFKCK FLHDGQEYTL
3260       3270       3280       3290       3300
LLIEAFPEDA AVYTCEAKND YGVATTSASL SVEVPEVVSP DQEMPVYPPA
3310       3320       3330       3340       3350
IITPLQDTVT SEGQPARFQC RVSGTDLKVS WYSKDKKIKP SRFFRMTQFE
3360       3370       3380       3390       3400
DTYQLEIAEA YPEDEGTYTF VASNAVGQVS STANLSLEVQ ALDRQSSGKD
3410       3420       3430       3440       3450
VRESTKSQAV ADSSFTKEES KISQKEIKSF QGSSYEYEVQ VFESVSQSSI
3460       3470       3480       3490       3500
HTAASVQDTQ LCHTASLSQI AESTELSKEC AKESTGEAPK IFLHLQDVTV
3510       3520       3530       3540       3550
KCGDTAQFLC VLKDDSFIDV TWTHEGAKIE ESERLKQSQN GNIQFLTICN
3560       3570       3580       3590       3600
VQLVDQGLYS CIVHNDCGER TTSAVLSVEG APESILHERI EQEIEMEMKE
3610       3620       3630       3640       3650
FSSSFLSAEE EGLHSAELQL SKINETLELL SESPVYPTKF DSEKEGTGPI
3660       3670       3680       3690       3700
FIKEVSNADI SMGDVATLSV TVIGIPKPKI QWFFNGVLLT PSADYKFVFD
3710       3720       3730       3740       3750
GDDHSLIILF TKLEDEGEYT CMASNDYGKT ICSAYLKINS KGEGHKDTET
3760       3770       3780       3790       3800
ESAVAKSLEK LGGPCPPHFL KELKPIRCAQ GLPAIFEYTV VGEPAPTVTW
3810       3820       3830       3840       3850
FKENKQLCTS VYYTIIHNPN GSGTFIVNDP QREDSGLYIC KAENMLGEST
3860       3870       3880       3890       3900
CAAELLVLLE DTDMTDTPCK AKSTPEAPED FPQTPLKGPA VEALDSEQEI
3910       3920       3930       3940       3950
ATFVKDTILK AALITEENQQ LSYEHIAKAN ELSSQLPLGA QELQSILEQD
3960       3970       3980       3990       4000
KLTPESTREF LCINGSIHFQ PLKEPSPNLQ LQIVQSQKTF SKEGILMPEE
4010       4020       4030       4040       4050
PETQAVLSDT EKIFPSAMSI EQINSLTVEP LKTLLAEPEG NYPQSSIEPP
4060       4070       4080       4090       4100
MHSYLTSVAE EVLSPKEKTV SDTNREQRVT LQKQEAQSAL ILSQSLAEGH
4110       4120       4130       4140       4150
VESLQSPDVM ISQVNYEPLV PSEHSCTEGG KILIESANPL ENAGQDSAVR
4160       4170       4180       4190       4200
IEEGKSLRFP LALEEKQVLL KEEHSDNVVM PPDQIIESKR EPVAIKKVQE
4210       4220       4230       4240       4250
VQGRDLLSKE SLLSGIPEEQ RLNLKIQICR ALQAAVASEQ PGLFSEWLRN
4260       4270       4280       4290       4300
IEKVEVEAVN ITQEPRHIMC MYLVTSAKSV TEEVTIIIED VDPQMANLKM
4310       4320       4330       4340       4350
ELRDALCAII YEEIDILTAE GPRIQQGAKT SLQEEMDSFS GSQKVEPITE
4360       4370       4380       4390       4400
PEVESKYLIS TEEVSYFNVQ SRVKYLDATP VTKGVASAVV SDEKQDESLK
4410       4420       4430       4440       4450
PSEEKEESSS ESGTEEVATV KIQEAEGGLI KEDGPMIHTP LVDTVSEEGD
4460       4470       4480       4490       4500
IVHLTTSITN AKEVNWYFEN KLVPSDEKFK CLQDQNTYTL VIDKVNTEDH
4510       4520       4530       4540       4550
QGEYVCEALN DSGKTATSAK LTVVKRAAPV IKRKIEPLEV ALGHLAKFTC
4560       4570       4580       4590       4600
EIQSAPNVRF QWFKAGREIY ESDKCSIRSS KYISSLEILR TQVVDCGEYT
4610       4620       4630       4640       4650
CKASNEYGSV SCTATLTVTV PGGEKKVRKL LPERKPEPKE EVVLKSVLRK
4660       4670       4680       4690       4700
RPEEEEPKVE PKKLEKVKKP AVPEPPPPKP VEEVEVPTVT KRERKIPEPT
4710       4720       4730       4740       4750
KVPEIKPAIP LPAPEPKPKP EAEVKTIKPP PVEPEPTPIA APVTVPVVGK
4760       4770       4780       4790       4800
KAEAKAPKEE AAKPKGPIKG VPKKTPSPIE AERRKLRPGS GGEKPPDEAP
4810       4820       4830       4840       4850
FTYQLKAVPL KFVKEIKDII LTESEFVGSS AIFECLVSPS TAITTWMKDG
4860       4870       4880       4890       4900
SNIRESPKHR FIADGKDRKL HIIDVQLSDA GEYTCVLRLG NKEKTSTAKL
4910       4920       4930       4940       4950
VVEELPVRFV KTLEEEVTVV KGQPLYLSCE LNKERDVVWR KDGKIVVEKP
4960       4970       4980       4990       5000
GRIVPGVIGL MRALTINDAD DTDAGTYTVT VENANNLECS SCVKVVEVIR
5010       5020       5030       5040       5050
DWLVKPIRDQ HVKPKGTAIF ACDIAKDTPN IKWFKGYDEI PAEPNDKTEI
5060       5070       5080       5090       5100
LRDGNHLYLK IKNAMPEDIA EYAVEIEGKR YPAKLTLGER EVELLKPIED
5110       5120       5130       5140       5150
VTIYEKESAS FDAEISEADI PGQWKLKGEL LRPSPTCEIK AEGGKRFLTL
5160       5170       5180       5190       5200
HKVKLDQAGE VLYQALNAIT TAILTVKEIE LDFAVPLKDV TVPERRQARF
5210       5220       5230       5240       5250
ECVLTREANV IWSKGPDIIK SSDKFDIIAD GKKHILVIND SQFDDEGVYT
5260       5270       5280       5290       5300
AEVEGKKTSA RLFVTGIRLK FMSPLEDQTV KEGETATFVC ELSHEKMHVV
5310       5320       5330       5340       5350
WFKNDAKLHT SRTVLISSEG KTHKLEMKEV TLDDISQIKA QVKELSSTAQ
5360       5370       5380       5390       5400
LKVLEADPYF TVKLHDKTAV EKDEITLKCE VSKDVPVKWF KDGEEIVPSP
5410       5420       5430       5440       5450
KYSIKADGLR RILKIKKADL KDKGEYVCDC GTDKTKANVT VEARLIKVEK
5460       5470       5480       5490       5500
PLYGVEVFVG ETAHFEIELS EPDVHGQWKL KGQPLTASPD CEIIEDGKKH
5510       5520       5530       5540       5550
ILILHNCQLG MTGEVSFQAA NAKSAANLKV KELPLIFITP LSDVKVFEKD
5560       5570       5580       5590       5600
EAKFECEVSR EPKTFRWLKG TQEITGDDRF ELIKDGTKHS MVIKSAAFED
5610       5620       5630       5640       5650
EAKYMFEAED KHTSGKLIIE GIRLKFLTPL KDVTAKEKES AVFTVELSHD
5660       5670       5680       5690       5700
NIRVKWFKND QRLHTTRSVS MQDEGKTHSI TFKDLSIDDT SQIRVEAMGM
5710       5720       5730       5740       5750
SSEAKLTVLE GDPYFTGKLQ DYTGVEKDEV ILQCEISKAD APVKWFKDGK
5760       5770       5780       5790       5800
EIKPSKNAVI KADGKKRMLI LKKALKSDIG QYTCDCGTDK TSGKLDIEDR
5810       5820       5830       5840       5850
EIKLVRPLHS VEVMETETAR FETEISEDDI HANWKLKGEA LLQTPDCEIK
5860       5870       5880       5890       5900
EEGKIHSLVL HNCRLDQTGG VDFQAANVKS SAHLRVKPRV IGLLRPLKDV
5910       5920       5930       5940       5950
TVTAGETATF DCELSYEDIP VEWYLKGKKL EPSDKVVPRS EGKVHTLTLR
5960       5970       5980       5990       6000
DVKLEDAGEV QLTAKDFKTH ANLFVKEPPV EFTKPLEDQT VEEGATAVLE
6010       6020       6030       6040       6050
CEVSRENAKV KWFKNGTEIL KSKKYEIVAD GRVRKLVIHD CTPEDIKTYT
6060       6070       6080       6090       6100
CDAKDFKTSC NLNVVPPHVE FLRPLTDLQV REKEMARFEC ELSRENAKVK
6110       6120       6130       6140       6150
WFKDGAEIKK GKKYDIISKG AVRILVINKC LLDDEAEYSC EVRTARTSGM
6160       6170       6180       6190       6200
LTVLEEEAVF TKNLANIEVS ETDTIKLVCE VSKPGAEVIW YKGDEEIIET
6210       6220       6230       6240       6250
GRYEILTEGR KRILVIQNAH LEDAGNYNCR LPSSRTDGKV KVHELAAEFI
6260       6270       6280       6290       6300
SKPQNLEILE GEKAEFVCSI SKESFPVQWK RDDKTLESGD KYDVIADGKK
6310       6320       6330       6340       6350
RVLVVKDATL QDMGTYVVMV GAARAAAHLT VIEKLRIVVP LKDTRVKEQQ
6360       6370       6380       6390       6400
EVVENCEVNT EGAKAKWFRN EEAIFDSSKY IILQKDLVYT LRIRDAHLDD
6410       6420       6430       6440       6450
QANYNVSLTN HRGENVKSAA NLIVEEEDLR IVEPLKDIET MEKKSVTFWC
6460       6470       6480       6490       6500
KVNRLNVTLK WTKNGEEVPF DNRVSYRVDK YKHMLTIKDC GFPDEGEYIV
6510       6520       6530       6540       6550
TAGQDKSVAE LLIIEAPTEF VEHLEDQTVT EFDDAVFSCQ LSREKANVKW
6560       6570       6580       6590       6600
YRNGREIKEG KKYKFEKDGS IHRLIIKDCR LDDECEYACG VEDRKSRARL
6610       6620       6630       6640       6650
FVEEIPVEII RPPQDILEAP GADVVFLAEL NKDKVEVQWL RNNMVVVQGD
6660       6670       6680       6690       6700
KHQMMSEGKI HRLQICDIKP RDQGEYRFIA KDKEARAKLE LAAAPKIKTA
6710       6720       6730       6740       6750
DQDLVVDVGK PLTMVVPYDA YPKAEAEWFK ENEPLSTKTI DTTAEQTSFR
6760       6770       6780       6790       6800
ILEAKKGDKG RYKIVLQNKH GKAEGFINLK VIDVPGPVRN LEVTETEDGE
6810       6820       6830       6840       6850
VSLAWEEPLT DGGSKIIGYV VERRDIKRKT WVLATDRAES CEFTVTGLQK
6860       6870       6880       6890       6900
GGVEYLFRVS ARNRVGTGEP VETDNPVEAR SKYDVPGPPL NVTITDVNRF
6910       6920       6930       6940       6950
GVSLTWEPPE YDGGAEITNY VIELRDKTSI RWDTAMTVRA EDLSATVTDV
6960       6970       6980       6990       7000
VEGQEYSFRV RAQNRIGVGK PSAATPFVKV ADPIERPSPP VNLTSSDQTQ
7010       7020       7030       7040       7050
SSVQLKWEPP LKDGGSPILG YIIERCEEGK DNWIRCNMKL VPELTYKVTG
7060       7070       7080       7090       7100
LEKGNKYLYR VSAENKAGVS DPSEILGPLT ADDAFVEPTM DLSAFKDGLE
7110       7120       7130       7140       7150
VIVPNPITIL VPSTGYPRPT ATWCFGDKVL ETGDRVKMKT LSAYAELVIS
7160       7170       7180       7190       7200
PSERSDKGIY TLKLENRVKT ISGEIDVNVI ARPSAPKELK FGDITKDSVH
7210       7220       7230       7240       7250
LTWEPPDDDG GSPLTGYVVE KREVSRKTWT KVMDFVTDLE FTVPDLVQGK
7260       7270       7280       7290       7300
EYLFKVCARN KCGPGEPAYV DEPVNMSTPA TVPDPPENVK WRDRTANSIF
7310       7320       7330       7340       7350
LTWDPPKNDG GSRIKGYIVE RCPRGSDKWV ACGEPVAETK MEVTGLEEGK
7360       7370       7380       7390       7400
WYAYRVKALN RQGASKPSRP TEEIQAVDTQ EAPEIFLDVK LLAGLTVKAG
7410       7420       7430       7440       7450
TKIELPATVT GKPEPKITWT KADMILKQDK RITIENVPKK STVTIVDSKR
7460       7470       7480       7490       7500
SDTGTYIIEA VNVCGRATAV VEVNVLDKPG PPAAFDITDV TNESCLLTWN
7510       7520       7530       7540       7550
PPRDDGGSKI TNYVVERRAT DSEVWHKLSS TVKDTNFKAT KLIPNKEYIF
7560       7570       7580       7590       7600
RVAAENMYGV GEPVQASPIT AKYQFDPPGP PTRLEPSDIT KDAVTLTWCE
7610       7620       7630       7640       7650
PDDDGGSPIT GYWVERLDPD TDKWVRCNKM PVKDTTYRVK GLTNKKKYRF
7660       7670       7680       7690       7700
RVLAENLAGP GKPSKSTEPI LIKDPIDPPW PPGKPTVKDV GKTSVRLNWT
7710       7720       7730       7740       7750
KPEHDGGAKI ESYVIEMLKT GTDEWVRVAE GVPTTQHLLP GLMEGQEYSF
7760       7770       7780       7790       7800
RVRAVNKAGE SEPSEPSDPV LCREKLYPPS PPRWLEVINI TKNTADLKWT
7810       7820       7830       7840       7850
VPEKDGGSPI TNYIVEKRDV RRKGWQTVDT TVKDTKCTVT PLTEGSLYVF
7860       7870       7880       7890       7900
RVAAENAIGQ SDYTEIEDSV LAKDTFTTPG PPYALAVVDV TKRHVDLKWE
7910       7920       7930       7940       7950
PPKNDGGRPI QRYVIEKKER LGTRWVKAGK TAGPDCNFRV TDVIEGTEVQ
7960       7970       7980       7990       8000
FQVRAENEAG VGHPSEPTEI LSIEDPTSPP SPPLDLHVTD AGRKHIAIAW
8010       8020       8030       8040       8050
KPPEKNGGSP IIGYHVEMCP VGTEKWMRVN SRPIKDLKFK VEEGVVPDKE
8060       8070       8080       8090       8100
YVLRVRAVNA IGVSEPSEIS ENVVAKDPDC KPTIDLETHD IIVIEGEKLS
8110       8120       8130       8140       8150
IPVPFRAVPV PTVSWHKDGK EVKASDRLTM KNDHISAHLE VPKSVRADAG
8160       8170       8180       8190       8200
IYTITLENKL GSATASINVK VIGLPGPCKD IKASDITKSS CKLTWEPPEF
8210       8220       8230       8240       8250
DGGTPILHYV LERREAGRRT YIPVMSGENK LSWTVKDLIP NGEYFFRVKA
8260       8270       8280       8290       8300
VNKVGGGEYI ELKNPVIAQD PKQPPDPPVD VEVHNPTAEA MTITWKPPLY
8310       8320       8330       8340       8350
DGGSKIMGYI IEKIAKGEER WKRCNEHLVP ILTYTAKGLE EGKEYQFRVR
8360       8370       8380       8390       8400
AENAAGISEP SRATPPTKAV DPIDAPKVIL RTSLEVKRGD EIALDASISG
8410       8420       8430       8440       8450
SPYPTITWIK DENVIVPEEI KKRAAPLVRR RKGEVQEEEP FVLPLTQRLS
8460       8470       8480       8490       8500
IDNSKKGESQ LRVRDSLRPD HGLYMIKVEN DHGIAKAPCT VSVLDTPGPP
8510       8520       8530       8540       8550
INFVFEDIRK TSVLCKWEPP LDDGGSEIIN YTLEKKDKTK PDSEWIVVTS
8560       8570       8580       8590       8600
TLRHCKYSVT KLIEGKEYLF RVRAENRFGP GPPCVSKPLV AKDPFGPPDA
8610       8620       8630       8640       8650
PDKPIVEDVT SNSMLVKWNE PKDNGSPILG YWLEKREVNS THWSRVNKSL
8660       8670       8680       8690       8700
LNALKANVDG LLEGLTYVFR VCAENAAGPG KFSPPSDPKT AHDPISPPGP
8710       8720       8730       8740       8750
PIPRVTDTSS TTIELEWEPP AFNGGGEIVG YFVDKQLVGT NEWSRCTEKM
8760       8770       8780       8790       8800
IKVRQYTVKE IREGADYKLR VSAVNAAGEG PPGETQPVTV AEPQEPPAVE
8810       8820       8830       8840       8850
LDVSVKGGIQ IMAGKTLRIP AVVTGRPVPT KVWTKEEGEL DKDRVVIDNV
8860       8870       8880       8890       8900
GTKSELIIKD ALRKDHGRYV ITATNSCGSK FAAARVEVFD VPGPVLDLKP
8910       8920       8930       8940       8950
VVTNRKMCLL NWSDPEDDGG SEITGFIIER KDAKMHTWRQ PIETERSKCD
8960       8970       8980       8990       9000
ITGLLEGQEY KFRVIAKNKF GCGPPVEIGP ILAVDPLGPP TSPERLTYTE
9010       9020       9030       9040       9050
RTKSTITLDW KEPRSNGGSP IQGYIIEKRR HDKPDFERVN KRLCPTTSFL
9060       9070       9080       9090       9100
VENLDEHQMY EFRVKAVNEI GESEPSLPLN VVIQDDEVPP TIKLRLSVRG
9110       9120       9130       9140       9150
DTIKVKAGEP VHIPADVTGL PMPKIEWSKN ETVIEKPTDA LQITKEEVSR
9160       9170       9180       9190       9200
SEAKTELSIP KAVREDKGTY TVTASNRLGS VFRNVHVEVY DRPSPPRNLA
9210       9220       9230       9240       9250
VTDIKAESCY LTWDAPLDNG GSEITHYVID KRDASRKKAE WEEVTNTAVE
9260       9270       9280       9290       9300
KRYGIWKLIP NGQYEFRVRA VNKYGISDEC KSDKVVIQDP YRLPGPPGKP
9310       9320       9330       9340       9350
KVLARTKGSM LVSWTPPLDN GGSPITGYWL EKREEGSPYW SRVSRAPITK
9360       9370       9380       9390       9400
VGLKGVEFNV PRLLEGVKYQ FRAMAINAAG IGPPSEPSDP EVAGDPIFPP
9410       9420       9430       9440       9450
GPPSCPEVKD KTKSSISLGW KPPAKDGGSP IKGYIVEMQE EGTTDWKRVN
9460       9470       9480       9490       9500
EPDKLITTCE CVVPNLKELR KYRFRVKAVN EAGESEPSDT TGEIPATDIQ
9510       9520       9530       9540       9550
EEPEVFIDIG AQDCLVCKAG SQIRIPAVIK GRPTPKSSWE FDGKAKKAMK
9560       9570       9580       9590       9600
DGVHDIPEDA QLETAENSSV IIIPECKRSH TGKYSITAKN KAGQKTANCR
9610       9620       9630       9640       9650
VKVMDVPGPP KDLKVSDITR GSCRLSWKMP DDDGGDRIKG YVIEKRTIDG
9660       9670       9680       9690       9700
KAWTKVNPDC GSTTFVVPDL LSEQQYFFRV RAENRFGIGP PVETIQRTTA
9710       9720       9730       9740       9750
RDPIYPPDPP IKLKIGLITK NTVHLSWKPP KNDGGSPVTH YIVECLAWDP
9760       9770       9780       9790       9800
TGTKKEAWRQ CNKRDVEELQ FTVEDLVEGG EYEFRVKAVN AAGVSKPSAT
9810       9820       9830       9840       9850
VGPVTVKDQT CPPSIDLKEF MEVEEGTNVN IVAKIKGVPF PTLTWFKAPP
9860       9870       9880       9890       9900
KKPDNKEPVL YDTHVNKLVV DDTCTLVIPQ SRRSDTGLYT ITAVNNLGTA
9910       9920       9930       9940       9950
SKEMRLNVLG RPGPPVGPIK FESVSADQMT LSWFPPKDDG GSKITNYVIE
9960       9970       9980       9990      10000
KREANRKTWV HVSSEPKECT YTIPKLLEGH EYVFRIMAQN KYGIGEPLDS
10010      10020      10030      10040      10050
EPETARNLFS VPGAPDKPTV SSVTRNSMTV NWEEPEYDGG SPVTGYWLEM
10060      10070      10080      10090      10100
KDTTSKRWKR VNRDPIKAMT LGVSYKVTGL IEGSDYQFRV YAINAAGVGP
10110      10120      10130      10140      10150
ASLPSDPATA RDPIAPPGPP FPKVTDWTKS SADLEWSPPL KDGGSKVTGY
10160      10170      10180      10190      10200
IVEYKEEGKE EWEKGKDKEV RGTKLVVTGL KEGAFYKFRV RAVNIAGIGE
10210      10220      10230      10240      10250
PGEVTDVIEM KDRLVSPDLQ LDASVRDRIV VHAGGVIRII AYVSGKPPPT
10260      10270      10280      10290      10300
VTWNMNERTL PQEATIETTA ISSSMVIKNC QRSHQGVYSL LAKNEAGERK
10310      10320      10330      10340      10350
KTIIVDVLDV PGPVGTPFLA HNLTNESCKL TWFSPEDDGG SPITNYVIEK
10360      10370      10380      10390      10400
RESDRRAWTP VTYTVTRQNA TVQGLIQGKA YFFRIAAENS IGMGPFVETS
10410      10420      10430      10440      10450
EALVIREPIT VPERPEDLEV KEVTKNTVTL TWNPPKYDGG SEIINYVLES
10460      10470      10480      10490      10500
RLIGTEKFHK VTNDNLLSRK YTVKGLKEGD TYEYRVSAVN IVGQGKPSFC
10510      10520      10530      10540      10550
TKPITCKDEL APPTLHLDER DKLTIRVGEA FALTGRYSGK PKPKVSWFKD
10560      10570      10580      10590      10600
EADVLEDDRT HIKTTPATLA LEKIKAKRSD SGKYCVVVEN STGSRKGFCQ
10610      10620      10630      10640      10650
VNVVDRPGPP VGPVSFDEVT KDYMVISWKP PLDDGGSKIT NYIIEKKEVG
10660      10670      10680      10690      10700
KDVWMPVTSA SAKTTCKVSK LLEGKDYIFR IHAENLYGIS DPLVSDSMKA
10710      10720      10730      10740      10750
KDRFRVPDAP DQPIVTEVTK DSALVTWNKP HDGGKPITNY ILEKRETMSK
10760      10770      10780      10790      10800
RWARVTKDPI HPYTKFRVPD LLEGCQYEFR VSAENEIGIG DPSPPSKPVF
10810      10820      10830      10840      10850
AKDPIAKPSP PVNPEAIDTT CNSVDLTWQP PRHDGGSKIL GYIVEYQKVG
10860      10870      10880      10890      10900
DEEWRRANHT PESCPETKYK VTGLRDGQTY KFRVLAVNAA GESDPAHVPE
10910      10920      10930      10940      10950
PVLVKDRLEP PELILDANMA REQHIKVGDT LRLSAIIKGV PFPKVTWKKE
10960      10970      10980      10990      11000
DRDAPTKARI DVTPVGSKLE IRNAAHEDGG IYSLTVENPA GSKTVSVKVL
11010      11020      11030      11040      11050
VLDKPGPPRD LEVSEIRKDS CYLTWKEPLD DGGSVITNYV VERRDVASAQ
11060      11070      11080      11090      11100
WSPLSATSKK KSHFAKHLNE GNQYLFRVAA ENQYGRGPFV ETPKPIKALD
11110      11120      11130      11140      11150
PLHPPGPPKD LHHVDVDKTE VSLVWNKPDR DGGSPITGYL VEYQEEGTQD
11160      11170      11180      11190      11200
WIKFKTVTNL ECVVTGLQQG KTYRFRVKAE NIVGLGLPDT TIPIECQEKL
11210      11220      11230      11240      11250
VPPSVELDVK LIEGLVVKAG TTVRFPAIIR GVPVPTAKWT TDGSEIKTDE
11260      11270      11280      11290      11300
HYTVETDNES SVLTIKNCLR RDTGEYQITV SNAAGSKTVA VHLTVLDVPG
11310      11320      11330      11340      11350
PPTGPINILD VTPEHMTISW QPPKDDGGSP VINYIVEKQD TRKDTWGVVS
11360      11370      11380      11390      11400
SGSSKTKLKI PHLQKGCEYV FRVRAENKIG VGPPLDSTPT VAKHKFSPPS
11410      11420      11430      11440      11450
PPGKPVVTDI TENAATVSWT LPKSDGGSPI TGYYMERREV TGKWVRVNKT
11460      11470      11480      11490      11500
PIADLKFRVT GLYEGNTYEF RVFAENLAGL SKPSPSSDPI KACRPIKPPG
11510      11520      11530      11540      11550
PPINPKLKDK SRETADLVWT KPLSDGGSPI LGYVVECQKP GTAQWNRINK
11560      11570      11580      11590      11600
DELIRQCAFR VPGLIEGNEY RFRIKAANIV GEGEPRELAE SVIAKDILHP
11610      11620      11630      11640      11650
PEVELDVTCR DVITVRVGQT IRILARVKGR PEPDITWTKE GKVLVREKRV
11660      11670      11680      11690      11700
DLIQDLPRVE LQIKEAVRAD HGKYIISAKN SSGHAQGSAI VNVLDRPGPC
11710      11720      11730      11740      11750
QNLKVTNVTK ENCTISWENP LDNGGSEITN FIVEYRKPNQ KGWSIVASDV
11760      11770      11780      11790      11800
TKRLIKANLL ANNEYYFRVC AENKVGVGPT IETKTPILAI NPIDRPGEPE
11810      11820      11830      11840      11850
NLHIADKGKT FVYLKWRRPD YDGGSPNLSY HVERRLKGSD DWERVHKGSI
11860      11870      11880      11890      11900
KETHYMVDRC VENQIYEFRV QTKNEGGESD WVKTEEVVVK EDLQKPVLDL
11910      11920      11930      11940      11950
KLSGVLTVKA GDTIRLEAGV RGKPFPEVAW TKDKDATDLT RSPRVKIDTR
11960      11970      11980      11990      12000
ADSSKFSLTK AKRSDGGKYV VTATNTAGSF VAYATVNVLD KPGPVRNLKI
12010      12020      12030      12040      12050
VDVSSDRCTV CWDPPEDDGG CEIQNYILEK CETKRMVWST YSATVLTPGT
12060      12070      12080      12090      12100
TVTRLIEGNE YIFRVRAENK IGTGPPTESK PVIAKTKYDK PGRPDPPEVT
12110      12120      12130      12140      12150
KVSKEEMTVV WNPPEYDGGK SITGYFLEKK EKHSTRWVPV NKSAIPERRM
12160      12170      12180      12190      12200
KVQNLLPDHE YQFRVKAENE IGIGEPSLPS RPVVAKDPIE PPGPPTNFRV
12210      12220      12230      12240      12250
VDTTKHSITL GWGKPVYDGG APIIGYVVEM RPKIADASPD EGWKRCNAAA
12260      12270      12280      12290      12300
QLVRKEFTVT SLDENQEYEF RVCAQNQVGI GRPAELKEAI KPKEILEPPE
12310      12320      12330      12340      12350
IDLDASMRKL VIVRAGCPIR LFAIVRGRPA PKVTWRKVGI DNVVRKGQVD
12360      12370      12380      12390      12400
LVDTMAFLVI PNSTRDDSGK YSLTLVNPAG EKAVFVNVRV LDTPGPVSDL
12410      12420      12430      12440      12450
KVSDVTKTSC HVSWAPPEND GGSQVTHYIV EKREADRKTW STVTPEVKKT
12460      12470      12480      12490      12500
SFHVTNLVPG NEYYFRVTAV NEYGPGVPTD VPKPVLASDP LSEPDPPRKL
12510      12520      12530      12540      12550
EVTEMTKNSA TLAWLPPLRD GGAKIDGYIT SYREEEQPAD RWTEYSVVKD
12560      12570      12580      12590      12600
LSLVVTGLKE GKKYKFRVAA RNAVGVSLPR EAEGVYEAKE QLLPPKILMP
12610      12620      12630      12640      12650
EQITIKAGKK LRIEAHVYGK PHPTCKWKKG EDEVVTSSHL AVHKADSSSI
12660      12670      12680      12690      12700
LIIKDVTRKD SGYYSLTAEN SSGTDTQKIK VVVMDAPGPP QPPFDISDID
12710      12720      12730      12740      12750
ADACSLSWHI PLEDGGSNIT NYIVEKCDVS RGDWVTALAS VTKTSCRVGK
12760      12770      12780      12790      12800
LIPGQEYIFR VRAENREGIS EPLTSPKMVA QFPFGVPSEP KNARVTKVNK
12810      12820      12830      12840      12850
DCIFVAWDRP DSDGGSPIIG YLIERKERNS LLWVKANDTL VRSTEYPCAG
12860      12870      12880      12890      12900
LVEGLEYSFR IYALNKAGSS PPSKPTEYVT ARMPVDPPGK PEVIDVTKST
12910      12920      12930      12940      12950
VSLIWARPKH DGGSKIIGYF VEACKLPGDK WVRCNTAPHQ IPQEEYTATG
12960      12970      12980      12990      13000
LEEKAQYQFR AIARTAVNIS PPSEPSDPVT ILAENVPPRI DLSVAMKSLL
13010      13020      13030      13040      13050
TVKAGTNVCL DATVFGKPMP TVSWKKDGTL LKPAEGIKMA MQRNLCTLEL
13060      13070      13080      13090      13100
FSVNRKDSGD YTITAENSSG SKSATIKLKV LDKPGPPASV KINKMYSDRA
13110      13120      13130      13140      13150
MLSWEPPLED GGSEITNYIV DKRETSRPNW AQVSATVPIT SCSVEKLIEG
13160      13170      13180      13190      13200
HEYQFRICAE NKYGVGDPVF TEPAIAKNPY DPPGRCDPPV ISNITKDHMT
13210      13220      13230      13240      13250
VSWKPPADDG GSPITGYLLE KRETQAVNWT KVNRKPIIER TLKATGLQEG
13260      13270      13280      13290      13300
TEYEFRVTAI NKAGPGKPSD ASKAAYARDP QYPPGPPAFP KVYDTTRSSV
13310      13320      13330      13340      13350
SLSWGKPAYD GGSPIIGYLV EVKRADSDNW VRCNLPQNLQ KTRFEVTGLM
13360      13370      13380      13390      13400
EDTQYQFRVY AVNKIGYSDP SDVPDKHYPK DILIPPEGEL DADLRKTLIL
13410      13420      13430      13440      13450
RAGVTMRLYV PVKGRPPPKI TWSKPNVNLR DRIGLDIKST DFDTFLRCEN
13460      13470      13480      13490      13500
VNKYDAGKYI LTLENSCGKK EYTIVVKVLD TPGPPVNVTV KEISKDSAYV
13510      13520      13530      13540      13550
TWEPPIIDGG SPIINYVVQK RDAERKSWST VTTECSKTSF RVANLEEGKS
13560      13570      13580      13590      13600
YFFRVFAENE YGIGDPGETR DAVKASQTPG PVVDLKVRSV SKSSCSIGWK
13610      13620      13630      13640      13650
KPHSDGGSRI IGYVVDFLTE ENKWQRVMKS LSLQYSAKDL TEGKEYTFRV
13660      13670      13680      13690      13700
SAENENGEGT PSEITVVARD DVVAPDLDLK GLPDLCYLAK ENSNFRLKIP
13710      13720      13730      13740      13750
IKGKPAPSVS WKKGEDPLAT DTRVSVESSA VNTTLIVYDC QKSDAGKYTI
13760      13770      13780      13790      13800
TLKNVAGTKE GTISIKVVGK PGIPTGPIKF DEVTAEAMTL KWAPPKDDGG
13810      13820      13830      13840      13850
SEITNYILEK RDSVNNKWVT CASAVQKTTF RVTRLHEGME YTFRVSAENK
13860      13870      13880      13890      13900
YGVGEGLKSE PIVARHPFDV PDAPPPPNIV DVRHDSVSLT WTDPKKTGGS
13910      13920      13930      13940      13950
PITGYHLEFK ERNSLLWKRA NKTPIRMRDF KVTGLTEGLE YEFRVMAINL
13960      13970      13980      13990      14000
AGVGKPSLPS EPVVALDPID PPGKPEVINI TRNSVTLIWT EPKYDGGHKL
14010      14020      14030      14040      14050
TGYIVEKRDL PSKSWMKANH VNVPECAFTV TDLVEGGKYE FRIRAKNTAG
14060      14070      14080      14090      14100
AISAPSESTE TIICKDEYEA PTIVLDPTIK DGLTIKAGDT IVLNAISILG
14110      14120      14130      14140      14150
KPLPKSSWSK AGKDIRPSDI TQITSTPTSS MLTIKYATRK DAGEYTITAT
14160      14170      14180      14190      14200
NPFGTKVEHV KVTVLDVPGP PGPVEISNVS AEKATLTWTP PLEDGGSPIK
14210      14220      14230      14240      14250
SYILEKRETS RLLWTVVSED IQSCRHVATK LIQGNEYIFR VSAVNHYGKG
14260      14270      14280      14290      14300
EPVQSEPVKM VDRFGPPGPP EKPEVSNVTK NTATVSWKRP VDDGGSEITG
14310      14320      14330      14340      14350
YHVERREKKS LRWVRAIKTP VSDLRCKVTG LQEGSTYEFR VSAENRAGIG
14360      14370      14380      14390      14400
PPSEASDSVL MKDAAYPPGP PSNPHVTDTT KKSASLAWGK PHYDGGLEIT
14410      14420      14430      14440      14450
GYVVEHQKVG DEAWIKDTTG TALRITQFVV PDLQTKEKYN FRISAINDAG
14460      14470      14480      14490      14500
VGEPAVIPDV EIVEREMAPD FELDAELRRT LVVRAGLSIR IFVPIKGRPA
14510      14520      14530      14540      14550
PEVTWTKDNI NLKNRANIEN TESFTLLIIP ECNRYDTGKF VMTIENPAGK
14560      14570      14580      14590      14600
KSGFVNVRVL DTPGPVLNLR PTDITKDSVT LHWDLPLIDG GSRITNYIVE
14610      14620      14630      14640      14650
KREATRKSYS TATTKCHKCT YKVTGLSEGC EYFFRVMAEN EYGIGEPTET
14660      14670      14680      14690      14700
TEPVKASEAP SPPDSLNIMD ITKSTVSLAW PKPKHDGGSK ITGYVIEAQR
14710      14720      14730      14740      14750
KGSDQWTHIT TVKGLECVVR NLTEGEEYTF QVMAVNSAGR SAPRESRPVI
14760      14770      14780      14790      14800
VKEQTMLPEL DLRGIYQKLV IAKAGDNIKV EIPVLGRPKP TVTWKKGDQI
14810      14820      14830      14840      14850
LKQTQRVNFE TTATSTILNI NECVRSDSGP YPLTARNIVG EVGDVITIQV
14860      14870      14880      14890      14900
HDIPGPPTGP IKFDEVSSDF VTFSWDPPEN DGGVPISNYV VEMRQTDSTT
14910      14920      14930      14940      14950
WVELATTVIR TTYKATRLTT GLEYQFRVKA QNRYGVGPGI TSACIVANYP
14960      14970      14980      14990      15000
FKVPGPPGTP QVTAVTKDSM TISWHEPLSD GGSPILGYHV ERKERNGILW
15010      15020      15030      15040      15050
QTVSKALVPG NIFKSSGLTD GIAYEFRVIA ENMAGKSKPS KPSEPMLALD
15060      15070      15080      15090      15100
PIDPPGKPVP LNITRHTVTL KWAKPEYTGG FKITSYIVEK RDLPNGRWLK
15110      15120      15130      15140      15150
ANFSNILENE FTVSGLTEDA AYEFRVIAKN AAGAISPPSE PSDAITCRDD
15160      15170      15180      15190      15200
VEAPKIKVDV KFKDTVILKA GEAFRLEADV SGRPPPTMEW SKDGKELEGT
15210      15220      15230      15240      15250
AKLEIKIADF STNLVNKDST RRDSGAYTLT ATNPGGFAKH IFNVKVLDRP
15260      15270      15280      15290      15300
GPPEGPLAVT EVTSEKCVLS WFPPLDDGGA KIDHYIVQKR ETSRLAWTNV
15310      15320      15330      15340      15350
ASEVQVTKLK VTKLLKGNEY IFRVMAVNKY GVGEPLESEP VLAVNPYGPP
15360      15370      15380      15390      15400
DPPKNPEVTT ITKDSMVVCW GHPDSDGGSE IINYIVERRD KAGQRWIKCN
15410      15420      15430      15440      15450
KKTLTDLRYK VSGLTEGHEY EFRIMAENAA GISAPSPTSP FYKACDTVFK
15460      15470      15480      15490      15500
PGPPGNPRVL DTSRSSISIA WNKPIYDGGS EITGYMVEIA LPEEDEWQIV
15510      15520      15530      15540      15550
TPPAGLKATS YTITGLTENQ EYKIRIYAMN SEGLGEPALV PGTPKAEDRM
15560      15570      15580      15590      15600
LPPEIELDAD LRKVVTIRAC CTLRLFVPIK GRPAPEVKWA RDHGESLDKA
15610      15620      15630      15640      15650
SIESTSSYTL LIVGNVNRFD SGKYILTVEN SSGSKSAFVN VRVLDTPGPP
15660      15670      15680      15690      15700
QDLKVKEVTK TSVTLTWDPP LLDGGSKIKN YIVEKRESTR KAYSTVATNC
15710      15720      15730      15740      15750
HKTSWKVDQL QEGCSYYFRV LAENEYGIGL PAETAESVKA SERPLPPGKI
15760      15770      15780      15790      15800
TLMDVTRNSV SLSWEKPEHD GGSRILGYIV EMQTKGSDKW ATCATVKVTE
15810      15820      15830      15840      15850
ATITGLIQGE EYSFRVSAQN EKGISDPRQL SVPVIAKDLV IPPAFKLLEN
15860      15870      15880      15890      15900
TFTVLAGEDL KVDVPFIGRP TPAVTWHKDN VPLKQTTRVN AESTENNSLL
15910      15920      15930      15940      15950
TIKDACREDV GHYVVKLINS AGEAIETLNV IVLDKPGPPT GPVKMDEVTA
15960      15970      15980      15990      16000
DSITLSWGPP KYDGGSSINN YIVEKRDTST TTWQIVSATV ARTTIKACRL
16010      16020      16030      16040      16050
KTGCEYQFRI AAENRYGKST YLNSEPTVAQ YPFKVPGPPG TPVVTLSSRD
16060      16070      16080      16090      16100
SMEVQWNEPI SDGGSRVIGY HLERKERNSI LWVKLNKTPI PQTKFKTTGL
16110      16120      16130      16140      16150
EEGVEYEFRV SAENIVGIGK PSKVSECYVA RDPCDPPGRP EAIIVTRNSV
16160      16170      16180      16190      16200
TLQWKKPTYD GGSKITGYIV EKKELPEGRW MKASFTNIID THFEVTGLVE
16210      16220      16230      16240      16250
DHRYEFRVIA RNAAGVFSEP SESTGAITAR DEVDPPRISM DPKYKDTIVV
16260      16270      16280      16290      16300
HAGESFKVDA DIYGKPIPTI QWIKGDQELS NTARLEIKST DFATSLSVKD
16310      16320      16330      16340      16350
AVRVDSGNYI LKAKNVAGER SVTVNVKVLD RPGPPEGPVV ISGVTAEKCT
16360      16370      16380      16390      16400
LAWKPPLQDG GSDIINYIVE RRETSRLVWT VVDANVQTLS CKVTKLLEGN
16410      16420      16430      16440      16450
EYTFRIMAVN KYGVGEPLES EPVVAKNPFV VPDAPKAPEV TTVTKDSMIV
16460      16470      16480      16490      16500
VWERPASDGG SEILGYVLEK RDKEGIRWTR CHKRLIGELR LRVTGLIENH
16510      16520      16530      16540      16550
DYEFRVSAEN AAGLSEPSPP SAYQKACDPI YKPGPPNNPK VIDITRSSVF
16560      16570      16580      16590      16600
LSWSKPIYDG GCEIQGYIVE KCDVSVGEWT MCTPPTGINK TNIEVEKLLE
16610      16620      16630      16640      16650
KHEYNFRICA INKAGVGEHA DVPGPIIVEE KLEAPDIDLD LELRKIINIR
16660      16670      16680      16690      16700
AGGSLRLFVP IKGRPTPEVK WGKVDGEIRD AAIIDVTSSF TSLVLDNVNR
16710      16720      16730      16740      16750
YDSGKYTLTL ENSSGTKSAF VTVRVLDTPS PPVNLKVTEI TKDSVSITWE
16760      16770      16780      16790      16800
PPLLDGGSKI KNYIVEKREA TRKSYAAVVT NCHKNSWKID QLQEGCSYYF
16810      16820      16830      16840      16850
RVTAENEYGI GLPAQTADPI KVAEVPQPPG KITVDDVTRN SVSLSWTKPE
16860      16870      16880      16890      16900
HDGGSKIIQY IVEMQAKHSE KWSECARVKS LQAVITNLTQ GEEYLFRVVA
16910      16920      16930      16940      16950
VNEKGRSDPR SLAVPIVAKD LVIEPDVKPA FSSYSVQVGQ DLKIEVPISG
16960      16970      16980      16990      17000
RPKPTITWTK DGLPLKQTTR INVTDSLDLT TLSIKETHKD DGGQYGITVA
17010      17020      17030      17040      17050
NVVGQKTASI EIVTLDKPDP PKGPVKFDDV SAESITLSWN PPLYTGGCQI
17060      17070      17080      17090      17100
TNYIVQKRDT TTTVWDVVSA TVARTTLKVT KLKTGTEYQF RIFAENRYGQ
17110      17120      17130      17140      17150
SFALESDPIV AQYPYKEPGP PGTPFATAIS KDSMVIQWHE PVNNGGSPVI
17160      17170      17180      17190      17200
GYHLERKERN SILWTKVNKT IIHDTQFKAQ NLEEGIEYEF RVYAENIVGV
17210      17220      17230      17240      17250
GKASKNSECY VARDPCDPPG TPEPIMVKRN EITLQWTKPV YDGGSMITGY
17260      17270      17280      17290      17300
IVEKRDLPDG RWMKASFTNV IETQFTVSGL TEDQRYEFRV IAKNAAGAIS
17310      17320      17330      17340      17350
KPSDSTGPIT AKDEVELPRI SMDPKFRDTI VVNAGETFRL EADVHGKPLP
17360      17370      17380      17390      17400
TIEWLRGDKE IEESARCEIK NTDFKALLIV KDAIRIDGGQ YILRASNVAG
17410      17420      17430      17440      17450
SKSFPVNVKV LDRPGPPEGP VQVTGVTSEK CSLTWSPPLQ DGGSDISHYV
17460      17470      17480      17490      17500
VEKRETSRLA WTVVASEVVT NSLKVTKLLE GNEYVFRIMA VNKYGVGEPL
17510      17520      17530      17540      17550
ESAPVLMKNP FVLPGPPKSL EVTNIAKDSM TVCWNRPDSD GGSEIIGYIV
17560      17570      17580      17590      17600
EKRDRSGIRW IKCNKRRITD LRLRVTGLTE DHEYEFRVSA ENAAGVGEPS
17610      17620      17630      17640      17650
PATVYYKACD PVFKPGPPTN AHIVDTTKNS ITLAWGKPIY DGGSEILGYV
17660      17670      17680      17690      17700
VEICKADEEE WQIVTPQTGL RVTRFEISKL TEHQEYKIRV CALNKVGLGE
17710      17720      17730      17740      17750
ATSVPGTVKP EDKLEAPELD LDSELRKGIV VRAGGSARIH IPFKGRPTPE
17760      17770      17780      17790      17800
ITWSREEGEF TDKVQIEKGV NYTQLSIDNC DRNDAGKYIL KLENSSGSKS
17810      17820      17830      17840      17850
AFVTVKVLDT PGPPQNLAVK EVRKDSAFLV WEPPIIDGGA KVKNYVIDKR
17860      17870      17880      17890      17900
ESTRKAYANV SSKCSKTSFK VENLTEGAIY YFRVMAENEF GVGVPVETVD
17910      17920      17930      17940      17950
AVKAAEPPSP PGKVTLTDVS QTSASLMWEK PEHDGGSRVL GYVVEMQPKG
17960      17970      17980      17990      18000
TEKWSIVAES KVCNAVVTGL SSGQEYQFRV KAYNEKGKSD PRVLGVPVIA
18010      18020      18030      18040      18050
KDLTIQPSLK LPFNTYSIQA GEDLKIEIPV IGRPRPNISW VKDGEPLKQT
18060      18070      18080      18090      18100
TRVNVEETAT STVLHIKEGN KDDFGKYTVT ATNSAGTATE NLSVIVLEKP
18110      18120      18130      18140      18150
GPPVGPVRED EVSADFVVIS WEPPAYTGGC QISNYIVEKR DTTTTTWHMV
18160      18170      18180      18190      18200
SATVARTTIK ITKLKTGTEY QFRIFAENRY GKSAPLDSKA VIVQYPFKEP
18210      18220      18230      18240      18250
GPPGTPFVTS ISKDQMLVQW HEPVNDGGTK IIGYHLEQKE KNSILWVKLN
18260      18270      18280      18290      18300
KTPIQDTKFK TTGLDEGLEY EFKVSAENIV GIGKPSKVSE CFVARDPCDP
18310      18320      18330      18340      18350
PGRPEAIVIT RNNVTLKWKK PAYDGGSKIT GYIVEKKDLP DGRWMKASFT
18360      18370      18380      18390      18400
NVLETEFTVS GLVEDQRYEF RVIARNAAGN FSEPSDSSGA ITARDEIDAP
18410      18420      18430      18440      18450
NASLDPKYKD VIVVHAGETF VLEADIRGKP IPDVVWSKDG KELEETAARM
18460      18470      18480      18490      18500
EIKSTIQKTT LVVKDCIRTD GGQYILKLSN VGGTKSIPIT VKVLDRPGPP
18510      18520      18530      18540      18550
EGPLKVTGVT AEKCYLAWNP PLQDGGANIS HYIIEKRETS RLSWTQVSTE
18560      18570      18580      18590      18600
VQALNYKVTK LLPGNEYIFR VMAVNKYGIG EPLESGPVTA CNPYKPPGPP
18610      18620      18630      18640      18650
STPEVSAITK DSMVVTWARP VDDGGTEIEG YILEKRDKEG VRWTKCNKKT
18660      18670      18680      18690      18700
LTDLRLRVTG LTEGHSYEFR VAAENAAGVG EPSEPSVFYR ACDALYPPGP
18710      18720      18730      18740      18750
PSNPKVTDTS RSSVSLAWSK PIYDGGAPVK GYVVEVKEAA ADEWTTCTPP
18760      18770      18780      18790      18800
TGLQGKQFTV TKLKENTEYN FRICAINSEG VGEPATLPGS VVAQERIEPP
18810      18820      18830      18840      18850
EIELDADLRK VVVLRASATL RLFVTIKGRP EPEVKWEKAE GILTDRAQIE
18860      18870      18880      18890      18900
VTSSFTMLVI DNVTRFDSGR YNLTLENNSG SKTAFVNVRV LDSPSAPVNL
18910      18920      18930      18940      18950
TIREVKKDSV TLSWEPPLID GGAKITNYIV EKRETTRKAY ATITNNCTKT
18960      18970      18980      18990      19000
TFRIENLQEG CSYYFRVLAS NEYGIGLPAE TTEPVKVSEP PLPPGRVTLV
19010      19020      19030      19040      19050
DVTRNTATIK WEKPESDGGS KITGYVVEMQ TKGSEKWSTC TQVKTLEATI
19060      19070      19080      19090      19100
SGLTAGEEYV FRVAAVNEKG RSDPRQLGVP VIARDIEIKP SVELPFHTEN
19110      19120      19130      19140      19150
VKAREQLKID VPFKGRPQAT VNWRKDGQTL KETTRVNVSS SKTVTSLSIK
19160      19170      19180      19190      19200
EASKEDVGTY ELCVSNSAGS ITVPITIIVL DRPGPPGPIR IDEVSCDSIT
19210      19220      19230      19240      19250
ISWNPPEYDG GCQISNYIVE KKETTSTTWH IVSQAVARTS IKIVRLTTGS
19260      19270      19280      19290      19300
EYQFRVCAEN RYGKSSYSES SAVVAEYPFS PPGPPGTPKV VHATKSTMLV
19310      19320      19330      19340      19350
TWQVPVNDGG SRVIGYHLEY KERSSILWSK ANKILIADTQ MKVSGLDEGL
19360      19370      19380      19390      19400
MYEYRVYAEN IAGIGKCSKS CEPVPARDPC DPPGQPEVTN ITRKSVSLKW
19410      19420      19430      19440      19450
SKPHYDGGAK ITGYIVERRE LPDGRWLKCN YTNIQETYFE VTELTEDQRY
19460      19470      19480      19490      19500
EFRVFARNAA DSVSEPSEST GPIIVKDDVE PPRVMMDVKF RDVIVVKAGE
19510      19520      19530      19540      19550
VLKINADIAG RPLPVISWAK DGIEIEERAR TEIISTDNHT LLTVKDCIRR
19560      19570      19580      19590      19600
DTGQYVLTLK NVAGTRSVAV NCKVLDKPGP PAGPLEINGL TAEKCSLSWG
19610      19620      19630      19640      19650
RPQEDGGADI DYYIVEKRET SHLAWTICEG ELQMTSCKVT KLLKGNEYIF
19660      19670      19680      19690      19700
RVTGVNKYGV GEPLESVAIK ALDPFTVPSP PTSLEITSVT KESMTLCWSR
19710      19720      19730      19740      19750
PESDGGSEIS GYIIERREKN SLRWVRVNKK PVYDLRVKST GLREGCEYEY
19760      19770      19780      19790      19800
RVYAENAAGL SLPSETSPLI RAEDPVFLPS PPSKPKIVDS GKTTITIAWV
19810      19820      19830      19840      19850
KPLFDGGAPI TGYTVEYKKS DDTDWKTSIQ SLRGTEYTIS GLTTGAEYVF
19860      19870      19880      19890      19900
RVKSVNKVGA SDPSDSSDPQ IAKEREEEPL FDIDSEMRKT LIVKAGASFT
19910      19920      19930      19940      19950
MTVPFRGRPV PNVLWSKPDT DLRTRAYVDT TDSRTSLTIE NANRNDSGKY
19960      19970      19980      19990      20000
TLTIQNVLSA ASLTLVVKVL DTPGPPTNIT VQDVTKESAV LSWDVPENDG
20010      20020      20030      20040      20050
GAPVKNYHIE KREASKKAWV SVINNCNRLS YKVTNLQEGA IYYFRVSGEN
20060      20070      20080      20090      20100
EFGVGIPAET KEGVKITEKP SPPEKLGVTS ISKDSVSLTW LKPEHDGGSR
20110      20120      20130      20140      20150
IVHYVVEALE KGQKNWVKCA VAKSTHHVVS GLRENSEYFF RVFAENQAGL
20160      20170      20180      20190      20200
SDPRELLLPV LIKEQLEPPE IDMKNFPSHT VYVRAGSNLK VDIPISGKPL
20210      20220      20230      20240      20250
PKVTLSRDGV PLKATMRENT EITAENLTIN LKESVTADAG RYEITAANSS
20260      20270      20280      20290      20300
GTTKAFINIV VLDRPGPPTG PVVISDITEE SVTLKWEPPK YDGGSQVTNY
20310      20320      20330      20340      20350
ILLKRETSTA VWTEVSATVA RTMMKVMKLT TGEEYQFRIK AENREGISDH
20360      20370      20380      20390      20400
IDSACVTVKL PYTTPGPPST PWVTNVTRES ITVGWHEPVS NGGSAVVGYH
20410      20420      20430      20440      20450
LEMKDRNSIL WQKANKLVIR TTHFKVTTIS AGLIYEFRVY AENAAGVGKP
20460      20470      20480      20490      20500
SHPSEPVLAI DACEPPRNVR ITDISKNSVS LSWQQPAFDG GSKITGYIVE
20510      20520      20530      20540      20550
RRDLPDGRWT KASFTNVTET QFIISGLTQN SQYEFRVFAR NAVGSISNPS
20560      20570      20580      20590      20600
EVVGPITCID SYGGPVIDLP LEYTEVVKYR AGTSVKLRAG ISGKPAPTIE
20610      20620      20630      20640      20650
WYKDDKELQT NALVCVENTT DLASILIKDA DRINSGCYEL KLRNAMGSAS
20660      20670      20680      20690      20700
ATIRVQILDK PGPPGGPIEF KTVTAEKITL LWRPPADDGG AKITHYIVEK
20710      20720      20730      20740      20750
RETSRVVWSM VSEHLEECII TTTKIIKGNE YIFRVRAVNK YGIGEPLESD
20760      20770      20780      20790      20800
SVVAKNAFVT PGPPGIPEVT KITKNSMTVV WSRPIADGGS DISGYFLEKR
20810      20820      20830      20840      20850
DKKSLGWFKV LKETIRDTRQ KVTGLTENSD YQYRVCAVNA AGQGPFSEPS
20860      20870      20880      20890      20900
EFYKAADPID PPGPPAKIRI ADSTKSSITL GWSKPVYDGG SAVTGYVVEI
20910      20920      20930      20940      20950
RQGEEEEWTT VSTKGEVRTT EYVVSNLKPG VNYYFRVSAV NCAGQGEPIE
20960      20970      20980      20990      21000
MNEPVQAKDI LEAPEIDLDV ALRTSVIAKA GEDVQVLIPF KGRPPPTVTW
21010      21020      21030      21040      21050
RKDEKNLGSD ARYSIENTDS SSLLTIPQVT RNDTGKYILT IENGVGEPKS
21060      21070      21080      21090      21100
STVSVKVLDT PAACQKLQVK HVSRGTVTLL WDPPLIDGGS PIINYVIEKR
21110      21120      21130      21140      21150
DATKRTWSVV SHKCSSTSFK LIDLSEKTPF FFRVLAENEI GIGEPCETTE
21160      21170      21180      21190      21200
PVKAAEVPAP IRDLSMKDST KTSVILSWTK PDFDGGSVIT EYVVERKGKG
21210      21220      21230      21240      21250
EQTWSHAGIS KTCEIEVSQL KEQSVLEFRV FAKNEKGLSD PVTIGPITVK
21260      21270      21280      21290      21300
ELIITPEVDL SDIPGAQVTV RIGHNVHLEL PYKGKPKPSI SWLKDGLPLK
21310      21320      21330      21340      21350
ESEFVRFSKT ENKITLSIKN AKKEHGGKYT VILDNAVCRI AVPITVITLG
21360      21370      21380      21390      21400
PPSKPKGPIR FDEIKADSVI LSWDVPEDNG GGEITCYSIE KRETSQTNWK
21410      21420      21430      21440      21450
MVCSSVARTT FKVPNLVKDA EYQFRVRAEN RYGVSQPLVS SIIVAKHQFR
21460      21470      21480      21490      21500
IPGPPGKPVI YNVTSDGMSL TWDAPVYDGG SEVTGFHVEK KERNSILWQK
21510      21520      21530      21540      21550
VNTSPISGRE YRATGLVEGL DYQFRVYAEN SAGLSSPSDP SKFTLAVSPV
21560      21570      21580      21590      21600
DPPGTPDYID VTRETITLKW NPPLRDGGSK IVGYSIEKRQ GNERWVRCNF
21610      21620      21630      21640      21650
TDVSECQYTV TGLSPGDRYE FRIIARNAVG TISPPSQSSG IIMTRDENVP
21660      21670      21680      21690      21700
PIVEFGPEYF DGLIIKSGES LRIKALVQGR PVPRVTWFKD GVEIEKRMNM
21710      21720      21730      21740      21750
EITDVLGSTS LFVRDATRDH RGVYTVEAKN ASGSAKAEIK VKVQDTPGKV
21760      21770      21780      21790      21800
VGPIRFTNIT GEKMTLWWDA PLNDGCAPIT HYIIEKRETS RLAWALIEDK
21810      21820      21830      21840      21850
CEAQSYTAIK LINGNEYQFR VSAVNKFGVG RPLDSDPVVA QIQYTVPDAP
21860      21870      21880      21890      21900
GIPEPSNITG NSITLTWARP ESDGGSEIQQ YILERREKKS TRWVKVISKR
21910      21920      21930      21940      21950
PISETRFKVT GLTEGNEYEF HVMAENAAGV GPASGISRLI KCREPVNPPG
21960      21970      21980      21990      22000
PPTVVKVTDT SKTTVSLEWS KPVEDGGMEI IGYIIEMCKA DLGDWHKVNA
22010      22020      22030      22040      22050
EACVKTRYTV TDLQAGEEYK FRVSAINGAG KGDSCEVTGT IKAVDRLTAP
22060      22070      22080      22090      22100
ELDIDANFKQ THVVRAGASI RLFIAYQGRP TPTAVWSKPD SNLSLRADIH
22110      22120      22130      22140      22150
TTDSFSTLTV ENCNRNDAGK YTLTVENNSG SKSITFTVKV LDTPGPPGPI
22160      22170      22180      22190      22200
TFKDVTRGSA TLMWDAPLLD GGARIHHYVV EKREASRRSW QVISEKCTRQ
22210      22220      22230      22240      22250
IFKVNDLAEG VPYYFRVSAV NEYGVGEPYE MPEPIVATEQ PAPPRRLDVV
22260      22270      22280      22290      22300
DTSKSSAVLA WLKPDHDGGS RITGYLLEMR QKGSDFWVEA GHTKQLTFTV
22310      22320      22330      22340      22350
ERLVEKTEYE FRVKAKNDAG YSEPREAFSS VIIKEPQIEP TADLTGITNQ
22360      22370      22380      22390      22400
LITCKAGSPF TIDVPISGRP APKVTWKLEE MRLKETDRVS ITTTKDRTTL
22410      22420      22430      22440      22450
TVKDSMRGDS GRYFLTLENT AGVKTFSVTV VVIGRPGPVT GPIEVSSVSA
22460      22470      22480      22490      22500
ESCVLSWGEP KDGGGTEITN YIVEKRESGT TAWQLVNSSV KRTQIKVTHL
22510      22520      22530      22540      22550
TKYMEYSFRV SSENRFGVSK PLESAPIIAE HPFVPPSAPT RPEVYHVSAN
22560      22570      22580      22590      22600
AMSIRWEEPY HDGGSKIIGY WVEKKERNTI LWVKENKVPC LECNYKVTGL
22610      22620      22630      22640      22650
VEGLEYQFRT YALNAAGVSK ASEASRPIMA QNPVDAPGRP EVTDVTRSTV
22660      22670      22680      22690      22700
SLIWSAPAYD GGSKVVGYII ERKPVSEVGD GRWLKCNYTI VSDNFFTVTA
22710      22720      22730      22740      22750
LSEGDTYEFR VLAKNAAGVI SKGSESTGPV TCRDEYAPPK AELDARLHGD
22760      22770      22780      22790      22800
LVTIRAGSDL VLDAAVGGKP EPKIIWTKGD KELDLCEKVS LQYTGKRATA
22810      22820      22830      22840      22850
VIKFCDRSDS GKYTLTVKNA SGTKAVSVMV KVLDSPGPCG KLTVSRVTQE
22860      22870      22880      22890      22900
KCTLAWSLPQ EDGGAEITHY IVERRETSRL NWVIVEGECP TLSYVVTRLI
22910      22920      22930      22940      22950
KNNEYIFRVR AVNKYGPGVP VESEPIVARN SFTIPSPPGI PEEVGTGKEH
22960      22970      22980      22990      23000
IIIQWTKPES DGGNEISNYL VDKREKKSLR WTRVNKDYVV YDTRLKVTSL
23010      23020      23030      23040      23050
MEGCDYQFRV TAVNAAGNSE PSEASNFISC REPSYTPGPP SAPRVVDTTK
23060      23070      23080      23090      23100
HSISLAWTKP MYDGGTDIVG YVLEMQEKDT DQWYRVHTNA TIRNTEFTVP
23110      23120      23130      23140      23150
DLKMGQKYSF RVAAVNVKGM SEYSESIAEI EPVERIEIPD LELADDLKKT
23160      23170      23180      23190      23200
VTIRAGASLR LMVSVSGRPP PVITWSKQGI DLASRAIIDT TESYSLLIVD
23210      23220      23230      23240      23250
KVNRYDAGKY TIEAENQSGK KSATVLVKVY DTPGPCPSVK VKEVSRDSVT
23260      23270      23280      23290      23300
ITWEIPTIDG GAPVNNYIVE KREAAMRAFK TVTTKCSKTL YRISGLVEGT
23310      23320      23330      23340      23350
MYYFRVLPEN IYGIGEPCET SDAVLVSEVP LVPAKLEVVD VTKSTVTLAW
23360      23370      23380      23390      23400
EKPLYDGGSR LTGYVLEACK AGTERWMKVV TLKPTVLEHT VTSLNEGEQY
23410      23420      23430      23440      23450
LFRIRAQNEK GVSEPRETVT AVTVQDLRVL PTIDLSTMPQ KTIHVPAGRP
23460      23470      23480      23490      23500
VELVIPIAGR PPPAASWFFA GSKLRESERV TVETHTKVAK LTIRETTIRD
23510      23520      23530      23540      23550
TGEYTLELKN VTGTTSETIK VIILDKPGPP TGPIKIDEID ATSITISWEP
23560      23570      23580      23590      23600
PELDGGAPLS GYVVEQRDAH RPGWLPVSES VTRSTFKFTR LTEGNEYVER
23610      23620      23630      23640      23650
VAATNRFGIG SYLQSEVIEC RSSIRIPGPP ETLQIFDVSR DGMTLTWYPP
23660      23670      23680      23690      23700
EDDGGSQVTG YIVERKEVRA DRWVRVNKVP VTMTRYRSTG LTEGLEYEHR
23710      23720      23730      23740      23750
VTAINARGSG KPSRPSKPIV AMDPIAPPGK PQNPRVTDTT RTSVSLAWSV
23760      23770      23780      23790      23800
PEDEGGSKVT GYLIEMQKVD QHEWTKCNTT PTKIREYTLT HLPQGAEYRF
23810      23820      23830      23840      23850
RVLACNAGGP GEPAEVPGTV KVTEMLEYPD YELDERYQEG IFVRQGGVIR
23860      23870      23880      23890      23900
LTIPIKGKPF PICKWTKEGQ DISKRAMIAT SETHTELVIK EADRGDSGTY
23910      23920      23930      23940      23950
DLVLENKCGK KAVYIKVRVI GSPNSPEGPL EYDDIQVRSV RVSWRPPADD
23960      23970      23980      23990      24000
GGADILGYIL ERREVPKAAW YTIDSRVRGT SLVVKGLKEN VEYHFRVSAE
24010      24020      24030      24040      24050
NQFGISKPLK SEEPVTPKTP LNPPEPPSNP PEVLDVTKSS VSLSWSRPKD
24060      24070      24080      24090      24100
DGGSRVTGYY IERKETSTDK WVRHNKTQIT TTMYTVTGLV PDAEYQFRII
24110      24120      24130      24140      24150
AQNDVGLSET SPASEPVVCK DPFDKPSQPG ELEILSISKD SVTLQWEKPE
24160      24170      24180      24190      24200
CDGGKEILGY WVEYRQSGDS AWKKSNKERI KDKQFTIGGL LEATEYEFRV
24210      24220      24230      24240      24250
FAENETGLSR PRRTAMSIKT KLTSGEAPGI RKEMKDVTTK LGEAAQLSCQ
24260      24270      24280      24290      24300
IVGRPLPDIK WYRFGKELIQ SRKYKMSSDG RTHTLTVMTE EQEDEGVYTC
24310      24320      24330      24340      24350
IATNEVGEVE TSSKLLLQAT PQFHPGYPLK EKYYGAVGST LRLHVMYIGR
24360      24370      24380      24390      24400
PVPAMTWFHG QKLLQNSENI TIENTEHYTH LVMKNVQRKT HAGKYKVQLS
24410      24420      24430      24440      24450
NVFGTVDAIL DVEIQDKPDK PTGPIVIEAL LKNSAVISWK PPADDGGSWI
24460      24470      24480      24490      24500
TNYVVEKCEA KEGAEWQLVS SAISVTTCRI VNLTENAGYY FRVSAQNTFG
24510      24520      24530      24540      24550
ISDPLEVSSV VIIKSPFEKP GAPGKPTITA VTKDSCVVAW KPPASDGGAK
24560      24570      24580      24590      24600
IRNYYLEKRE KKQNKWISVT TEEIRETVFS VKNLIEGLEY EFRVKCENLG
24610      24620      24630      24640      24650
GESEWSEISE PITPKSDVPI QAPHFKEELR NLNVRYQSNA TLVCKVTGHP
24660      24670      24680      24690      24700
KPIVKWYRQG KEIIADGLKY RIQEFKGGYH QLIIASVTDD DATVYQVRAT
24710      24720      24730      24740      24750
NQGGSVSGTA SLEVEVPAKI HLPKTLEGMG AVHALRGEVV SIKIPFSGKP
24760      24770      24780      24790      24800
DPVITWQKGQ DLIDNNGHYQ VIVTRSFTSL VFPNGVERKD AGFYVVCAKN
24810      24820      24830      24840      24850
RFGIDQKTVE LDVADVPDPP RGVKVSDVSR DSVNLTWTEP ASDGGSKITN
24860      24870      24880      24890      24900
YIVEKCATTA ERWLRVGQAR ETRYTVINLF GKTSYQFRVI AENKFGLSKP
24910      24920      24930      24940      24950
SEPSEPTITK EDKTRAMNYD EEVDETREVS MTKASHSSTK ELYEKYMIAE
24960      24970      24980      24990      25000
DLGRGEFGIV HRCVETSSKK TYMAKFVKVK GTDQVLVKKE ISILNIARHR
25010      25020      25030      25040      25050
NILHLHESFE SMEELVMIFE FISGLDIFER INTSAFELNE REIVSYVHQV
25060      25070      25080      25090      25100
CEALQFLHSH NIGHFDIRPE NIIYQTRRSS TIKIIEFGQA RQLKPGDNFR
25110      25120      25130      25140      25150
LLFTAPEYYA PEVHQHDVVS TATDMWSLGT LVYVLLSGIN PFLAETNQQI
25160      25170      25180      25190      25200
IENIMNAEYT FDEEAFKEIS IEAMDFVDRL LVKERKSRMT ASEALQHPWL
25210      25220      25230      25240      25250
KQKIERVSTK VIRTLKHRRY YHTLIKKDLN MVVSAARISC GGAIRSQKGV
25260      25270      25280      25290      25300
SVAKVKVASI EIGPVSGQIM HAVGEEGGHV KYVCKIENYD QSTQVTWYFG
25310      25320      25330      25340      25350
VRQLENSEKY EITYEDGVAI LYVKDITKLD DGTYRCKVVN DYGEDSSYAE
25360      25370      25380      25390      25400
LFVKGVREVY DYYCRRTMKK IKRRTDTMRL LERPPEFTLP LYNKTAYVGE
25410      25420      25430      25440      25450
NVRFGVTITV HPEPHVTWYK SGQKIKPGDN DKKYTFESDK GLYQLTINSV
25460      25470      25480      25490      25500
TTDDDAEYTV VARNKYGEDS CKAKLTVTLH PPPTDSTLRP MFKRLLANAE
25510      25520      25530      25540      25550
CQEGQSVCFE IRVSGIPPPT LKWEKDGQPL SLGPNIEIIH EGLDYYALHI
25560      25570      25580      25590      25600
RDTLPEDTGY YRVTATNTAG STSCQAHLQV ERLRYKKQEF KSKEEHERHV
25610      25620      25630      25640      25650
QKQIDKTLRM AEILSGTESV PLTQVAKEAL REAAVLYKPA VSTKTVKGEF
25660      25670      25680      25690      25700
RLEIEEKKEE RKLRMPYDVP EPRKYKQTTI EEDQRIKQFV PMSDMKWYKK
25710      25720      25730      25740      25750
IRDQYEMPGK LDRVVQKRPK RIRLSRWEQF YVMPLPRITD QYRPKWRIPK
25760      25770      25780      25790      25800
LSQDDLEIVR PARRRTPSPD YDFYYRPRRR SLGDISDEEL LLPIDDYLAM
25810      25820      25830      25840      25850
KRTEEERLRL EEELELGFSA SPPSRSPPHF ELSSLRYSSP QAHVKVEETR
25860      25870      25880      25890      25900
KDFRYSTYHI PTKAEASTSY AELRERHAQA AYRQPKQRQR IMAEREDEEL
25910      25920      25930      25940      25950
LRPVTTTQHL SEYKSELDEM SKEEKSRKKS RRQREVTEIT EIEEEYEISK
25960      25970      25980      25990      26000
HAQRESSSSA SRLLRRRRSL SPTYIELMRP VSELIRSRPQ PAEEYEDDTE
26010      26020      26030      26040      26050
RRSPTPERTR PRSPSPVSSE RSLSRFERSA RFDIFSRYES MKAALKTQKT
26060      26070      26080      26090      26100
SERKYEVLSQ QPFTLDHAPR ITLRMRSHRV PCGQNTRFIL NVQSKPTAEV
26110      26120      26130      26140      26150
KWYHNGVELQ ESSKIHYTNT SGVLTLEILD CHTDDSGTYR AVCTNYKGEA
26160      26170      26180      26190      26200
SDYATLDVTG GDYTTYASQR RDEEVPRSVF PELTRTEAYA VSSFKKTSEM
26210      26220      26230      26240      26250
EASSSVREVK SQMTETRESL SSYEHSASAE MKSAALEEKS LEEKSTTRKI
26260      26270      26280      26290      26300
KTTLAARILT KPRSMTVYEG ESARFSCDTD GEPVPTVTWL RKGQVLSTSA
26310      26320      26330      26340      26350
RHQVTTTKYK STFEISSVQA SDEGNYSVVV ENSEGKQEAE FTLTIQKARV
26360      26370      26380      26390      26400
TEKAVTSPPR VKSPEPRVKS PEAVKSPKRV KSPEPSHPKA VSPTETKPTP
26410      26420      26430      26440      26450
TEKVQHLPVS APPKITQFLK AEASKEIAKL TCVVESSVLR AKEVTWYKDG
26460      26470      26480      26490      26500
KKLKENGHFQ FHYSADGTYE LKINNLTESD QGEYVCEISG EGGTSKTNLQ
26510      26520      26530      26540      26550
FMGQAFKSIH EKVSKISETK KSDQKTTEST VTRKTEPKAP EPISSKPVIV
26560      26570      26580      26590      26600
TGLQDTTVSS DSVAKFAVKA TGEPRPTAIW TKDGKAITQG GKYKLSEDKG
26610      26620      26630      26640      26650
GFFLEIHKTD TSDSGLYTCT VKNSAGSVSS SCKLTIKAIK DTEAQKVSTQ
26660      26670      26680      26690      26700
KTSEITPQKK AVVQEEISQK ALRSEEIKMS EAKSQEKLAL KEEASKVLIS
26710      26720      26730      26740      26750
EEVKKSAATS LEKSIVHEEI TKTSQASEEV RTHAEIKAFS TQMSINEGQR
26760      26770      26780      26790      26800
LVLKANIAGA TDVKWVLNGV ELTNSEEYRY GVSGSDQTLT IKQASHRDEG
26810      26820      26830      26840      26850
ILTCISKTKE GIVKCQYDLT LSKELSDAPA FISQPRSQNI NEGQNVLFTC
26860      26870      26880      26890      26900
EISGEPSPEI EWFKNNLPIS ISSNVSISRS RNVYSLEIRN ASVSDSGKYT
26910      26920      26930      26940      26950
IKAKNFRGQC SATASLMVLP LVEEPSREVV LRTSGDTSLQ GSFSSQSVQM
26960      26970      26980      26990      27000
SASKQEASFS SFSSSSASSM TEMKFASMSA QSMSSMQESF VEMSSSSFMG
27010      27020      27030      27040      27050
ISNMTQLESS TSKMLKAGIR GIPPKIEALP SDISIDEGKV LTVACAFTGE
27060      27070      27080      27090      27100
PTPEVTWSCG GRKIHSQEQG RFHIENTDDL TTLIIMDVQK QDGGLYTLSL
27110
GNEFGSDSAT VNIHIRSI

Another example of a titin protein sequence is provided by UniProt as accession number Q8WZ42.

The N2-B splice variant of titin encodes the major N2-B cardiac muscle isoform, which lacks multiple exons in the region encoding PEVK repeats. This results in a shortened PEVK region in isoform N2-B compared to isoform IC. A sequence for such a human N2-B titin isoform is shown below (NCBI accession number NP 003310.4), provided below as SEQ ID NO:2.

1     MTTQAPTFTQ PLQSVVVLEG STATFEAHIS GFPVPEVSWF
41    RDGQVISTST LPGVQISFSD GRAKLTIPAV TKANSGRYSL
81    KATNGSGQAT STAELLVKAE TAPPNFVQRL QSMTVRQGSQ
121   VRLQVRVTGI PTPVVKFYRD GAEIQSSLDF QISQEGDLYS
161   LLIAEAYPED SGTYSVNATN SVGRATSTAE LLVQGEEEVP
201   AKKTKTIVST AQISESRQTR IEKKIEAHFD ARSIATVEMV
241   IDGAAGQQLP HKTPPRIPPK PKSRSPTPPS IAAKAQLARQ
281   QSPSPIRHSP SPVRHVRAPT PSPVRSVSPA ARISTSPIRS
321   VRSPLLMRKT QASTVATGPE VPPPWKQEGY VASSSEAEMR
361   ETTLTTSTQI RTEERWEGRY GVQEQVTISG AAGAAASVSA
401   SASYAAEAVA TGAKEVKQDA DKSAAVATVV AAVDMARVRE
441   PVISAVEQTA QRTTTTAVHI QPAQEQVRKE AEKTAVTKVV
481   VAADKAKEQE LKSRTKEVIT TKQEQMHVTH EQIRKETEKT
521   FVPKVVISAA KAKEQETRIS EEITKKQKQV TQEAIMKETR
561   KTVVPKVIVA TPKVKEQDLV SRGREGITTK REQVQITQEK
601   MRKEAEKTAL STIAVATAKA KEQETILRTR ETMATRQEQI
641   QVTHGKVDVG KKAEAVATVV AAVDQARVRE PREPGHLEES
681   YAQQTTLEYG YKERISAAKV AEPPQRPASE PHVVPKAVKP
721   RVIQAPSETH IKTTDQKGMH ISSQIKKTTD LTTERLVHVD
761   KRPRTASPHF TVSKISVPKT EHGYEASIAG SAIATLQKEL
781   SATSSAQKIT KSVKAPTVKP SETRVRAEPT PLPQFPFADT
841   PDTYKSEAGV EVKKEVGVSI TGTTVREERF EVLHGREAKV
881   TETARVPAPV EIPVTPPTLVVSGLKNVTVIE GESVTLECHI
921   SGYPSPTVTW YREDYQIESS IDFQITFQSG IARLMIREAF
961   AEDSGRFTCS AVNEAGTVST SCYLAVQVSE EFEKETTAVT
1001  EKFTTEEKRF VESRDVVMTD TSLTEEQAGP GEPAAPYFIT
1041  KPVVQKLVEG GSVVFGCQVG GNPKPHVYWK KSGVPLTTGY
1081  RYKVSYNKQT GECKLVISMT FADDAGEYTI VVRNKHGETS
1121  ASASLLEEAD YELLMKSQQE MLYQTQVTAF VQEPKVGETA
1161  PGFVYSEYEK EYEKEQALIR KKMAKDTVVV RTYVEDQEFH
1201  ISSFEERLIK EIEYRIIKTT LEELLEEDGE EKMAVDISES
1241  EAVESGFDSR IKNYRILEGM GVTFHCKMSG YPLPKIAWYK
1281  DGKRIKHGER YQMDFLQDGR ASLRIPVVLP EDEGIYTAFA
1321  SNIKGNAICS GKLYVEPAAP LGAPTYIPTL EPVSRIRSLS
1361  PRSVSRSPIR MSPARMSPAR MSPARMSPAR MSPGRRLEET
1401  DESQLERLYK PVFVLKPVSF KCLEGQTARF DLKVVGRPMP
1441  ETFWFHDGQQ IVNDYTHKVV IKEDGTQSLI IVPATPSDSG
1481  EWTVVAQNRA GRSSISVILT VEAVEHQVKP MFVEKLKNVN
1521  IKEGSRLEMK VRATGNPNPD IVWLKNSDII VPHKYPKIRI
1561  EGTKGEAALK IDSTVSQDSA WYTATAINKA GRDTTRCKVN
1601  VEVEFAEPEP ERKLIIPRGT YRAKEIAAPE LEPLHLRYGQ
1641  EQWEEGDLYD KEKQQKPFFK KKLTSLRLKR FGPAHFECRL
1681  TPIGDPTMVV EWLHDGKPLE AANRLRMINE FGYCSLDYGV
1721  AYSRDSGIIT CRATNKYGTD HTSATLIVKD EKSLVEESQL
1761  PEGRKGLQRI EELERMAHEG ALTGVTTDQK EKQKPDIVLY
1801  PEPVRVLEGE TARFRCRVTG YPQPKVNWYL NGQLIRKSKR
1841  FRVRYDGIHY LDIVDCKSYD TGEVKVTAEN PEGVIEHKVK
1881  LEIQQREDFR SVLRRAPEPR PEFHVHEPGK LQFEVQKVDR
1921  PVDTTETKEV VKLKRAERIT HEKVPEESEE LRSKFKRRTE
1961  EGYYEAITAV ELKSRKKDES YEELLRKTKD ELLHWTKELT
2001  EEEKKALAEE GKITIPTFKP DKIELSPSME APKIFERIQS
2041  QTVGQGSDAH FRVRVVGKPD PECEWYKNGV KIERSDRIYW
2081  YWPEDNVCEL VIRDVTAEDS ASIMVKAINI AGETSSHAFL
2121  LVQAKQLITF TQELQDVVAK EKDTMATFEC ETSEPFVKVK
2161  WYKDGMEVHE GDKYRMHSDR KVHFLSILTI DTSDAEDYSC
2201  VLVEDENVKT TAKLIVEGAV VEFVKELQDI EVPESYSGEL
2241  ECIVSPENIE GKWYHNDVEL KSNGKYTITS RRGRQNLTVK
2281  DVTKEDQGEY SFVIDGKKTT CKLKMKPRPI AILQGLSDQK
2321  VCEGDIVQLE VKVSLESVEG VWMKDGQEVQ PSDRVHIVID
2361  KQSHMLLIED MTKEDAGNYS FTIPALGLST SGRVSVYSVD
2401  VITPLKDVNV IEGTKAVLEC KVSVPDVTSV KWYLNDEQIK
2441  PDDRVQAIVK GTKQRLVINR THASDEGPYK LIVGRVETNC
2481  NLSVEKIKII RGLRDLTCTE TQNVVFEVEL SHSGIDVLWN
2521  FKDKEIKPSS KYKIEAHGKI YKLTVLNMMK DDEGKYTFYA
2561  GENMTSGKLT VAGGAISKPL TDQTVAESQE AVFECEVANP
2601  DSKGEWLRDG KHLPLTNNIR SESDGHKRRL IIAATKLDDI
2641  GEYTYKVATS KTSAKLKVEA VKIKKTLKNL TVTETQDAVE
2681  TVELTHPNVK GVQWIKNGVV LESNEKYAIS VKGTIYSLRI
2721  KNCAIVDESV YGFRLGRLGA SARLHVETVK IIKKPKDVTA
2761  LENATVAFEV SVSHDTVPVK WFHKSVEIKP SDKHRLVSER
2801  KVHKLMLQNI SPSDAGEYTA VVGQLECKAK LFVETLHITK
2841  TMKNIEVPET KTASFECEVS HFNVPSMWLK NGVEIEMSEK
2881  FKIVVQGKLH QLIIMNTSTE DSAEYTFVCG NDQVSATLTV
2921  TPIMITSMLK DINAEEKDTI TFEVTVNYEG ISYKWLKNGV
2961  EIKSTDKCQM RTKKLTHSLN IRNVHFGDAA DYTFVAGKAT
3001  STATLYVEAR HIEFRKHIKD IKVLEKKRAM FECEVSEPDI
3041  TVQWMKDDQE LQITDRIKIQ KEKYVHRLLI PSTRMSDAGK
3081  YTVVAGGNVS TAKLFVEGRD VRIRSIKKEV QVIEKQRAVV
3121  EFEVNEDDVD AHWYKDGIEI NFQVQERHKY VVERRIHRMF
3161  ISETRQSDAG EYTFVAGRNR SSVTLYVNAP EPPQVLQELQ
3201  PVTVQSGKPA RFCAVISGRP QPKISWYKEE QLLSTGFKCK
3241  FLHDGQEYTL LLIEAFPEDA AVYTCEAKND YGVATTSASL
3281  SVEVPEVVSP DQEMPVYPPA IITPLQDTVT SEGQPARFQC
3321  RVSGTDLKVS WYSKDKKIKP SRFFRMTQFE DTYQLEIAEA
3361  YPEDEGTYTF VASNAVGQVS STANLSLEAP ESILHERIEQ
3401  EIEMEMKEFS SSFLSAEEEG LHSAELQLSK INETLELLSE
3441  SPVYPTKFDS EKEGTGPIFI KEVSNADISM GDVATLSVTV
3481  IGIPKPKIQW FFNGVLLTPS ADYKFVFDGD DHSLIILFTK
3521  LEDEGEYTCM ASNDYGKTIC SAYLKINSKG EGHKDTETES
3561  AVAKSLEKLG GPCPPHFLKE LKPIRCAQGL PAIFEYTVVG
3601  EPAPTVTWFK ENKQLCTSVY YTIIHNPNGS GTFIVNDPQR
3641  EDSGLYICKA ENMLGESTCA AELLVLLEDT DMTDTPCKAK
3681  STPEAPEDFP QTPLKGPAVE ALDSEQEIAT FVKDTILKAA
3721  LITEENQQLS YEHIAKANEL SSQLPLGAQE LQSILEQDKL
3761  TPESTREFLC INGSIHFQPL KEPSPNLQLQ IVQSQKTFSK
3801  EGILMPEEPE TQAVLSDTEK IFPSAMSIEQ INSLTVEPLK
3841  TLLAEPEGNY PQSSIEPPMH SYLTSVAEEV LSPKEKTVSD
3881  TNREQRVTLQ KQEAQSALIL SQSLAEGHVE SLQSPDVMIS
3921  QVNYEPLVPS EHSCTEGGKI LIESANPLEN AGQDSAVRIE
3961  EGKSLRFPLA LEEKQVLLKE EHSDNVVMPP DQIIESKREP
4001  VAIKKVQEVQ GRDLLSKESL LSGIPEEQRL NLKIQICRAL
4041  QAAVASEQPG LFSEWLRNIE KVEVEAVNIT QEPRHIMCMY
4081  LVTSAKSVTE EVTIIIEDVD PQMANLKMEL RDALCAIIYE
4121  EIDILTAEGP RIQQGAKTSL QEEMDSFSGS QKVEPITEPE
4161  VESKYLISTE EVSYFNVQSR VKYLDATPVT KGVASAVVSD
4201  EKQDESLKPS EEKEESSSES GTEEVATVKI QEAEGGLIKE
4241  DGPMIHTPLV DTVSEEGDIV HLTTSITNAK EVNWYFENKL
4281  VPSDEKFKCL QDQNTYTLVI DKVNTEDHQG EYVCEALNDS
4321  GKTATSAKLT VVKRAAPVIK RKIEPLEVAL GHLAKFTCEI
4361  QSAPNVRFQW FKAGREIYES DKCSIRSSKY ISSLEILRTQ
4401  VVDCGEYTCK ASNEYGSVSC TATLTVTVPG GEKKVRKLLP
4441  ERKPEPKEEV VLKSVLRKRP EEEEPKVEPK KLEKVKKPAV
4481  PEPPPPKPVE EVEVPTVTKR ERKIPEPTKV PEIKPAIPLP
4521  APEPKPKPEA EVKTIKPPPV EPEPTPIAAP VTVPVVGKKA
4561  EAKAPKEEAA KPKGPIKGVP KKTPSPIEAE RRKLRPGSGG
4601  EKPPDEAPFT YQLKAVPLKF VKEIKDIILT ESEFVGSSAI
4641  FECLVSPSTA ITTWMKDGSN IRESPKHRFI ADGKDRKLHI
4681  IDVQLSDAGE YTCVLRLGNK EKTSTAKLVV EELPVRFVKT
4721  LEEEVTVVKG QPLYLSCELN KERDVVWRKD GKIVVEKPGR
4761  IVPGVIGLMR ALTINDADDT DAGTYTVTVE NANNLECSSC
4801  VKVVEVIRDW LVKPIRDQHV KPKGTAIFAC DIAKDTPNIK
4841  WFKGYDEIPA EPNDKTEILR DGNHLYLKIK NAMPEDIAEY
4881  AVEIEGKRYP AKLTLGEREV ELLKPIEDVT IYEKESASFD
4921  AEISEADIPG QWKLKGELLR PSPTCEIKAE GGKRFLTLHK
4961  VKLDQAGEVL YQALNAITTA ILTVKEIELD FAVPLKDVTV
5001  PERRQARFEC VLTREANVIW SKGPDIIKSS DKFDIIADGK
5041  KHILVINDSQ FDDEGVYTAE VEGKKTSARL FVTGIRLKFM
5081  SPLEDQTVKE GETATFVCEL SHEKMHVVWF KNDAKLHTSR
5121  TVLISSEGKT HKLEMKEVTL DDISQIKAQV KELSSTAQLK
5161  VLEADPYFTV KLHDKTAVEK DEITLKCEVS KDVPVKWFKD
5201  GEEIVPSPKY SIKADGLRRI LKIKKADLKD KGEYVCDCGT
5241  DKTKANVTVE ARLIKVEKPL YGVEVFVGET AHFEIELSEP
5281  DVHGQWKLKG QPLTASPDCE IIEDGKKHIL ILHNCQLGMT
5321  GEVSFQAANA KSAANLKVKE LPLIFITPLS DVKVFEKDEA
5361  KFECEVSREP KTFRWLKGTQ EITGDDRFEL IKDGTKHSMV
5401  IKSAAFEDEA KYMFEAEDKH TSGKLIIEGI RLKFLTPLKD
5441  VTAKEKESAV FTVELSHDNI RVKWFKNDQR LHTTRSVSMQ
5481  DEGKTHSITF KDLSIDDTSQ IRVEAMGMSS EAKLTVLEGD
5521  PYFTGKLQDY TGVEKDEVIL QCEISKADAP VKWFKDGKEI
5561  KPSKNAVIKA DGKKRMLILK KALKSDIGQY TCDCGTDKTS
5601  GKLDIEDREI KLVRPLHSVE VMETETARFE TEISEDDIHA
5641  NWKLKGEALL QTPDCEIKEE GKIHSLVLHN CRLDQTGGVD
5681  FQAANVKSSA HLRVKPRVIG LLRPLKDVTV TAGETATFDC
5721  ELSYEDIPVE WYLKGKKLEP SDKVVPRSEG KVHTLTLRDV
5761  KLEDAGEVQL TAKDFKTHAN LFVKEPPVEF TKPLEDQTVE
5801  EGATAVLECE VSRENAKVKW FKNGTEILKS KKYEIVADGR
5841  VRKLVIHDCT PEDIKTYTCD AKDFKTSCNL NVVPPHVEFL
5881  RPLTDLQVRE KEMARFECEL SRENAKVKWF KDGAEIKKGK
5921  KYDIISKGAV RILVINKCLL DDEAEYSCEV RTARTSGMLT
5961  VLEEEAVFTK NLANIEVSET DTIKLVCEVS KPGAEVIWYK
6001  GDEEIIETGR YEILTEGRKR ILVIQNAHLE DAGNYNCRLP
6041  SSRTDGKVKV HELAAEFISK PQNLEILEGE KAEFVCSISK
6081  ESFPVQWKRD DKTLESGDKY DVIADGKKRV LVVKDATLQD
6121  MGTYVVMVGA ARAAAHLTVI EKLRIVVPLK DTRVKEQQEV
6161  VFNCEVNTEG AKAKWFRNEE AIFDSSKYII LQKDLVYTLR
6201  IRDAHLDDQA NYNVSLINHR GENVKSAANL IVEEEDLRIV
6241  EPLKDIETME KKSVTFWCKV NRLNVTLKWT KNGEEVPFDN
6281  RVSYRVDKYK HMLTIKDCGF PDEGEYIVTA GQDKSVAELL
6321  IIEAPTEFVE HLEDQTVTEF DDAVFSCQLS REKANVKWYR
6361  NGREIKEGKK YKFEKDGSIH RLIIKDCRLD DECEYACGVE
6401  DRKSRARLFV EEIPVEIIRP PQDILEAPGA DVVFLAELNK
6441  DKVEVQWLRN NMVVVQGDKH QMMSEGKIHR LQICDIKPRD
6481  QGEYRFIAKD KEARAKLELA AAPKIKTADQ DLVVDVGKPL
6521  TMVVPYDAYP KAEAEWFKEN EPLSTKTIDT TAEQTSFRIL
6561  EAKKGDKGRY KIVLQNKHGK AEGFINLKVI DVPGPVRNLE
6601  VTETFDGEVS LAWEEPLTDG GSKIIGYVVE RRDIKRKTWV
6641  LATDRAESCE FTVTGLQKGG VEYLFRVSAR NRVGTGEPVE
6681  TDNPVEARSK YDVPGPPLNV TITDVNRFGV SLTWEPPEYD
6721  GGAEITNYVI ELRDKTSIRW DTAMTVRAED LSATVTDVVE
6761  GQEYSFRVRA QNRIGVGKPS AATPFVKVAD PIERPSPPVN
6801  LTSSDQTQSS VQLKWEPPLK DGGSPILGYI IERCEEGKDN
6841  WIRCNMKLVP ELTYKVTGLE KGNKYLYRVS AENKAGVSDP
6881  SEILGPLTAD DAFVEPTMDL SAFKDGLEVI VPNPITILVP
6921  STGYPRPTAT WCFGDKVLET GDRVKMKTLS AYAELVISPS
6961  ERSDKGIYTL KLENRVKTIS GEIDVNVIAR PSAPKELKFG
7001  DITKDSVHLT WEPPDDDGGS PLTGYVVEKR EVSRKTWTKV
7041  MDFVTDLEFT VPDLVQGKEY LFKVCARNKC GPGEPAYVDE
7081  PVNMSTPATV PDPPENVKWR DRTANSIFLT WDPPKNDGGS
7121  RIKGYIVERC PRGSDKWVAC GEPVAETKME VTGLEEGKWY
7161  AYRVKALNRQ GASKPSRPTE EIQAVDTQEA PEIFLDVKLL
7201  AGLTVKAGTK IELPATVTGK PEPKITWTKA DMILKQDKRI
7241  TIENVPKKST VTIVDSKRSD TGTYIIEAVN VCGRATAVVE
7281  VNVLDKPGPP AAFDITDVTN ESCLLTWNPP RDDGGSKITN
7321  YVVERRATDS EVWHKLSSTV KDTNFKATKL IPNKEYIFRV
7361  AAENMYGVGE PVQASPITAK YQFDPPGPPT RLEPSDITKD
7401  AVTLTWCEPD DDGGSPITGY WVERLDPDTD KWVRCNKMPV
7441  KDTTYRVKGL TNKKKYRFRV LAENLAGPGK PSKSTEPILI
7481  KDPIDPPWPP GKPTVKDVGK TSVRLNWTKP EHDGGAKIES
7521  YVIEMLKTGT DEWVRVAEGV PTTQHLLPGL MEGQEYSFRV
7561  RAVNKAGESE PSEPSDPVLC REKLYPPSPP RWLEVINITK
7601  NTADLKWTVP EKDGGSPITN YIVEKRDVRR KGWQTVDTTV
7641  KDTKCTVTPL TEGSLYVFRV AAENAIGQSD YTEIEDSVLA
7681  KDTFTTPGPP YALAVVDVTK RHVDLKWEPP KNDGGRPIQR
7721  YVIEKKERLG TRWVKAGKTA GPDCNFRVTD VIEGTEVQFQ
7761  VRAENEAGVG HPSEPTEILS IEDPTSPPSP PLDLHVTDAG
7801  RKHIAIAWKP PEKNGGSPII GYHVEMCPVG TEKWMRVNSR
7841  PIKDLKFKVE EGVVPDKEYV LRVRAVNAIG VSEPSEISEN
7881  VVAKDPDCKP TIDLETHDII VIEGEKLSIP VPFRAVPVPT
7921  VSWHKDGKEV KASDRLTMKN DHISAHLEVP KSVRADAGIY
7961  TITLENKLGS ATASINVKVI GLPGPCKDIK ASDITKSSCK
8001  LTWEPPEFDG GTPILHYVLE RREAGRRTYI PVMSGENKLS
8041  WTVKDLIPNG EYFFRVKAVN KVGGGEYIEL KNPVIAQDPK
8081  QPPDPPVDVE VHNPTAEAMT ITWKPPLYDG GSKIMGYIIE
8121  KIAKGEERWK RCNEHLVPIL TYTAKGLEEG KEYQFRVRAE
8161  NAAGISEPSR ATPPTKAVDP IDAPKVILRT SLEVKRGDEI
8201  ALDASISGSP YPTITWIKDE NVIVPEEIKK RAAPLVRRRK
8241  GEVQEEEPFV LPLTQRLSID NSKKGESQLR VRDSLRPDHG
8281  LYMIKVENDH GIAKAPCTVS VLDTPGPPIN FVFEDIRKTS
8321  VLCKWEPPLD DGGSEIINYT LEKKDKTKPD SEWIVVTSTL
8361  RHCKYSVTKL IEGKEYLFRV RAENRFGPGP PCVSKPLVAK
8401  DPFGPPDAPD KPIVEDVTSN SMLVKWNEPK DNGSPILGYW
8441  LEKREVNSTH WSRVNKSLLN ALKANVDGLL EGLTYVFRVC
8481  AENAAGPGKF SPPSDPKTAH DPISPPGPPI PRVTDTSSTT
8521  IELEWEPPAF NGGGEIVGYF VDKQLVGTNE WSRCTEKMIK
8561  VRQYTVKEIR EGADYKLRVS AVNAAGEGPP GETQPVTVAE
8601  PQEPPAVELD VSVKGGIQIM AGKTLRIPAV VTGRPVPTKV
8641  WTKEEGELDK DRVVIDNVGT KSELIIKDAL RKDHGRYVIT
8681  ATNSCGSKFA AARVEVFDVP GPVLDLKPVV TNRKMCLLNW
8721  SDPEDDGGSE ITGFIIERKD AKMHTWRQPI ETERSKCDIT
8761  GLLEGQEYKF RVIAKNKFGC GPPVEIGPIL AVDPLGPPTS
8801  PERLTYTERT KSTITLDWKE PRSNGGSPIQ GYIIEKRRHD
8841  KPDFERVNKR LCPTTSFLVE NLDEHQMYEF RVKAVNEIGE
8881  SEPSLPLNVV IQDDEVPPTI KLRLSVRGDT IKVKAGEPVH
8921  IPADVTGLPM PKIEWSKNET VIEKPTDALQ ITKEEVSRSE
8961  AKTELSIPKA VREDKGTYTV TASNRLGSVF RNVHVEVYDR
9001  PSPPRNLAVT DIKAESCYLT WDAPLDNGGS EITHYVIDKR
9041  DASRKKAEWE EVTNTAVEKR YGIWKLIPNG QYEFRVRAVN
9081  KYGISDECKS DKVVIQDPYR LPGPPGKPKV LARTKGSMLV
9121  SWTPPLDNGG SPITGYWLEK REEGSPYWSR VSRAPITKVG
9161  LKGVEFNVPR LLEGVKYQFR AMAINAAGIG PPSEPSDPEV
9201  AGDPIFPPGP PSCPEVKDKT KSSISLGWKP PAKDGGSPIK
9241  GYIVEMQEEG TTDWKRVNEP DKLITTCECV VPNLKELRKY
9281  RFRVKAVNEA GESEPSDTTG EIPATDIQEE PEVFIDIGAQ
9321  DCLVCKAGSQ IRIPAVIKGR PTPKSSWEFD GKAKKAMKDG
9361  VHDIPEDAQL ETAENSSVII IPECKRSHTG KYSITAKNKA
9401  GQKTANCRVK VMDVPGPPKD LKVSDITRGS CRLSWKMPDD
9441  DGGDRIKGYV IEKRTIDGKA WTKVNPDCGS TTFVVPDLLS
9481  EQQYFFRVRA ENRFGIGPPV ETIQRTTARD PIYPPDPPIK
9521  LKIGLITKNT VHLSWKPPKN DGGSPVTHYI VECLAWDPTG
9561  TKKEAWRQCN KRDVEELQFT VEDLVEGGEY EFRVKAVNAA
9601  GVSKPSATVG PVTVKDQTCP PSIDLKEFME VEEGTNVNIV
9641  AKIKGVPFPT LTWFKAPPKK PDNKEPVLYD THVNKLVVDD
9681  TCTLVIPQSR RSDTGLYTIT AVNNLGTASK EMRLNVLGRP
9721  GPPVGPIKFE SVSADQMTLS WFPPKDDGGS KITNYVIEKR
9761  EANRKTWVHV SSEPKECTYT IPKLLEGHEY VFRIMAQNKY
9801  GIGEPLDSEP ETARNLFSVP GAPDKPTVSS VTRNSMTVNW
9841  EEPEYDGGSP VTGYWLEMKD TTSKRWKRVN RDPIKAMTLG
9881  VSYKVTGLIE GSDYQFRVYA INAAGVGPAS LPSDPATARD
9921  PIAPPGPPFP KVTDWTKSSA DLEWSPPLKD GGSKVTGYIV
9961  EYKEEGKEEW EKGKDKEVRG TKLVVTGLKE GAFYKFRVRA
10001 VNIAGIGEPG EVTDVIEMKD RLVSPDLQLD ASVRDRIVVH
10041 AGGVIRIIAY VSGKPPPTVT WNMNERTLPQ EATIETTAIS
10081 SSMVIKNCQR SHQGVYSLLA KNEAGERKKT IIVDVLDVPG
10121 PVGTPFLAHN LTNESCKLTW FSPEDDGGSP ITNYVIEKRE
10161 SDRRAWTPVT YTVTRQNATV QGLIQGKAYF FRIAAENSIG
10201 MGPFVETSEA LVIREPITVP ERPEDLEVKE VTKNTVTLTW
10241 NPPKYDGGSE IINYVLESRL IGTEKFHKVT NDNLLSRKYT
10281 VKGLKEGDTY EYRVSAVNIV GQGKPSFCTK PITCKDELAP
10321 PTLHLDERDK LTIRVGEAFA LTGRYSGKPK PKVSWFKDEA
10361 DVLEDDRTHI KTTPATLALE KIKAKRSDSG KYCVVVENST
10401 GSRKGFCQVN VVDRPGPPVG PVSFDEVTKD YMVISWKPPL
10441 DDGGSKITNY IIEKKEVGKD VWMPVTSASA KTTCKVSKLL
10481 EGKDYIFRIH AENLYGISDP LVSDSMKAKD RFRVPDAPDQ
10521 PIVTEVTKDS ALVTWNKPHD GGKPITNYIL EKRETMSKRW
10561 ARVTKDPIHP YTKFRVPDLL EGCQYEFRVS AENEIGIGDP
10601 SPPSKPVFAK DPIAKPSPPV NPEAIDTTCN SVDLTWQPPR
10641 HDGGSKILGY IVEYQKVGDE EWRRANHTPE SCPETKYKVT
10681 GLRDGQTYKF RVLAVNAAGE SDPAHVPEPV LVKDRLEPPE
10721 LILDANMARE QHIKVGDTLR LSAIIKGVPF PKVTWKKEDR
10761 DAPTKARIDV TPVGSKLEIR NAAHEDGGIY SLTVENPAGS
10801 KTVSVKVLVL DKPGPPRDLE VSEIRKDSCY LTWKEPLDDG
10841 GSVITNYVVE RRDVASAQWS PLSATSKKKS HFAKHLNEGN
10881 QYLFRVAAEN QYGRGPFVET PKPIKALDPL HPPGPPKDLH
10921 HVDVDKTEVS LVWNKPDRDG GSPITGYLVE YQEEGTQDWI
10961 KFKTVTNLEC VVTGLQQGKT YRFRVKAENI VGLGLPDTTI
11001 PIECQEKLVP PSVELDVKLI EGLVVKAGTT VRFPAIIRGV
11041 PVPTAKWTTD GSEIKTDEHY TVETDNFSSV LTIKNCLRRD
11081 TGEYQITVSN AAGSKTVAVH LTVLDVPGPP TGPINILDVT
11121 PEHMTISWQP PKDDGGSPVI NYIVEKQDTR KDTWGVVSSG
11161 SSKTKLKIPH LQKGCEYVFR VRAENKIGVG PPLDSTPTVA
11201 KHKFSPPSPP GKPVVTDITE NAATVSWTLP KSDGGSPITG
11241 YYMERREVTG KWVRVNKTPI ADLKFRVTGL YEGNTYEFRV
11281 FAENLAGLSK PSPSSDPIKA CRPIKPPGPP INPKLKDKSR
11321 ETADLVWTKP LSDGGSPILG YVVECQKPGT AQWNRINKDE
11361 LIRQCAFRVP GLIEGNEYRF RIKAANIVGE GEPRELAESV
11401 IAKDILHPPE VELDVTCRDV ITVRVGQTIR ILARVKGRPE
11441 PDITWTKEGK VLVREKRVDL IQDLPRVELQ IKEAVRADHG
11481 KYIISAKNSS GHAQGSAIVN VLDRPGPCQN LKVTNVTKEN
11521 CTISWENPLD NGGSEITNFI VEYRKPNQKG WSIVASDVTK
11561 RLIKANLLAN NEYYFRVCAE NKVGVGPTIE TKTPILAINP
11601 IDRPGEPENL HIADKGKTFV YLKWRRPDYD GGSPNLSYHV
11641 ERRLKGSDDW ERVHKGSIKE THYMVDRCVE NQIYEFRVQT
11681 KNEGGESDWV KTEEVVVKED LQKPVLDLKL SGVLTVKAGD
11721 TIRLEAGVRG KPFPEVAWTK DKDATDLTRS PRVKIDTRAD
11761 SSKFSLTKAK RSDGGKYVVT ATNTAGSFVA YATVNVLDKP
11801 GPVRNLKIVD VSSDRCTVCW DPPEDDGGCE IQNYILEKCE
11841 TKRMVWSTYS ATVLTPGTTV TRLIEGNEYI FRVRAENKIG
11881 TGPPTESKPV IAKTKYDKPG RPDPPEVTKV SKEEMTVVWN
11921 PPEYDGGKSI TGYFLEKKEK HSTRWVPVNK SAIPERRMKV
11961 QNLLPDHEYQ FRVKAENEIG IGEPSLPSRP VVAKDPIEPP
12001 GPPTNFRVVD TTKHSITLGW GKPVYDGGAP IIGYVVEMRP
12041 KIADASPDEG WKRCNAAAQL VRKEFTVTSL DENQEYEFRV
12081 CAQNQVGIGR PAELKEAIKP KEILEPPEID LDASMRKLVI
12121 VRAGCPIRLF AIVRGRPAPK VTWRKVGIDN VVRKGQVDLV
12161 DTMAFLVIPN STRDDSGKYS LTLVNPAGEK AVFVNVRVLD
12201 TPGPVSDLKV SDVTKTSCHV SWAPPENDGG SQVTHYIVEK
12241 READRKTWST VTPEVKKTSF HVTNLVPGNE YYFRVTAVNE
12281 YGPGVPTDVP KPVLASDPLS EPDPPRKLEV TEMTKNSATL
12321 AWLPPLRDGG AKIDGYITSY REEEQPADRW TEYSVVKDLS
12361 LVVTGLKEGK KYKFRVAARN AVGVSLPREA EGVYEAKEQL
12401 LPPKILMPEQ ITIKAGKKLR IEAHVYGKPH PTCKWKKGED
12441 EVVTSSHLAV HKADSSSILI IKDVTRKDSG YYSLTAENSS
12481 GTDTQKIKVV VMDAPGPPQP PFDISDIDAD ACSLSWHIPL
12521 EDGGSNITNY IVEKCDVSRG DWVTALASVT KTSCRVGKLI
12561 PGQEYIFRVR AENRFGISEP LTSPKMVAQF PFGVPSEPKN
12601 ARVTKVNKDC IFVAWDRPDS DGGSPIIGYL IERKERNSLL
12641 WVKANDTLVR STEYPCAGLV EGLEYSFRIY ALNKAGSSPP
12681 SKPTEYVTAR MPVDPPGKPE VIDVTKSTVS LIWARPKHDG
12721 GSKIIGYFVE ACKLPGDKWV RCNTAPHQIP QEEYTATGLE
12761 EKAQYQFRAI ARTAVNISPP SEPSDPVTIL AENVPPRIDL
12801 SVAMKSLLTV KAGTNVCLDA TVFGKPMPTV SWKKDGTLLK
12841 PAEGIKMAMQ RNLCTLELFS VNRKDSGDYT ITAENSSGSK
12881 SATIKLKVLD KPGPPASVKI NKMYSDRAML SWEPPLEDGG
12921 SEITNYIVDK RETSRPNWAQ VSATVPITSC SVEKLIEGHE
12961 YQFRICAENK YGVGDPVFTE PAIAKNPYDP PGRCDPPVIS
13001 NITKDHMTVS WKPPADDGGS PITGYLLEKR ETQAVNWTKV
13041 NRKPIIERTL KATGLQEGTE YEFRVTAINK AGPGKPSDAS
13081 KAAYARDPQY PPGPPAFPKV YDTTRSSVSL SWGKPAYDGG
13121 SPIIGYLVEV KRADSDNWVR CNLPQNLQKT RFEVTGLMED
13161 TQYQFRVYAV NKIGYSDPSD VPDKHYPKDI LIPPEGELDA
13201 DLRKTLILRA GVTMRLYVPV KGRPPPKITW SKPNVNLRDR
13241 IGLDIKSTDF DTFLRCENVN KYDAGKYILT LENSCGKKEY
13281 TIVVKVLDTP GPPVNVTVKE ISKDSAYVTW EPPIIDGGSP
13321 IINYVVQKRD AERKSWSTVT TECSKTSFRV ANLEEGKSYF
13361 FRVFAENEYG IGDPGETRDA VKASQTPGPV VDLKVRSVSK
13401 SSCSIGWKKP HSDGGSRIIG YVVDFLTEEN KWQRVMKSLS
13441 LQYSAKDLTE GKEYTFRVSA ENENGEGTPS EITVVARDDV
13481 VAPDLDLKGL PDLCYLAKEN SNFRLKIPIK GKPAPSVSWK
13521 KGEDPLATDT RVSVESSAVN TTLIVYDCQK SDAGKYTITL
13561 KNVAGTKEGT ISIKVVGKPG IPTGPIKFDE VTAEAMTLKW
13601 APPKDDGGSE ITNYILEKRD SVNNKWVTCA SAVQKTTFRV
13641 TRLHEGMEYT FRVSAENKYG VGEGLKSEPI VARHPFDVPD
13681 APPPPNIVDV RHDSVSLTWT DPKKTGGSPI TGYHLEFKER
13721 NSLLWKRANK TPIRMRDFKV TGLTEGLEYE FRVMAINLAG
13761 VGKPSLPSEP VVALDPIDPP GKPEVINITR NSVTLIWTEP
13801 KYDGGHKLTG YIVEKRDLPS KSWMKANHVN VPECAFTVTD
13841 LVEGGKYEFR IRAKNTAGAI SAPSESTETI ICKDEYEAPT
13881 IVLDPTIKDG LTIKAGDTIV LNAISILGKP LPKSSWSKAG
13921 KDIRPSDITQ ITSTPTSSML TIKYATRKDA GEYTITATNP
13961 FGTKVEHVKV TVLDVPGPPG PVEISNVSAE KATLTWTPPL
14001 EDGGSPIKSY ILEKRETSRL LWTVVSEDIQ SCRHVATKLI
14041 QGNEYIFRVS AVNHYGKGEP VQSEPVKMVD RFGPPGPPEK
14081 PEVSNVTKNT ATVSWKRPVD DGGSEITGYH VERREKKSLR
14121 WVRAIKTPVS DLRCKVTGLQ EGSTYEFRVS AENRAGIGPP
14161 SEASDSVLMK DAAYPPGPPS NPHVTDTTKK SASLAWGKPH
14201 YDGGLEITGY VVEHQKVGDE AWIKDTTGTA LRITQFVVPD
14241 LQTKEKYNFR ISAINDAGVG EPAVIPDVEI VEREMAPDFE
14281 LDAELRRTLV VRAGLSIRIF VPIKGRPAPE VTWTKDNINL
14321 KNRANIENTE SFTLLIIPEC NRYDTGKFVM TIENPAGKKS
14361 GFVNVRVLDT PGPVLNLRPT DITKDSVTLH WDLPLIDGGS
14401 RITNYIVEKR EATRKSYSTA TTKCHKCTYK VTGLSEGCEY
14441 FFRVMAENEY GIGEPTETTE PVKASEAPSP PDSLNIMDIT
14481 KSTVSLAWPK PKHDGGSKIT GYVIEAQRKG SDQWTHITTV
14521 KGLECVVRNL TEGEEYTFQV MAVNSAGRSA PRESRPVIVK
14561 EQTMLPELDL RGIYQKLVIA KAGDNIKVEI PVLGRPKPTV
14601 TWKKGDQILK QTQRVNFETT ATSTILNINE CVRSDSGPYP
14641 LTARNIVGEV GDVITIQVHD IPGPPTGPIK FDEVSSDFVT
14681 FSWDPPENDG GVPISNYVVE MRQTDSTTWV ELATTVIRTT
14721 YKATRLTTGL EYQFRVKAQN RYGVGPGITS ACIVANYPFK
14761 VPGPPGTPQV TAVTKDSMTI SWHEPLSDGG SPILGYHVER
14801 KERNGILWQT VSKALVPGNI FKSSGLTDGI AYEFRVIAEN
14841 MAGKSKPSKP SEPMLALDPI DPPGKPVPLN ITRHTVTLKW
14881 AKPEYTGGFK ITSYIVEKRD LPNGRWLKAN FSNILENEFT
14921 VSGLTEDAAY EFRVIAKNAA GAISPPSEPS DAITCRDDVE
14961 APKIKVDVKF KDTVILKAGE AFRLEADVSG RPPPTMEWSK
15001 DGKELEGTAK LEIKIADFST NLVNKDSTRR DSGAYTLTAT
15041 NPGGFAKHIF NVKVLDRPGP PEGPLAVTEV TSEKCVLSWF
15081 PPLDDGGAKI DHYIVQKRET SRLAWTNVAS EVQVTKLKVT
15121 KLLKGNEYIF RVMAVNKYGV GEPLESEPVL AVNPYGPPDP
15161 PKNPEVTTIT KDSMVVCWGH PDSDGGSEII NYIVERRDKA
15201 GQRWIKCNKK TLTDLRYKVS GLTEGHEYEF RIMAENAAGI
15241 SAPSPTSPFY KACDTVFKPG PPGNPRVLDT SRSSISIAWN
15281 KPIYDGGSEI TGYMVEIALP EEDEWQIVTP PAGLKATSYT
15321 ITGLTENQEY KIRIYAMNSE GLGEPALVPG TPKAEDRMLP
15361 PEIELDADLR KVVTIRACCT LRLFVPIKGR PAPEVKWARD
15401 HGESLDKASI ESTSSYTLLI VGNVNRFDSG KYILTVENSS
15441 GSKSAFVNVR VLDTPGPPQD LKVKEVTKTS VTLTWDPPLL
15481 DGGSKIKNYI VEKRESTRKA YSTVATNCHK TSWKVDQLQE
15521 GCSYYFRVLA ENEYGIGLPA ETAESVKASE RPLPPGKITL
15561 MDVTRNSVSL SWEKPEHDGG SRILGYIVEM QTKGSDKWAT
15601 CATVKVTEAT ITGLIQGEEY SFRVSAQNEK GISDPRQLSV
15641 PVIAKDLVIP PAFKLLFNTF TVLAGEDLKV DVPFIGRPTP
15681 AVTWHKDNVP LKQTTRVNAE STENNSLLTI KDACREDVGH
15721 YVVKLTNSAG EAIETLNVIV LDKPGPPTGP VKMDEVTADS
15761 ITLSWGPPKY DGGSSINNYI VEKRDTSTTT WQIVSATVAR
15801 TTIKACRLKT GCEYQFRIAA ENRYGKSTYL NSEPTVAQYP
15841 FKVPGPPGTP VVTLSSRDSM EVQWNEPISD GGSRVIGYHL
15881 ERKERNSILW VKLNKTPIPQ TKFKTTGLEE GVEYEFRVSA
15921 ENIVGIGKPS KVSECYVARD PCDPPGRPEA IIVTRNSVTL
15961 QWKKPTYDGG SKITGYIVEK KELPEGRWMK ASFTNIIDTH
16001 FEVTGLVEDH RYEFRVIARN AAGVFSEPSE STGAITARDE
16041 VDPPRISMDP KYKDTIVVHA GESFKVDADI YGKPIPTIQW
16081 IKGDQELSNT ARLEIKSTDF ATSLSVKDAV RVDSGNYILK
16121 AKNVAGERSV TVNVKVLDRP GPPEGPVVIS GVTAEKCTLA
16161 WKPPLQDGGS DIINYIVERR ETSRLVWTVV DANVQTLSCK
16201 VTKLLEGNEY TFRIMAVNKY GVGEPLESEP VVAKNPFVVP
16241 DAPKAPEVTT VTKDSMIVVW ERPASDGGSE ILGYVLEKRD
16281 KEGIRWTRCH KRLIGELRLR VTGLIENHDY EFRVSAENAA
16321 GLSEPSPPSA YQKACDPIYK PGPPNNPKVI DITRSSVFLS
16361 WSKPIYDGGC EIQGYIVEKC DVSVGEWTMC TPPTGINKTN
16401 IEVEKLLEKH EYNFRICAIN KAGVGEHADV PGPIIVEEKL
16441 EAPDIDLDLE LRKIINIRAG GSLRLFVPIK GRPTPEVKWG
16481 KVDGEIRDAA IIDVTSSFTS LVLDNVNRYD SGKYTLTLEN
16521 SSGTKSAFVT VRVLDTPSPP VNLKVTEITK DSVSITWEPP
16561 LLDGGSKIKN YIVEKREATR KSYAAVVTNC HKNSWKIDQL
16601 QEGCSYYFRV TAENEYGIGL PAQTADPIKV AEVPQPPGKI
16641 TVDDVTRNSV SLSWTKPEHD GGSKIIQYIV EMQAKHSEKW
16681 SECARVKSLQ AVITNLTQGE EYLFRVVAVN EKGRSDPRSL
16721 AVPIVAKDLV IEPDVKPAFS SYSVQVGQDL KIEVPISGRP
16761 KPTITWTKDG LPLKQTTRIN VTDSLDLTTL SIKETHKDDG
16801 GQYGITVANV VGQKTASIEI VTLDKPDPPK GPVKFDDVSA
16841 ESITLSWNPP LYTGGCQITN YIVQKRDTTT TVWDVVSATV
16881 ARTTLKVTKL KTGTEYQFRI FAENRYGQSF ALESDPIVAQ
16921 YPYKEPGPPG TPFATAISKD SMVIQWHEPV NNGGSPVIGY
16961 HLERKERNSI LWTKVNKTII HDTQFKAQNL EEGIEYEFRV
17001 YAENIVGVGK ASKNSECYVA RDPCDPPGTP EPIMVKRNEI
17041 TLQWTKPVYD GGSMITGYIV EKRDLPDGRW MKASFTNVIE
17081 TQFTVSGLTE DQRYEFRVIA KNAAGAISKP SDSTGPITAK
17121 DEVELPRISM DPKFRDTIVV NAGETFRLEA DVHGKPLPTI
17161 EWLRGDKEIE ESARCEIKNT DFKALLIVKD AIRIDGGQYI
17201 LRASNVAGSK SFPVNVKVLD RPGPPEGPVQ VTGVTSEKCS
17241 LTWSPPLQDG GSDISHYVVE KRETSRLAWT VVASEVVTNS
17281 LKVTKLLEGN EYVFRIMAVN KYGVGEPLES APVLMKNPFV
17321 LPGPPKSLEV TNIAKDSMTV CWNRPDSDGG SEIIGYIVEK
17361 RDRSGIRWIK CNKRRITDLR LRVTGLTEDH EYEFRVSAEN
17401 AAGVGEPSPA TVYYKACDPV FKPGPPTNAH IVDTTKNSIT
17441 LAWGKPIYDG GSEILGYVVE ICKADEEEWQ IVTPQTGLRV
17481 TRFEISKLTE HQEYKIRVCA LNKVGLGEAT SVPGTVKPED
17521 KLEAPELDLD SELRKGIVVR AGGSARIHIP FKGRPTPEIT
17561 WSREEGEFTD KVQIEKGVNY TQLSIDNCDR NDAGKYILKL
17601 ENSSGSKSAF VTVKVLDTPG PPQNLAVKEV RKDSAFLVWE
17641 PPIIDGGAKV KNYVIDKRES TRKAYANVSS KCSKTSFKVE
17681 NLTEGAIYYF RVMAENEFGV GVPVETVDAV KAAEPPSPPG
17721 KVTLTDVSQT SASLMWEKPE HDGGSRVLGY VVEMQPKGTE
17761 KWSIVAESKV CNAVVTGLSS GQEYQFRVKA YNEKGKSDPR
17801 VLGVPVIAKD LTIQPSLKLP FNTYSIQAGE DLKIEIPVIG
17841 RPRPNISWVK DGEPLKQTTR VNVEETATST VLHIKEGNKD
17881 DFGKYTVTAT NSAGTATENL SVIVLEKPGP PVGPVREDEV
17921 SADFVVISWE PPAYTGGCQI SNYIVEKRDT TTTTWHMVSA
17961 TVARTTIKIT KLKTGTEYQF RIFAENRYGK SAPLDSKAVI
18001 VQYPFKEPGP PGTPFVTSIS KDQMLVQWHE PVNDGGTKII
18041 GYHLEQKEKN SILWVKLNKT PIQDTKFKTT GLDEGLEYEF
18081 KVSAENIVGI GKPSKVSECF VARDPCDPPG RPEAIVITRN
18121 NVTLKWKKPA YDGGSKITGY IVEKKDLPDG RWMKASFTNV
18161 LETEFTVSGL VEDQRYEFRV IARNAAGNES EPSDSSGAIT
18201 ARDEIDAPNA SLDPKYKDVI VVHAGETFVL EADIRGKPIP
18241 DVVWSKDGKE LEETAARMEI KSTIQKTTLV VKDCIRTDGG
18281 QYILKLSNVG GTKSIPITVK VLDRPGPPEG PLKVTGVTAE
18321 KCYLAWNPPL QDGGANISHY IIEKRETSRL SWTQVSTEVQ
18361 ALNYKVTKLL PGNEYIFRVM AVNKYGIGEP LESGPVTACN
18401 PYKPPGPPST PEVSAITKDS MVVTWARPVD DGGTEIEGYI
18441 LEKRDKEGVR WTKCNKKTLT DLRLRVTGLT EGHSYEFRVA
18481 AENAAGVGEP SEPSVFYRAC DALYPPGPPS NPKVTDTSRS
18521 SVSLAWSKPI YDGGAPVKGY VVEVKEAAAD EWTTCTPPTG
18561 LQGKQFTVTK LKENTEYNFR ICAINSEGVG EPATLPGSVV
18601 AQERIEPPEI ELDADLRKVV VLRASATLRL FVTIKGRPEP
18641 EVKWEKAEGI LTDRAQIEVT SSFTMLVIDN VTRFDSGRYN
18681 LTLENNSGSK TAFVNVRVLD SPSAPVNLTI REVKKDSVTL
18721 SWEPPLIDGG AKITNYIVEK RETTRKAYAT ITNNCTKTTF
18761 RIENLQEGCS YYFRVLASNE YGIGLPAETT EPVKVSEPPL
18801 PPGRVTLVDV TRNTATIKWE KPESDGGSKI TGYVVEMQTK
18841 GSEKWSTCTQ VKTLEATISG LTAGEEYVFR VAAVNEKGRS
18881 DPRQLGVPVI ARDIEIKPSV ELPFHTFNVK AREQLKIDVP
18921 FKGRPQATVN WRKDGQTLKE TTRVNVSSSK TVTSLSIKEA
18961 SKEDVGTYEL CVSNSAGSIT VPITIIVLDR PGPPGPIRID
19001 EVSCDSITIS WNPPEYDGGC QISNYIVEKK ETTSTTWHIV
19041 SQAVARTSIK IVRLTTGSEY QFRVCAENRY GKSSYSESSA
19081 VVAEYPFSPP GPPGTPKVVH ATKSTMLVTW QVPVNDGGSR
19121 VIGYHLEYKE RSSILWSKAN KILIADTQMK VSGLDEGLMY
19161 EYRVYAENIA GIGKCSKSCE PVPARDPCDP PGQPEVTNIT
19201 RKSVSLKWSK PHYDGGAKIT GYIVERRELP DGRWLKCNYT
19241 NIQETYFEVT ELTEDQRYEF RVFARNAADS VSEPSESTGP
19281 IIVKDDVEPP RVMMDVKFRD VIVVKAGEVL KINADIAGRP
19321 LPVISWAKDG IEIEERARTE IISTDNHTLL TVKDCIRRDT
19361 GQYVLTLKNV AGTRSVAVNC KVLDKPGPPA GPLEINGLTA
19401 EKCSLSWGRP QEDGGADIDY YIVEKRETSH LAWTICEGEL
19441 QMTSCKVTKL LKGNEYIFRV TGVNKYGVGE PLESVAIKAL
19481 DPFTVPSPPT SLEITSVTKE SMTLCWSRPE SDGGSEISGY
19521 IIERREKNSL RWVRVNKKPV YDLRVKSTGL REGCEYEYRV
19561 YAENAAGLSL PSETSPLIRA EDPVFLPSPP SKPKIVDSGK
19601 TTITIAWVKP LFDGGAPITG YTVEYKKSDD TDWKTSIQSL
19641 RGTEYTISGL TTGAEYVFRV KSVNKVGASD PSDSSDPQIA
19681 KEREEEPLFD IDSEMRKTLI VKAGASFTMT VPFRGRPVPN
19721 VLWSKPDTDL RTRAYVDTTD SRTSLTIENA NRNDSGKYTL
19761 TIQNVLSAAS LTLVVKVLDT PGPPTNITVQ DVTKESAVLS
19801 WDVPENDGGA PVKNYHIEKR EASKKAWVSV TNNCNRLSYK
19841 VTNLQEGAIY YFRVSGENEF GVGIPAETKE GVKITEKPSP
19881 PEKLGVTSIS KDSVSLTWLK PEHDGGSRIV HYVVEALEKG
19921 QKNWVKCAVA KSTHHVVSGL RENSEYFFRV FAENQAGLSD
19961 PRELLLPVLI KEQLEPPEID MKNFPSHTVY VRAGSNLKVD
20001 IPISGKPLPK VTLSRDGVPL KATMRENTEI TAENLTINLK
20041 ESVTADAGRY EITAANSSGT TKAFINIVVL DRPGPPTGPV
20081 VISDITEESV TLKWEPPKYD GGSQVTNYIL LKRETSTAVW
20121 TEVSATVART MMKVMKLTTG EEYQFRIKAE NRFGISDHID
20161 SACVTVKLPY TTPGPPSTPW VTNVTRESIT VGWHEPVSNG
20201 GSAVVGYHLE MKDRNSILWQ KANKLVIRTT HFKVTTISAG
20241 LIYEFRVYAE NAAGVGKPSH PSEPVLAIDA CEPPRNVRIT
20281 DISKNSVSLS WQQPAFDGGS KITGYIVERR DLPDGRWTKA
20321 SFTNVTETQF IISGLTQNSQ YEFRVFARNA VGSISNPSEV
20361 VGPITCIDSY GGPVIDLPLE YTEVVKYRAG TSVKLRAGIS
20401 GKPAPTIEWY KDDKELQTNA LVCVENTTDL ASILIKDADR
20441 LNSGCYELKL RNAMGSASAT IRVQILDKPG PPGGPIEFKT
20481 VTAEKITLLW RPPADDGGAK ITHYIVEKRE TSRVVWSMVS
20521 EHLEECIITT TKIIKGNEYI FRVRAVNKYG IGEPLESDSV
20561 VAKNAFVTPG PPGIPEVTKI TKNSMTVVWS RPIADGGSDI
20601 SGYFLEKRDK KSLGWFKVLK ETIRDTRQKV TGLTENSDYQ
20641 YRVCAVNAAG QGPFSEPSEF YKAADPIDPP GPPAKIRIAD
20681 STKSSITLGW SKPVYDGGSA VTGYVVEIRQ GEEEEWTTVS
20721 TKGEVRTTEY VVSNLKPGVN YYFRVSAVNC AGQGEPIEMN
20761 EPVQAKDILE APEIDLDVAL RTSVIAKAGE DVQVLIPFKG
20801 RPPPTVTWRK DEKNLGSDAR YSIENTDSSS LLTIPQVTRN
20841 DTGKYILTIE NGVGEPKSST VSVKVLDTPA ACQKLQVKHV
20881 SRGTVTLLWD PPLIDGGSPI INYVIEKRDA TKRTWSVVSH
20921 KCSSTSFKLI DLSEKTPFFF RVLAENEIGI GEPCETTEPV
20961 KAAEVPAPIR DLSMKDSTKT SVILSWTKPD FDGGSVITEY
21001 VVERKGKGEQ TWSHAGISKT CEIEVSQLKE QSVLEFRVFA
21041 KNEKGLSDPV TIGPITVKEL IITPEVDLSD IPGAQVTVRI
21081 GHNVHLELPY KGKPKPSISW LKDGLPLKES EFVRFSKTEN
21121 KITLSIKNAK KEHGGKYTVI LDNAVCRIAV PITVITLGPP
21161 SKPKGPIRFD EIKADSVILS WDVPEDNGGG EITCYSIEKR
21201 ETSQTNWKMV CSSVARTTFK VPNLVKDAEY QFRVRAENRY
21241 GVSQPLVSSI IVAKHQFRIP GPPGKPVIYN VTSDGMSLTW
21281 DAPVYDGGSE VTGFHVEKKE RNSILWQKVN TSPISGREYR
21321 ATGLVEGLDY QFRVYAENSA GLSSPSDPSK FTLAVSPVDP
21361 PGTPDYIDVT RETITLKWNP PLRDGGSKIV GYSIEKRQGN
21401 ERWVRCNFTD VSECQYTVTG LSPGDRYEFR IIARNAVGTI
21441 SPPSQSSGII MTRDENVPPI VEFGPEYFDG LIIKSGESLR
21481 IKALVQGRPV PRVTWFKDGV EIEKRMNMEI TDVLGSTSLF
21521 VRDATRDHRG VYTVEAKNAS GSAKAEIKVK VQDTPGKVVG
21561 PIRFTNITGE KMTLWWDAPL NDGCAPITHY IIEKRETSRL
21601 AWALIEDKCE AQSYTAIKLI NGNEYQFRVS AVNKFGVGRP
21641 LDSDPVVAQI QYTVPDAPGI PEPSNITGNS ITLTWARPES
21681 DGGSEIQQYI LERREKKSTR WVKVISKRPI SETRFKVTGL
21721 TEGNEYEFHV MAENAAGVGP ASGISRLIKC REPVNPPGPP
21761 TVVKVTDTSK TTVSLEWSKP VEDGGMEIIG YIIEMCKADL
21801 GDWHKVNAEA CVKTRYTVTD LQAGEEYKFR VSAINGAGKG
21841 DSCEVTGTIK AVDRLTAPEL DIDANFKQTH VVRAGASIRL
21881 FIAYQGRPTP TAVWSKPDSN LSLRADIHTT DSFSTLTVEN
21921 CNRNDAGKYT LTVENNSGSK SITFTVKVLD TPGPPGPITF
21961 KDVTRGSATL MWDAPLLDGG ARIHHYVVEK REASRRSWQV
22001 ISEKCTRQIF KVNDLAEGVP YYFRVSAVNE YGVGEPYEMP
22041 EPIVATEQPA PPRRLDVVDT SKSSAVLAWL KPDHDGGSRI
22081 TGYLLEMRQK GSDFWVEAGH TKQLTFTVER LVEKTEYEFR
22121 VKAKNDAGYS EPREAFSSVI IKEPQIEPTA DLTGITNQLI
22161 TCKAGSPFTI DVPISGRPAP KVTWKLEEMR LKETDRVSIT
22201 TTKDRTTLTV KDSMRGDSGR YFLTLENTAG VKTFSVTVVV
22241 IGRPGPVTGP IEVSSVSAES CVLSWGEPKD GGGTEITNYI
22281 VEKRESGTTA WQLVNSSVKR TQIKVTHLTK YMEYSFRVSS
22321 ENRFGVSKPL ESAPIIAEHP FVPPSAPTRP EVYHVSANAM
22361 SIRWEEPYHD GGSKIIGYWV EKKERNTILW VKENKVPCLE
22401 CNYKVTGLVE GLEYQFRTYA LNAAGVSKAS EASRPIMAQN
22441 PVDAPGRPEV TDVTRSTVSL IWSAPAYDGG SKVVGYIIER
22481 KPVSEVGDGR WLKCNYTIVS DNFFTVTALS EGDTYEFRVL
22521 AKNAAGVISK GSESTGPVTC RDEYAPPKAE LDARLHGDLV
22561 TIRAGSDLVL DAAVGGKPEP KIIWTKGDKE LDLCEKVSLQ
22601 YTGKRATAVI KFCDRSDSGK YTLTVKNASG TKAVSVMVKV
22641 LDSPGPCGKL TVSRVTQEKC TLAWSLPQED GGAEITHYIV
22681 ERRETSRLNW VIVEGECPTL SYVVTRLIKN NEYIFRVRAV
22721 NKYGPGVPVE SEPIVARNSF TIPSPPGIPE EVGTGKEHII
22761 IQWTKPESDG GNEISNYLVD KREKKSLRWT RVNKDYVVYD
22801 TRLKVTSLME GCDYQFRVTA VNAAGNSEPS EASNFISCRE
22841 PSYTPGPPSA PRVVDTTKHS ISLAWTKPMY DGGTDIVGYV
22881 LEMQEKDTDQ WYRVHTNATI RNTEFTVPDL KMGQKYSFRV
22921 AAVNVKGMSE YSESIAEIEP VERIEIPDLE LADDLKKTVT
22961 IRAGASLRLM VSVSGRPPPV ITWSKQGIDL ASRAIIDTTE
23001 SYSLLIVDKV NRYDAGKYTI EAENQSGKKS ATVLVKVYDT
23041 PGPCPSVKVK EVSRDSVTIT WEIPTIDGGA PVNNYIVEKR
23081 EAAMRAFKTV TTKCSKTLYR ISGLVEGTMY YFRVLPENIY
23121 GIGEPCETSD AVLVSEVPLV PAKLEVVDVT KSTVTLAWEK
23161 PLYDGGSRLT GYVLEACKAG TERWMKVVTL KPTVLEHTVT
23201 SLNEGEQYLF RIRAQNEKGV SEPRETVTAV TVQDLRVLPT
23241 IDLSTMPQKT IHVPAGRPVE LVIPIAGRPP PAASWFFAGS
23281 KLRESERVTV ETHTKVAKLT IRETTIRDTG EYTLELKNVT
23321 GTTSETIKVI ILDKPGPPTG PIKIDEIDAT SITISWEPPE
23361 LDGGAPLSGY VVEQRDAHRP GWLPVSESVT RSTFKFTRLT
23401 EGNEYVFRVA ATNRFGIGSY LQSEVIECRS SIRIPGPPET
23441 LQIFDVSRDG MTLTWYPPED DGGSQVTGYI VERKEVRADR
23481 WVRVNKVPVT MTRYRSTGLT EGLEYEHRVT AINARGSGKP
23521 SRPSKPIVAM DPIAPPGKPQ NPRVTDTTRT SVSLAWSVPE
23561 DEGGSKVTGY LIEMQKVDQH EWTKCNTTPT KIREYTLTHL
23601 PQGAEYRFRV LACNAGGPGE PAEVPGTVKV TEMLEYPDYE
23641 LDERYQEGIF VRQGGVIRLT IPIKGKPFPI CKWTKEGQDI
23681 SKRAMIATSE THTELVIKEA DRGDSGTYDL VLENKCGKKA
23721 VYIKVRVIGS PNSPEGPLEY DDIQVRSVRV SWRPPADDGG
23761 ADILGYILER REVPKAAWYT IDSRVRGTSL VVKGLKENVE
23801 YHFRVSAENQ FGISKPLKSE EPVTPKTPLN PPEPPSNPPE
23841 VLDVTKSSVS LSWSRPKDDG GSRVTGYYIE RKETSTDKWV
23881 RHNKTQITTT MYTVTGLVPD AEYQFRIIAQ NDVGLSETSP
23921 ASEPVVCKDP FDKPSQPGEL EILSISKDSV TLQWEKPECD
23961 GGKEILGYWV EYRQSGDSAW KKSNKERIKD KQFTIGGLLE
24001 ATEYEFRVFA ENETGLSRPR RTAMSIKTKL TSGEAPGIRK
24041 EMKDVTTKLG EAAQLSCQIV GRPLPDIKWY RFGKELIQSR
24081 KYKMSSDGRT HTLTVMTEEQ EDEGVYTCIA TNEVGEVETS
24121 SKLLLQATPQ FHPGYPLKEK YYGAVGSTLR LHVMYIGRPV
24161 PAMTWFHGQK LLQNSENITI ENTEHYTHLV MKNVQRKTHA
24201 GKYKVQLSNV FGTVDAILDV EIQDKPDKPT GPIVIEALLK
24241 NSAVISWKPP ADDGGSWITN YVVEKCEAKE GAEWQLVSSA
24281 ISVTTCRIVN LTENAGYYFR VSAQNTFGIS DPLEVSSVVI
24321 IKSPFEKPGA PGKPTITAVT KDSCVVAWKP PASDGGAKIR
24361 NYYLEKREKK QNKWISVTTE EIRETVFSVK NLIEGLEYEF
24401 RVKCENLGGE SEWSEISEPI TPKSDVPIQA PHFKEELRNL
24441 NVRYQSNATL VCKVTGHPKP IVKWYRQGKE IIADGLKYRI
24481 QEFKGGYHQL IIASVTDDDA TVYQVRATNQ GGSVSGTASL
24521 EVEVPAKIHL PKTLEGMGAV HALRGEVVSI KIPFSGKPDP
24561 VITWQKGQDL IDNNGHYQVI VTRSFTSLVF PNGVERKDAG
24601 FYVVCAKNRF GIDQKTVELD VADVPDPPRG VKVSDVSRDS
24641 VNLTWTEPAS DGGSKITNYI VEKCATTAER WLRVGQARET
24681 RYTVINLFGK TSYQFRVIAE NKFGLSKPSE PSEPTITKED
24721 KTRAMNYDEE VDETREVSMT KASHSSTKEL YEKYMIAEDL
24761 GRGEFGIVHR CVETSSKKTY MAKFVKVKGT DQVLVKKEIS
24801 ILNIARHRNI LHLHESFESM EELVMIFEFI SGLDIFERIN
24841 TSAFELNERE IVSYVHQVCE ALQFLHSHNI GHFDIRPENI
24881 IYQTRRSSTI KIIEFGQARQ LKPGDNFRLL FTAPEYYAPE
24921 VHQHDVVSTA TDMWSLGTLV YVLLSGINPF LAETNQQIIE
24961 NIMNAEYTFD EEAFKEISIE AMDFVDRLLV KERKSRMTAS
25001 EALQHPWLKQ KIERVSTKVI RTLKHRRYYH TLIKKDLNMV
25041 VSAARISCGG AIRSQKGVSV AKVKVASIEI GPVSGQIMHA
25081 VGEEGGHVKY VCKIENYDQS TQVTWYFGVR QLENSEKYEI
25121 TYEDGVAILY VKDITKLDDG TYRCKVVNDY GEDSSYAELF
25161 VKGVREVYDY YCRRTMKKIK RRTDTMRLLE RPPEFTLPLY
25201 NKTAYVGENV RFGVTITVHP EPHVTWYKSG QKIKPGDNDK
25241 KYTFESDKGL YQLTINSVTT DDDAEYTVVA RNKYGEDSCK
25281 AKLTVTLHPP PTDSTLRPMF KRLLANAECQ EGQSVCFEIR
25321 VSGIPPPTLK WEKDGQPLSL GPNIEIIHEG LDYYALHIRD
25361 TLPEDTGYYR VTATNTAGST SCQAHLQVER LRYKKQEFKS
25401 KEEHERHVQK QIDKTLRMAE ILSGTESVPL TQVAKEALRE
25441 AAVLYKPAVS TKTVKGEFRL EIEEKKEERK LRMPYDVPEP
25481 RKYKQTTIEE DQRIKQFVPM SDMKWYKKIR DQYEMPGKLD
25521 RVVQKRPKRI RLSRWEQFYV MPLPRITDQY RPKWRIPKLS
25561 QDDLEIVRPA RRRTPSPDYD FYYRPRRRSL GDISDEELLL
25601 PIDDYLAMKR TEEERLRLEE ELELGFSASP PSRSPPHFEL
25641 SSLRYSSPQA HVKVEETRKD FRYSTYHIPT KAEASTSYAE
25681 LRERHAQAAY RQPKQRQRIM AEREDEELLR PVTTTQHLSE
25721 YKSELDFMSK EEKSRKKSRR QREVTEITEI EEEYEISKHA
25761 QRESSSSASR LLRRRRSLSP TYIELMRPVS ELIRSRPQPA
25801 EEYEDDTERR SPTPERTRPR SPSPVSSERS LSRFERSARF
25841 DIFSRYESMK AALKTQKTSE RKYEVLSQQP FTLDHAPRIT
25881 LRMRSHRVPC GQNTRFILNV QSKPTAEVKW YHNGVELQES
25921 SKIHYTNTSG VLTLEILDCH TDDSGTYRAV CTNYKGEASD
25961 YATLDVTGGD YTTYASQRRD EEVPRSVFPE LTRTEAYAVS
26001 SFKKTSEMEA SSSVREVKSQ MTETRESLSS YEHSASAEMK
26041 SAALEEKSLE EKSTTRKIKT TLAARILTKP RSMTVYEGES
26081 ARFSCDTDGE PVPTVTWLRK GQVLSTSARH QVTTTKYKST
26121 FEISSVQASD EGNYSVVVEN SEGKQEAEFT LTIQKARVTE
26161 KAVTSPPRVK SPEPRVKSPE AVKSPKRVKS PEPSHPKAVS
26201 PTETKPTPTE KVQHLPVSAP PKITQFLKAE ASKEIAKLTC
26241 VVESSVLRAK EVTWYKDGKK LKENGHFQFH YSADGTYELK
26281 INNLTESDQG EYVCEISGEG GTSKTNLQFM GQAFKSIHEK
26321 VSKISETKKS DQKTTESTVT RKTEPKAPEP ISSKPVIVTG
26361 LQDTTVSSDS VAKFAVKATG EPRPTAIWTK DGKAITQGGK
26401 YKLSEDKGGF FLEIHKTDTS DSGLYTCTVK NSAGSVSSSC
26441 KLTIKAIKDT EAQKVSTQKT SEITPQKKAV VQEEISQKAL
26481 RSEEIKMSEA KSQEKLALKE EASKVLISEE VKKSAATSLE
26521 KSIVHEEITK TSQASEEVRT HAEIKAFSTQ MSINEGQRLV
26561 LKANIAGATD VKWVLNGVEL TNSEEYRYGV SGSDQTLTIK
26601 QASHRDEGIL TCISKTKEGI VKCQYDLTLS KELSDAPAFI
26641 SQPRSQNINE GQNVLFTCEI SGEPSPEIEW FKNNLPISIS
26681 SNVSISRSRN VYSLEIRNAS VSDSGKYTIK AKNFRGQCSA
26721 TASLMVLPLV EEPSREVVLR TSGDTSLQGS FSSQSVQMSA
26761 SKQEASFSSF SSSSASSMTE MKFASMSAQS MSSMQESFVE
26801 MSSSSFMGIS NMTQLESSTS KMLKAGIRGI PPKIEALPSD
26841 ISIDEGKVLT VACAFTGEPT PEVTWSCGGR KIHSQEQGRF
26881 HIENTDDLTT LIIMDVQKQD GGLYTLSLGN EFGSDSATVN
26921 IHIRSI

In some cases, SARS-CoV-2 infection can be monitored by observing cleavage of titin in the C-terminal region. For example, such cleavage can occur in the M-band (also called the M-line) region of titin. The M band is at the C-terminal end 35 of the titin protein and in the center of the A band, which is in the center of the sarcomere. The approximate 250 kilodalton M band is an attachment site for the thick filaments, and it is encoded by six exons, exons 359 to 364, which are also termed M-band exons 1 to 6 (Mex1 to Mex6). The M band region interacts with several sarcomeric proteins including myosin-binding protein C, calmodulin 1, CAPN3, obscurin, and MURF1.

Cleavage of titin can be observed within the C-terminal 2000-4000 amino acids, or the 2000-3000 amino acids of the titin protein. Such cleavage is observed when SARS-CoV-2 infection occurs. A test agent that causes a reduction in titin cleavage (e.g., compared to a control) can be useful for treating and/or preventing SARS-CoV-2 infection.

COVID-19

Initial descriptions of COVID-19, the pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), characterized it as a primarily respiratory syndrome (see website at pubmed.ncbi.nlm.nih.gov/32031570/). However, increasing clinical evidence now implicates multiple organ systems in COVID-19 infection, including the heart, gastrointestinal tract, and kidneys (Wang, see websites at sciencedirect.com/science/article/pii/S0140673620302117; ahajournals.org/doi/10.1161/CIRCULATIONAHA.120.047164; jamanetwork.com/journals/jama/fullarticle/2763485; jamanetwork.com/joumals/jama/fullarticle/2765184).

As illustrated herein, multiple COVID-19 patients frequently present with significant myocardial damage (see also websites at jamanetwork.com/journals/jamacardiology/fullarticle/2763845; academic.oup.com/cardiovascres/article/116/10/1666/5826160; nature.com/articles/s41569-020-0413-9), even when they exhibited no prior cardiovascular disease (CVD) (jamanetwork.com/journals/jamacardiology/fullarticle/2763524), indicating that viral infection may be directly responsible for the cardiac damage. Meta-analyses identify elevated high-sensitivity troponin-I or natriuretic peptides as the strongest predictor of mortality in hospitalized patients, eclipsing both cardiovascular disease and congestive obstructive pulmonary disease (see websites at thelancet.com/journals/lancet/article/PIIS0140-6736(20)30566-3/fulltext; pubmed.ncbi.nlm.nih.gov/32362922/; pubmed.ncbi.nlm.nih.gov/32125452/; jamanetwork.com/joumals/jamacardiology/fullarticle/2763524). Alarmingly, evidence of elevated troponin can be found even in mild cases of COVID-19, and a recent study observed that the majority of recovered patients in the studied cohort presented with impaired cardiac function, indicating that long-term heart sequelae from COVID-19 may not be limited to intensive care unit cases (see website atjamanetwork.com/joumals/jamacardiology/fullarticle/2768916).

Identifying therapeutic strategies to prevent or manage myocardial injury in COVID-19 patients is hindered by limited understanding of the mechanisms by which SARS-CoV-2 induces cardiac damage. Besides direct myocardial infection, cardiac damage may be caused by other systemic impacts of SARS-CoV-2, such as hypoxic stress due to pulmonary damage, microvascular thrombosis, and/or the systemic immune response to viral infection (see website at ncbi.nlm.nih.gov/pmc/articles/PMC7270045/). Recent histological results from deceased COVID-19 patients detect viral RNA in the myocardium without inflammatory cell infiltrates (see website at jamanetwork.com/joumals/jamacardiology/fullarticle/2768914), but whether these transcripts arise from infected myocytes, cardiac stroma, or blood vessels was previously unknown (see website at onlinelibrary.wiley.com/doi/abs/10.1002/ejhf.1828). Cardiomyocytes are known to express the primary receptor for viral entry, ACE2 (see website at sciencedirect.com/science/article/pii/S0092867420302294) and may be infectable by SARS-CoV-2 (see website at ahajournals.org/doi/full/10.1161/CIRCULATIONAHA.120.047549). Developing effective interventions for cardiac injury in COVID-19 requires identification of the key molecules and cell types involved in mediating viral infection and cellular anomalies.

As described herein, ex vivo studies employed using human cell-based models of the heart were used to afford the most direct route for the prospective and clinically relevant study of the effects of cardiac viral infection. Human induced pluripotent stem cells (iPSCs) can be used as described herein to obtain functional cardiac tissue models for disease modeling and discovery, overcoming the infeasibility of using primary human hearts. Stem-cell derived models have already demonstrated the susceptibility of hepatocytes (see website at sciencedirect.com/science/article/pii/S1934590920302824), intestinal epithelium (see website at nature.com/articles/s41591-020-0912-6; see website at ncbi.nlm.nih.gov/pmc/articles/PMC7199907/), and lung organoids (see website at biorxiv.org/content/10.1101/2020.05.05.079095v1. abstract) to SARS-CoV-2 infection.

While two recent reports indicated that human iPSC-cardiomyocytes are susceptible to SARS-CoV-2 infection (see websites at cell.com/cell-reports-medicine/fulltext/S2666-3791(20)30068-9, biorxiv.org/content/10.1101/2020.06.01.127605v1), clear indications of specific cardiac cytopathic features have not been identified. In addition, the relative viral tropism for other cardiac cell types that may be involved in microthrombosis or weakening of the ventricular wall has previously not been explored, nor has there been direct correlation of in vitro results to clinical pathology specimens.

Identifying phenotypic biomarkers of SARS-CoV-2 infection and cardiac cytopathy that recapitulate features of patient tissue is critical for rapidly developing novel cardioprotective therapies efficacious against COVID-19. As described herein, the inventors have examined the relative susceptibility of three iPS-derived cardiac cell types: cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs), to SARS-CoV-2 infection, and identify clear hallmarks of infection and cardiac cytopathy that predict pathologic features found in human COVID-19 tissue specimens.

Definitions

The term “about” as used herein when referring to a measurable value such as an amount, a length, and the like, is meant to encompass variations of 20% or +10%, more preferably 5%, even more preferably 1%, and still more preferably 0.1% from the specified value.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosed subject matter.

As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a nucleic acid” or “a protein” or “a cell” includes a plurality of such nucleic acids, proteins, or cells (for example, a solution or dried preparation of nucleic acids or expression cassettes, a solution of proteins, or a population of cells), and so forth. In this document, the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated.

“Recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, bacterial, mammalian, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.

The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms. The host organism expresses the foreign gene to produce the protein under expression conditions.

As used herein, a “cell” refers to any type of cell. The cell can be in an organism or it can be maintained outside of an organism. The cell can be within a living organism and be in its normal (native) state. The term “cell” includes an individual cell or a group or population of cells. The cell(s) can be a prokaryotic, eukaryotic, or archaeon cell(s), such as a bacterial, archaeal, fungal, protist, plant, or animal cell(s). The cell(s) can be from or be within tissues, organs, and biopsies. The cell(s) can be a recombinant cell(s), a cell(s) from a cell line cultured in vitro. The cell(s) can include cellular fragments, cell components, or organelles comprising nucleic acids. In some cases, the cell(s) are human cells. The term cell(s) also encompasses artificial cells, such as nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids. The methods described herein can be performed, for example, on a sample comprising a single cell or a population of cells. The term also includes genetically modified cells.

The term “transformation” refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transfection, or transduction are included. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

“Recombinant host cells,” “host cells”, “cells”, “cell lines”, “cell cultures”, and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.

A “coding sequence” or a sequence which “encodes” a selected RNA or a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”). The boundaries of the coding sequence can be determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3′ to the coding sequence.

Typical “control elements,” include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3′ to the translation stop codon), sequences for optimization of initiation of translation (located 5′ to the coding sequence), and translation termination sequences.

“Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

“Encoded by” refers to a nucleic acid sequence which codes for a polypeptide or RNA sequence. For example, the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. The RNA sequence or a portion thereof contains a nucleotide sequence of at least 3 to 5 nucleotides, more preferably at least 8 to 10 nucleotides, and even more preferably at least 15 to 20 nucleotides.

The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

“Expression” refers to detectable production of a gene product by a cell. The gene product may be a transcription product (i.e., RNA), which may be referred to as “gene expression”, or the gene product may be a translation product of the transcription product (i.e., a protein), depending on the context.

“Purified polynucleotide” refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein and/or nucleic acids with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are available in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.

“Substantially purified” generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically, in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

The term “transfection” is used to refer to the uptake of foreign DNA by a cell. A cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material and includes uptake of peptide-linked or antibody-linked DNAs.

The term “transduction” refers to the introduction of foreign nucleic acid to a cell through a replication-incompetent viral vector.

A “vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

“Mammalian cell” refers to any cell derived from a mammalian subject suitable for transfection with an engineered vector system comprising an expression system described herein. The cell may be xenogeneic, autologous, or allogeneic. The cell can be a primary cell obtained directly from a mammalian subject. The cell may also be a cell derived from the culture and expansion of a cell obtained from a mammalian subject. Immortalized cells are also included within this definition. In some embodiments, the cell has been genetically engineered to express a recombinant protein and/or nucleic acid.

The term “subject” includes animals, including both vertebrates and invertebrates, including, without limitation, invertebrates such as arthropods, mollusks, annelids, and cnidarians; and vertebrates such as amphibians, including frogs, salamanders, and caecillians; reptiles, including lizards, snakes, turtles, crocodiles, and alligators; fish; mammals, including human and non-human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows; and birds such as domestic, wild and game birds, including chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. In some cases, the disclosed methods find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; primates, and transgenic animals.

“Gene transfer” or “gene delivery” refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non-viral vectors, alphaviruses, pox viruses and vaccinia viruses.

The term “derived from” is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.

A polynucleotide “derived from” a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence. The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.

As used herein, the terms “complementary” or “complementarity” refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated. “Complementarity” may exist between two RNA strands, two DNA strands, or between an RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be “complementary” and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are “perfectly complementary” or “100% complementary” if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered “perfectly complementary” or “100% complementary” even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. “Less than perfect” complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art.

The following example illustrate some of the experiments used in the development of the invention and some features of the invention.

Example 1: Materials and Methods

This Example describes some of the materials and methods used in developing and practicing the invention.

hiPSC Maintenance; iPS-Cardiomyocyte Differentiation and Purification

Human iPS cells (WTC11 line; see website at ncbi.nlm.nih.gov/pmc/articles/PMC4063274/) were maintained in mTESR or mTESR+(STEMCELL Technologies) on Matrigel (8 μg/ml, BD Biosciences)-coated cell culture plates at 37° C., 5% CO2. Cells were passaged every 3 days using Relesr (STEMCELL Technologies) and supplemented with Rock Inhibitor Y-27632 (SelleckChem) for 24 hours after each passaging. hiPSCs were differentiated into cardiomyocytes following a modified Wnt pathway modulation-based GiWi protocol (see website at ncbi.nlm.nih.gov/pmc/articles/PMC3612968/). Briefly, hiPSCs cultures were harvested using Accutase (STEMCELL Technologies) and seeded onto Matrigel-coated 12-well plates. Three days later, cells were exposed to 12 uM CHIR99021 (Tocris) in RPMI1640 (Gibco, 11875093) supplemented with B27 without insulin (Gibco, A1895601) (R/B media) for 24 hours. After an additional 48 hours, media was changed to R/B media supplemented with 5 uM IWP2 (Tocris) for 48 hours. On day 7, media was changed to RPMI1640 medium supplemented with B27 with insulin (Gibco, 17504044) (R/B*) and refreshed every 3 days thereafter. Beating was generally observed around day 8-11. At day 15, cells were cryopreserved using CryoStor CS10 (STEMCELL Technologies). After thawing, cell cultures were enriched for iPS-cardiomyocytes following metabolic switch purification (see website at pubmed.ncbi.nlm.nih.gov/23168164/). Briefly, cells were washed once with saline buffer and incubated in DMEM (without glucose, without sodium pyruvate; Gibco, 11966025) supplemented with GlutaMax (Gibco, 35050061), MEM Non-Essential Amino Acids (Gibco, 11140050) and sodium L-lactate (4 mM, Sigma-Aldrich). Lactate media was refreshed every other day for a total of 6 days. Four to six days later (day 28-30), iPS-CMs were replated into assay plates for infection using 0.25% Trypsin (Gibco, 15050065) at a density of approximately 60,000 cells/cm².

scRNAseq Analysis of SARS-CoV-2 Entry Factors

A historic single cell RNA sequencing data set consisting of iPSC-derived cardiomyocytes, primary fetal cardiac fibroblasts, and iPSC-derived endothelial cells was re-analyzed to compare relative expression levels of SARS-CoV-2 relevant receptors and proteases (GSE155226) (see web at biorxiv.org/content/10.1101/2020.07.06.190504v1). Briefly, day 30 lactate purified cardiomyocytes were force aggregated either alone or with a single supporting cell type. The cardiomyocytes were then cultured in suspension culture. Aggregates were dissociated and libraries prepared using the Chromium 3′ v2 library preparation platform (10× Genomics). Libraries were sequenced on a NextSeq 550 sequencer (Illumina) to a depth of at least 30 million reads per sample. Samples were demultiplexed and aligned to GRCh38 with CellRanger v3.0.2. Samples were normalized and clustered with Seurat v3.2.0, yielding four primary clusters corresponding to each cell type, which were used to profile cell-type specific expression of SARS-CoV-2 relevant factors.

Cardiac Fibroblast Differentiation

Second heart field-derived cardiac fibroblasts (SHF-CFs) were differentiated following the GiFGF protocol, as described by (website at nature.com/articles/s41467-019-09831-5). Briefly, hiPSCs were seeded at 15,000 cells/cm² in mTeSR1 medium. Once they reached 100% confluency, they were treated with R/B media supplemented with 12 μM CHIR99021 (day 0) and refreshed with R/B media 24 hours later (day 1). From days 2-20, cells were fed every 2 days with cardiac fibroblast basal media (CFBM) (Lonza, CC-3131) supplemented with 75 ng/mL bFGF. On day 20, CFs were singularized with Accutase for 10 minutes and replated at 7,000 cells/cm² onto tissue culture plastic 10 cm dishes in FibroGRO medium (Millipore Sigma, SCMF001). FibroGRO media was changed every two days until the CFs reached approximately 80-90% confluency, at which point they were passaged with Accutase. SHF-CFs were validated to be >80% double-positive for TE-7 and vimentin by flow cytometry.

Endothelial Cell Differentiation

WTC11 iPSCs were directed towards an endothelial cell (EC) lineage by the addition of E8 media supplemented with BMP4 (5 ng/ml) and Activin A (25 ng/ml) for 48 hours followed by E7BVi media, consisting of E6 medium supplemented with bFGF (50 ng/ml), VEGF-A (50 ng/ml), BMP4 (50 ng/ml) and a TGFβ inhibitor, SB431542, (5 μM) for 72 hours. After 5 days of successive media changes, ECs were split and plated at high density in EGM media (Lonza, CC-3162) on tissue culture flasks coated with fibronectin (1:100, Sigma Aldrich F0895). On day 8, all cells were cryo-preserved and a fraction of ECs were assayed for >95% purity by flow cytometry using antibodies against mature EC markers CD31 and CDH5.

Mixed Cultures of CMs, CFs, and ECs

Mixed cultures of induced pluripotent stem cell derived cardiomyocytes (iPS-CMs), induced pluripotent stem cell derived endothelial cells (iPS-ECs), and induced pluripotent stem cell derived cardiac fibroblasts (iPS-CFs) were created by combining single cell suspensions of each cell types in a ratio of 60:30:10 CM:EC:CF at a density of 200,000 cells/mL. The mixed suspension was replated onto Matrigel-coated tissue culture plates 48 hours prior to infection at a density of 62,500 cells/cm².

Reagents

TABLE 1A
Drugs
Drug	Concentration	Provider
DMSO	0.1% (1:1000)	Fisher Scientific (BP231-100)
Interferon α	2500	U/mL	Sigma Aldrich (SRP4596-100UG)
Interferon β	2500	U/mL	Sigma Aldrich (IF014)
Interferon γ	2500	U/mL	BioRad (PHP050A)
Interferon λ	2500	U/mL	Cedarlane Laboratories (CLY100-
			169-5UG)
Ruxolitinib	500	nM	Thermo (NC1399519)
Doxorubicin	20 nM and	Sigma (D1515)
	200 nM
Bortezomib	1 uM and 10 uM	Sigma (CAS 179324-69-7)
Dutasteride	2	uM	Cayman Chemical (164656-23-9)
Albendazole	2	uM	Cayman Chemical (54965-21-8)
Bafilomycin	100	nM	Cayman Chemical (98813-13-9)
IL-32 blocking antibody	50	ng/mL	R&D Systems (AF3040)
IL-8 recombinant	50	ng/mL	Sigma (I1645-10UG)
IL-32 recombinant	100	ng/mL	Thermo Fisher (4690IL025CF)
IL-8 blocking antibody	100	ng/mL	R&D Systems (MAB208-100)
Repaxirin	1	uM	MedChem Express (HY-15252)
Aprotinin	50	uM	Cayman Chemical (9087-70-1)
Camostat mesilate	2	uM	Cayman Chemical (59721-29-8)
CA074	30	uM	Cayman Chemical (134448-10-5)
E-64d	25	uM	Cayman Chemical (88321-09-9)
Z-Phe-Tyr(tBu)-	30	uM	Cayman Chemical (114014-15-2)
diazomethylketone
ACE2 blocking antibody	20	ug/ml	R&D Systems (AF933)
Cardiac Troponin antibody	1:200	Abcam (ab45932-100 ug)
ACTN2 polyclonal antibody	1:200	Life Technologies Corporation
		(14221-1-AP)
Apilimod	1	uM	SelleckChem Chemicals (S6414)
Anti-SARS-CoV-2 Spike	1:200	Provided by BEI resources (NR-616)
protein antibody
Anti-dsRNA antibody (J2)	1:200	Absolute Antibody (Ab01299-2.0)

SARS-CoV-2 Infection

The WA-1 strain (BEI resources) of SARS-CoV-2 was used for all experiments. SARS-CoV-2 stocks were passaged in Vero cells (ATCC) and titer was determined via plaque assay on Vero cells as previously described (Honko et al ref). Briefly, virus was diluted 1:10²-1:10⁶ and incubated for 1 hour on Vero cells before an overlay of Avicel and complete DMEM (Sigma Aldrich, SLM-241) was added. After incubation at 37° C. for 72 hours, the overlay was removed and cells were fixed with 10% formalin, stained with crystal violet, and counted for plaque formation. SARS-CoV-2 infections of iPS-derived cardiac cells were done at a multiplicity of infection of 0.006 for 48 hours unless otherwise specified. For heat inactivation, SARS-CoV-2 stocks were incubated at 85° C. for 5 min.

Immunocytochemistry

Infected and mock-treated cell cultures were washed with Phosphate Buffered Solution (PBS) and fixed in 4% paraformaldehyde (PFA) overnight, followed by blocking and permeabilization with 0.1% Triton-X 100 (T8787, Sigma) and 5% BSA (A4503, Sigma) for one hour at RT. Antibody dilution buffer (Ab buffer) was comprised of PBS supplemented with 0.1% Triton-X 100 and 1% BSA. Samples were incubated with primary antibodies overnight at 4° C. (Table 1), followed by 3 washes with PBS and incubation with fluorescent-conjugated secondary antibodies at 1:250 in Ab buffer for 1 hour at room temperature (Table 1). For immunofluorescence staining, epitopes were retrieved through 35 min incubation at 95° C. in citrate solution (pH 6) or TE buffer (pH 9) and coverslips were mounted onto SuperFrost Slides (FisherBrand, 12-550-15) with ProLong Antifade mounting solution with DAPI (Invitrogen, P36931). Primary antibodies and nuclear stains were used as follows: J2 (Absolute Antibody Ab02199-2.0, 1:200), Spike (Ms, BEI Resources NR-616, 1:200), ACE2 (ProteinTech 21115-1-AP, 1:200), TNNT2 (Abcam ab45932, 1:400), ACTN2 (Sigma A7732, 1:200), PECAM-1 (Santa Cruz sc1506, 1:50), GFP (Abcam ab13970, 1:200), MTCO2 (Abcam ab110258, 1:200), Hoechst 33342 (ThermoFisher 62249, 1:10,000). Images were acquired with a Zeiss Axio Observer Z.1 Spinning Disk Confocal (Carl Zeiss) or with an ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices) and processed using ZenBlue and ImageJ.

Histology

Paraffin sections of healthy and COVID-19 patient hearts were deparaffinized using xylene, re-hydrated through a decrease series of ethanol solutions (100%, 100%, 95%, 80%, 70%) and rinsed in PB1X. Hematoxylin and eosin staining was performed according manufacturer instructions and the slides were mounted with Cytoseal 60 (Richard-Allan Scientific) and glass coverslips. For immunofluorescence staining, epitopes were retrieved by immersing slides through 35 min incubation at 95° C. in citrate buffer (Vector Laboratories, pH 6) or Tris-EDTA buffer (Cellgro, pH 9). Slides were cooled for 20 min at RT and washed with PBS. Samples were permeabilized in 0.2% Triton X-100 (Sigma) in PBS by slide immersion and washed in PBS. Blocking was performed in 1.5% normal donkey serum (NDS; Jackson ImmunoResearch) and PBS solution for 1 h at RT. Primary and secondary antibody cocktails were diluted in blocking solution (Table 1). PBS washes were performed after primary (overnight, 4° C.) and secondary antibody (1 h, RT) incubations. Nuclei were stained with Hoechst and coverslips were mounted on slides using ProLong™ Gold Antifade Mountant. Samples were imaged on the Zeiss Axio Observer Z1.

TABLE 1B
Reagents
Protein	Source	Catalog #	Dilution
ACE2	ProteinTech	21115-1-AP	1:200
ACTN2	Sigma	A7732	1:200
Alexa Fluor 488 Donkey	Invitrogen		1:400
anti-mouse IgG
Alexa Fluor 555 Donkey	Invitrogen		1:400
anti-rabbit IgG
Alexa Fluor 647 Donkey	Invitrogen		1:400
anti-rabbit IgG
Alexa Fluor 647 Donkey	Invitrogen		1:400
anti-goat IgG
Collagen IV	Millipore	AB769	1:100
GFP	Abcam	ab13970	1:200
Hoechst 33342	Thermo Scientific	62246	1:10000
J2	Absolute Antibody	Ab02199-2.0	1:200
MTCO2	Abcam	ab110258	1:200
PECAM-1	Santa Cruz	sc1506	1:50
SARS Nucleoprotein	Thermo	MA1-7404	1:100
Spike	BEI Resources	NR-616	1:200
Troponin T	Abcam	ab45932	1:400

RT-qPCR

Cultured cells were lysed with Qiagen buffer RLT (Qiagen, 79216) supplemented with 1% β-mercaptoethanol (Bio-Rad, 1610710) and RNA was isolated using the RNeasy Mini Kit (Qiagen 74104) or Quick-RNA MicroPrep (ThermoFisher, 50444593) and quantified using the NanoDrop 2000c (ThermoFisher). Viral load was measured by detection of the viral Nucleocapsid (N5) transcript through one-step quantitative real-time PCR, performed using PrimeTime Gene Expression Master Mix (Integrated DNA Technologies, 1055772) with primers and probes specific to N5 and RPP30 as in internal reference. RT-qPCR reactions were performed on a CFX384 (BioRad) and delta cycle threshold (ΔCt) was determined relative to RPP30 levels. Viral detection levels in pharmacologically treated samples were normalized to DMSO-treated controls.

TABLE 2
Primers
	Name	Direction	Sequence (5′-3′)
	RNAse P	Forward	AGA TTT GGA CCT
	PF_030_RP_F	(F)	GCG AGC G
			(SEQ ID NO: 3)
	RNAse P	Reverse	GAG CGG CTG TCT
	PF_031_RP_R	(R)	CCA CAA GT
			(SEQ ID NO: 4)
	RNAse P	Forward	FAM-TTC TGA CCT
	PF_032_RP_P	(probe)	GAA GGC TCT GCG
			CG-BHQ1
			(SEQ ID NO: 5)
	SARS-CoV-2	Forward	AAATTTTGGGGACCAG
	N gene		GAAC
	PF_039_nCoV_N5_F		(SEQ ID NO: 6)
	SARS-CoV-2	Reverse	TGGCACCTGTGTAGGT
	N gene		CAAC
	PF_040_nCoV_N5_R		(SEQ ID NO: 7)
	SARS-CoV-2	Forward	ATGTCGCGCATTGGCA
	N gene	(probe)	TGGA
	PF_041_nCoV_N5_P		(SEQ ID NO: 8)
	SARS-CoV-2	Forward	ACAGGTACGTTAATAG
	E gene		TTAATAGCGT
	PF_042_nCoV_E_F		(SEQ ID NO: 9)
	SARS-CoV-2	Reverse	ATATTGCAGCAGTACG
	E gene		CACACA
	PF_043_nCoV_E_R		(SEQ ID NO: 10)
	SARS-CoV-2	Forward	ACACTAGCCATCCTTA
	E gene	(probe)	CTGCGCTTCG
	PF_044_nCoV_E_P		(SEQ ID NO: 11)
	RNAse P	Forward	AGA TTT GGA CCT
	PF_030_RP_F		GCG AGC G
			(SEQ ID NO: 12)
	RNAse P	Reverse	GAG CGG CTG TCT
	PF_031_RP_R		CCA CAA GT
			(SEQ ID NO: 13)
	RNAse P	Forward	FAM-TTC TGA CCT
	PF_032_RP_P	(probe)	GAA GGC TCT GCG
			CG-BHQ1
			(SEQ ID NO: 14)
	SARS-CoV-2	Forward	AAATTTTGGGGACCAG
	N gene		GAAC
	PF_039_nCoV_N5_F		(SEQ ID NO: 15)
	SARS-CoV-2	Reverse	TGGCACCTGTGTAGGT
	N gene		CAAC
	PF_040_nCoV_N5_R		(SEQ ID NO: 16)
	SARS-CoV-2	Forward	ATGTCGCGCATTGGCA
	N gene	(probe)	TGGA
	PF_041_nCoV_N5_P		(SEQ ID NO: 17)

RNA-Seq

For generating libraries for RNA-sequencing, RNA isolate quality was assessed with an Agilent Bioanalyzer 2100 on using the RNA Pico Kit (Agilent, 5067-1513). 10 ng of each RNA isolate was then prepared using the Takara SMARTer Stranded Total RNA-Seq Kit v2—Pico Input Mammalian (Takara, 634412). Transcripts were fragmented for 3.5 minutes and amplified for 12 cycles. Library concentrations were quantified with the Qubit dsDNA HS Assay Kit (Thermo Fisher, Q32851) and pooled for sequencing. Sequencing was performed on an Illumina NextSeq 550 system, using the NextSeq 500/550 High Output Kit v2.5 (150 Cycles) (Illumina, 20024907) to a depth of at least 10 million reads per sample.

Bioinformatic Analyses

Samples were demultiplexed using bcl2fastq v2.20.0 and aligned to both GRCh38 and the SARS-CoV-2 reference sequence (NC_045512) using hisat2 v2.1.0 (see website at nature.com/articles/nmeth.3317). Aligned reads were converted to counts using featureCounts v1.6.2 (see website at pubmed.ncbi.nlm.nih.gov/24227677/). Cell-type clustering, gene loadings, and technical replication were assessed using the PCA and MDS projections implemented in scikit-learn v0.23.1 (see website at scikit-learn.org/stable/about.html#citing-scikit-leam). Differential expression analysis was performed using limma v3.44.3 with voom normalization (see website at genomebiology.biomedcentral.com/articles/10.1186/gb-2014-15-2-r29) and GO term enrichment analysis was performed using clusterProfiler v3.16.0 (see website at liebertpub.com/doi/10.1089/omi.2011.0118). Unbiased GO term selection was performed by non-negative matrix factorization using scikit-learn.

TEM/CLEM

Cells grown on gridded 35 mm MatTek glass-bottom dishes (MatTek Corp., Ashland, MA, USA) were fixed in 2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.1M sodium cacodylate buffer, pH 7.4 (EMS, Hatfield, PA, USA) following fluorescence imaging. Samples were rinsed 3×5 min at RT in 0.1M sodium cacodylate buffer, pH 7.2, and immersed in 1% osmium tetroxide with 1.6% potassium ferricyanide in 0.1M sodium cacodylate buffer for 30 minutes. Samples were rinsed (3×5 min, RT) in buffer and briefly washed with distilled water (1×1 min, RT) before sample were then subjected to an ascending ethanol gradient (7 min; 35%, 50%, 70%, 80%, 90%) followed by pure ethanol. Samples were progressively infiltrated (using ethanol as the solvent) with Epon resin (EMS, Hatfield, PA, USA) and polymerized at 60° C. for 24-48 hours. Care was taken to ensure only a thin amount of resin remained within the glass bottom dishes to enable the best possible chance for separation of the glass coverslip. Following polymerization, the glass coverslips were removed using ultra-thin Personna razor blades (EMS, Hatfield, PA, USA) and liquid nitrogen exposure as needed. The regions of interest, identified by the gridded alpha-numerical labeling, were carefully removed and mounted with cyanoacrylate glue for sectioning on a blank block. Serial thin sections (100 nm) were cut using a Leica UC 6 ultramicrotome (Leica, Wetzlar, Germany) from the surface of the block until approximately 4-5 microns in to ensure complete capture of the cell volumes. Section-ribbons were then collected sequentially onto formvar-coated 50 mesh copper grids. The grids were post-stained with 2% uranyl acetate followed by Reynold's lead citrate, for 5 min each. The sections were imaged using a Tecnai 12 120 kV TEM (FEI, Hillsboro, OR, USA), data were recorded using an UltraScan 1000 with Digital Micrograph 3 software (Gatan Inc., Pleasanton, CA, USA), and montaged datasets were collected with SerialEM (bio3d.colorado.edu/SerialEM) and reconstructed using IMOD eTOMO (bio3d.colorado.edu/imod).

Example 2: Relative Susceptibility of Cardiac Cells to SARS-CoV-2 Infection

The relative infectability of different cardiac cell types had not previously been characterized for SARS-CoV-2, leading to ambiguity over the sources of cardiac damage and relevant therapeutic targets. The inventors determined the tropism of SARS-CoV-2 for different cardiac cell types by infecting cardiomyocytes (CMs), cardiac fibroblasts (CFs), endothelial cells (ECs), or a mix of all three with SARS-CoV-2 at a relatively low MOI (MOI=0.006).

Viral infection load was measured by qPCR detection of the SARS-CoV-2 nucleocapsid transcript (N5) at 48 hours (FIG. 1A) or by immunostaining for double-stranded viral RNA (dsRNA) or Spike protein at 24, 48, and 72 hours (FIG. 1C-1E).

Viral replication measured in each cell type after 48 h largely correlated with corresponding ACE2 expression levels. Undifferentiated iPSCs were not infectable (FIG. 1F-1G). CFs and ECs also showed little to no viral N5 transcript detection (FIG. 1A, 1H), whereas CMs exhibited >10⁴ greater levels of viral RNA than any other cell type (FIG. 1A, 1C-1E, 1H). There was no significant difference in viral detection between CMs and mixed cultures, indicating that CMs are exclusively responsible for viral infection in the mixed cell condition that mimics native myocardial cellularity.

To further study if cardiac cells enable productive infection by SARS-CoV-2, plaque assays were performed on Vero cells from the supernatants of exposed cells that confirmed CFs, ECs, and iPSCs did not support productive infection, but CMs robustly produced new replication competent virions (FIG. 1H).

Immunostaining for replicating virus in the form of double-stranded viral RNA (dsRNA) or Spike protein further confirmed that infected CMs support viral replication. Positive dsRNA and Spike staining were only detected throughout infected CM cultures. Consistent with our qPCR results and plaque assays, CFs and ECs showed no dsRNA or Spike staining. However, all three cultures showed significant cytopathic effects after 48 hours of viral exposure, characterized by significant cell loss in all cell types (FIG. 1B-1E), fragmented cell bodies and dissociation from neighboring cells, with cytopathic effects most prevalent in CFs and particularly ECs (FIG. 1C-IE). Interestingly, despite cytopathic effects resulting from viral exposure without detectable infection, inoculation with heat-inactivated SARS-CoV-2 did not cause cell death or dissociation in any of the cell types assayed (FIG. 1B), suggesting the observed toxicity is due to live viral exposure.

Replication of (+)ssRNA viruses, including SARS-CoV and MERS-CoV, involves budding of double-membrane vesicles (DMVs) from the endoplasmic reticulum, with viral particle assembly occurring in the ER-Golgi intermediate compartment (ERGIC) cisternae (see website at biorxiv.org/content/10.1101/2020.06.23.167064v1). In CMs infected with SARS-CoV-2, dsRNA and Spike signals initially (24 h post infection) accumulated near the nucleus in small perinuclear puncta, closely matching the typical location of this ERGIC region, indicating potential active centers of replication. After 48 h post infection, an increase in the number of cells was observed with dsRNA signals throughout their cytoplasm, potentially correlating with breakdown of the ER-Golgi membrane as viral replication accelerates and the cell deteriorates, as evidenced by a decrease in sarcomeric integrity and intensity. By 72 h post infection, SARS-CoV-2 had spread throughout the culture and large swathes of the CMs had died, with the remaining cells displaying dispersed viral stain localization, dissociation from neighboring cells, and heavily reduced sarcomeric signal (FIG. 1C-1E).

Using transmission electron microscopy of infected CMs, the inventors readily identified the remnants of the ER-Golgi membranes and large vesicles in the proximity of the nucleus (FIG. 1C-1E). These vesicles, about 500-750 nanometers in diameter, contained multiple complete viral particles approximately 50-60 nm in diameter (FIG. 1D-1E), consistent with the dsRNA/Spike+ aggregates detected in infected CMs.

These results demonstrate that SARS-CoV-2 is able to readily infect, replicate in, and rapidly propagate through CMs.

Example 3: Pharmacological Modulation of SARS-CoV-2 Cardiomyocytes Infection

Cardiomyocytes (CMs) were the only type of cell that proved infectable by SARS-CoV-2, from amongst the cell types tested (cardiomyocytes, cardiac fibroblasts, endothelial cells, and stem cells). This Example describes experiments for elucidating the mechanism of viral entry into CMs by using exogenous inhibition of CM factors.

Cells pretreated with an ACE2 blocking antibody, cathepsin inhibitor E-64-D, or serine protease inhibitor aprotinin were able to significantly reduce the detection of viral transcripts in infected CMs (FIG. 2A-2B). Despite detection of FURIN in CMs, inhibition of FURIN (FURUNi) did not lead to a reduction in infection (FIG. 2B). Further probing revealed that cathepsin-L inhibition via Z-Phe-Tyr(tBu)-diazomethylketone (Z-FY-DK) was able to decrease viral detection in infected cells to about 10% of vehicle levels, but inhibition of cathepsin-B with CA-074 did not (FIG. 2A). In addition, the PIKfyve inhibitor apilimod and autolysosome acidification blocker bafilomycin also successfully reduced viral infection to ˜0.1% and 1% viral RNA detection compared to vehicle, respectively (FIG. 2A). In contrast, inhibition of TMPRSS2 with aprotinin or camostat mesylate did not significantly inhibit viral infection (FIG. 2A).

Taken altogether, these results strongly indicate that the SARS-CoV-2 virus employs the ACE2 receptor to bind to iPS-CMs and is able to utilize a cathepsin-L (CTSL)-dependent endolysosomal route, but not a cathepsin-B (CSTB)-dependent endolysosomal route, to infection without TMPRSS2/serine protease-mediated activation at the cellular membrane.

Based on the ability of SARS-CoV-2 to robustly infect and propagate through CMs, the inventors examined whether priming the innate immune response could effectively combat SARS-CoV-2 infection. CMs were primed with IFNα, IFNβ, IFNγ, or IFNλ, in addition to a combination of IFNs and a JAK/Stat inhibitor (ruxolitinib; ruxo) prior to infection. Only pre-exposure to IFNs was able to prevent infection, and this phenotype was reversed by JAK/Stat inhibition (FIG. 2C). Surprisingly, none of the other interferon exposures were able to prevent infection (FIG. 2C). Single-cell RNA-sequencing data indicated that CMs express undetectable levels of IFNβ, perhaps indicating that their high infectivity may be due to an intrinsic inability to appropriately trigger a sufficient immune response to combat viral infection.

Example 4: Transcriptomic Response to SARS-CoV-2 Exposure

This Example describes experiments for evaluating the transcriptional response of cardiac cells exposed to SARS-CoV-2, and in particular to identify differences in the level of immune suppression or cytokine activation across different levels of viral load. The experiments involved RNA-sequencing of infected and mock-treated CFs, ECs, and iPSCs at a MOI of 0.006, or a range of MOIs (0.001, 0.01, and 0.1) for CMs.

Sequencing recovered a high proportion of SARS-CoV-2 transcripts in an MOI and cell-type dependent fashion (FIG. 3A), with CMs at the highest MOI reaching >50% SARS-CoV-2 recovered reads (FIG. 3A). Principal component analysis (PCA) of the biological conditions revealed the expected clustering primarily based on cell type, with CFs and ECs clustering near together and CMs and iPSCs clustering separately (FIG. 3B). Loading plots of the principal components complemented this interpretation: the genes determining the spectrum of variation between CMs and CF/ECs were associated with CMs (MYH7, MYH6, TNNT2) at one pole (FIG. 3C) and anti-correlated with CF/EC specific genes at the other (FN1, COL1A2, TFPI2, MME). Notably, the distance between mock CMs and the furthest infected CMs was slightly further than the distance between CMs and CFs or ECs, indicating that viral infection altered cellular expression profiles at least as strongly as cellular identity. Along this axis, however, the inventors also observed that the level of transcriptional disruption correlated poorly with MOI across all CM samples, potentially due to natural stochasticity in the kinetics of infection. Regrouping conditions by the level of transcriptional disruption showed transcriptional trends resulting from viral exposure more clearly.

However, the significant distance between infected and mock conditions indicates that viral infection impacted the variation in expression profiles at least as strongly as the differences in cell type. Individual samples within the low, middle, and high MOI conditions correlated poorly with the degree of transcriptional disruption observed, potentially due to natural stochasticity in the kinetics of infection.

Regrouping conditions by the level of transcriptional disruption allowed transcriptional trends to be deduced as a function of viral impact. Loading plots of the principal components indicated that the main axis of variation aligned along a CM, CF/EC spectrum with CM specific genes (MYH7, MYH6, TNNT2) at one pole (FIG. 3C), anti-correlated with CF/EC specific genes (FN1, COL1A2).

Analysis of differential regulation of genes involved in inflammation and innate immunity for infected CFs, ECs, and CMs agree with the observed infectivity of CMs. Infected CFs and ECs have a depressed cytokine response compared to all three levels of disrupted CMs, which are enriched for genes involved in cytokine production and T-cell activation (OAS2, MX1, IFIT1, IL1B, IL6, TNF) (FIG. 3D) in addition to olfactory receptor (OR) genes, the ectopic expression of which may reflect a stress response (see websites at link.springer.com/chapter/10.1007/978-3-319-26932-0_33; www.nature.com/articles/s41573-018-0002-3?WT.feed_name=subjects_neuroscience).

Interestingly, the inventors noted that CMs at each MOI showed very clear dysregulation of genes involved in contractile machinery and proteasome homeostasis. All MOI conditions tested showed very clear dysregulation of genes involved in contractile machinery and proteasome homeostasis. In particular, sarcomeric structural proteins, myosin light chains, and proteasome kinases and chaperones were strongly downregulated, and most myosin heavy chains were significantly upregulated (FIG. 3D), indicating a potential effect of SARS-CoV-2 infection in the contractile and structural integrity of CMs.

In light of observations that impairment of cardiac function can occur even in mild cases of COVID-19 (which were mimicked by low MOIs), these results illustrate that SARS-CoV-2 may have unique interactions with structural features of CMs that can potentially cause cardiac dysfunction. Deeper analyses of the individual genes driving the GO terms revealed significant downregulation of mitochondrial metabolism networks, decreased regulation of protein degradation, and loss of genes associated with sarcomere formation and maintenance.

Example 5: Differential Expression of Viral Entry Factors in Cardiac Cells

Historical single-cell RNA-Seq data was first analyzed to determine the expression of putative viral entry host factors in CMs, ECs, and primary cardiac fibroblasts (see website at biorxiv.org/content/10.1101/2020.07.06.190504v1).

The primary SARS-CoV-2 receptor, ACE2, was detected at low levels in all cells, but ACE2 displayed greater than 10-fold higher expression in cardiomyocytes than in cardiac fibroblasts or endothelial cells, indicating that cardiomyocytes are more susceptible to infection than other cardiac cell types (FIG. 3E). Of the proteases thought to cleave the viral Spike protein to prime SARS-CoV-2 entry, TMPRSS2 was not detected in any cell types, but FURIN was ubiquitously expressed (FIG. 3F). It has also been proposed that SARS-CoV-2 can infect cells via endocytosis (see website at nature.com/articles/s41467-020-15562-9), similar to SARS-CoV. Endosomal entry factors for SARS-CoV include cathepsin-L (CTSL), cathepsin-B (CTSB), and the endosomal kinase PIKfyve (see website at pnas.org/content/102/33/11876.short). Protein structural similarity studies predict that these factors can also act on SARS-CoV-2 (see website at mdpi.com/2076-0817/9/3/186), and all three were expressed in all the examined cell types, with elevated PIKfyve in CMs (FIG. 3G-3I. In addition, DPP4, the surface protease used by the closely related MERS-CoV (see website at nature.com/articles/cr201392) and speculated to facilitate SARS-CoV-2 invasion (see website at ncbi.nlm.nih.gov/pmc/articles/PMC7103712/), was also detected, though at higher levels in primary cardiac fibroblasts (FIG. 3J).

These data support the viability of SARS-CoV-2 infection of cardiac cells via an ACE2-endocytosis axis.

To validate expression of the ACE2 receptor in CMs, the inventors directly examined ACE2 transcript and protein expression. While ACE2 transcripts were undetected in iPSCs by qPCR, differentiated and purified CMs exhibited robust expression (FIG. 1F). Heterotypic tissues comprising CMs and iPS-derived stromal non-myocytes were also examined, and strong expression was observed of ACE2 protein in cardiac muscle troponin T (cTnT)+ CMs while low to no expression in the surrounding cTnT-non-myocytes.

These results demonstrate that CMs are susceptible to SARS-CoV-2 infection.

Example 6: SARS-CoV-2 Infection Disrupts Multiple Intracellular CM Features

As described in this Example, motivated by the discovery of disruptions to various structural and contractile genes in our transcriptomic data, the inventors performed high content imaging of CMs following SARS-CoV-2 infection.

A number of abnormal structural features were immediately observed in many of the infected CMs that were not seen in parallel mock samples. Widespread myofibrillar disruption throughout the cytoplasm was the most common feature observed, which manifested as a unique pattern of very specific and periodic cleavage of myofibrils into individual sarcomeric units of identical size but without any alignment (FIG. 4A). Evidence of sarcomeric fragmentation was generally identified as early as 24 hours after infection, but was more widespread and common after 48 hours, and also observed in many of the CMs that remained after 72 hours. At a single time point 48 h post infection, up to 20% of cells exposed to virus displayed similar phenotypes of this rapid fragmentation (FIG. 4B), indicating this is a pervasive and continuous phenomenon. Curiously, myofibrillar fragmentation was more prevalent in bystander CMs that lacked signs of active viral infection (as per viral dsRNA staining), while cells positive for dsRNA rarely showed signs of myofibrillar fragmentation. The inventors found an inverse correlation (p-value<0.01) between the number of viral RNA positive cells in a well and the number of cells presenting sarcomere fragmentation (FIG. 4A-4C).

Since transcriptomic profiling data indicated viral infection altered the proteasome system (FIG. 3), CMs were exposed to the proteasome inhibitor bortezomib and observed that only high doses of bortezomib (but not the well-known cardiotoxic drug doxorubicin) induced myofibril fragmentations in CMs. However, bortezomib treatment induced fragmentation much more infrequently and less severely than SARS-CoV-2 and was generally accompanied by diffuse cTnT staining throughout the cell cytoplasm.

Altogether, these results indicate that the observed fragmentation of the sarcomere is dependent on SARS-CoV-2 infection of neighboring CMs. Reducing productive infection of CMs by means of IFN-β pre-treatment or E64D treatment did not reduce the incidence of myofibrillar disruption. However, ACE2 blocking did reduce the incident of myofibrillar disruption, potentially indicating an immediate response to viral exposure to the cell surface.

Co-staining SARS-CoV-2-exposed CMs with cTnT and the Z-disk marker α-actinin 2 revealed the myofibrillar fragments observed upon SARS-CoV-2 exposure consisted of two cTnT-positive bands flanking a single α-actinin 2 band, indicating cleavage at the M-line or a separation of thick and thin filaments (FIG. 4D). To examine sarcomeric fragmentation in greater detail, the inventors employed TEM imaging of SARS-CoV-2 infected and mock-treated cardiomyocytes. While intact sarcomeres were clearly identifiable with a classic dark Z-disk, light I-band, and dark A-band, single fragmented sarcomeres displayed an extended I-band and complete absence of the A-band (FIG. 4E), suggesting a mechanism by which thick filaments are liberated from sarcomere subunits. The intracellular network of mitochondria in CMs exposed to SARS-CoV-2 also appeared to be disrupted relative to normal mitochondrial organization.

In addition, the inventors observed that CMs with intact or moderately disrupted myofibrils often appeared to lack nuclear DNA staining (FIG. 4F). This phenomenon was observed most frequently in localized patches, with numerous cells lacking dsRNA staining along with stark nuclear absence (FIG. 4F).

Example 7: Intracellular Disruption in Myocardium of COVID-19 Patients

Based on the in vitro findings, the inventors sought to identify whether similar features were contributing to COVID-19 myocardial damage in vivo. The sarcomere fragmentation observed in COVID-19 patients appears to present some extreme features even compared to in vitro system.

Patient specimens were obtained from four COVID-19 positive patients—one diagnosed with viral myocarditis. Compared to healthy myocardial tissue (FIG. 5A), significant histological alterations were observed of the myocardium in the COVID-19 myocarditis case (FIG. 5B), in addition to moderate levels of immune infiltration. Mononuclear cells that appeared to be immune cells were detected, as well as nuclei with loss of nuclear material. Intercalated disks between cardiomyocytes were broken.

The tissues from the COVID-19 myocarditis case exhibited signs of edema with increased spacing between adjacent cardiomyocytes (FIG. 5B) and highly uneven staining for cardiac troponin-T, indicating sarcomere disruption (FIG. 5B, 5E) and there was evidence of troponin-T positive cells in the blood vessels, indicating phagocytosis of compromised myocytes (FIG. 5B, 5E). Some of the observed cardiomyocytes lacked hematoxylin staining for nuclei, showing that the in vitro phenotype of nuclear loss was also observed in patients infected with COVID-19 (FIG. 5B).

In COVID-19 infected patients that were not diagnosed with myocarditis (FIG. 5D), clear evidence was observed of nuclear loss (FIG. 5D) as well as evidence of myocyte compaction (FIG. 5D), and large regions exhibiting significant disruption of intercalated disk connections between cardiomyocytes (FIG. 5D). Strikingly, immunohistochemical labeling of the myofibrils revealed regions of extreme myofibrillar anomalies. Patients without diagnoses of myocarditis present large regions of myofibrils (ACTN2+) within cardiomyocytes that were entirely missing or collapsed (FIG. 5D).

The results described herein demonstrate that the in vitro phenotypes are able to predict previously unobserved disruptions in myocardium. Therefore, the in vitro methods described herein can be used to dissect the mechanisms of COVID-19 cardiovascular injury and identify agents that reduce or inhibit such injury.

REFERENCES

• Honko, A. N.; Storm, N.; Bean, D. J.; Henao Vasquez, J.; Downs, S. N.; Griffiths, A. Rapid Quantification and Neutralization Assays for Novel Coronavirus SARS-CoV-2 Using Avicel RC-591 Semi-Solid Overlay. Preprints 2020, 2020050264 (doi: 10.20944/preprints202005.0264.v1)
• Lian, X. et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. PNAS (2012) doi:10.1073/pnas.1200250109.
• Tohyama, S. et al. Distinct Metabolic Flow Enables Large-Scale Purification of Mouse and Human Pluripotent Stem Cell-Derived Cardiomyocytes. Cell Stem Cell 12, 127-137 (2013).

All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.

The following statements are intended to describe and summarize various embodiments of the invention according to the foregoing description in the specification.

Statements:

• • 1. A method comprising: incubating one or more test agents with cardiomyocytes in the presence of SARS-CoV-2 virus; and identifying any of the one or more test agents that reduce any of myofibrillar disruption, sarcomeric fragmentation, nuclear material, nuclear staining, enucleation, cardiac troponin solute levels, herniated mitochondria, apoptotic mitochondria, or a combination thereof in the cardiomyocytes compared to a control assay comprising with cardiomyocytes in the presence of SARS-CoV-2 virus without the test agent(s).
  • 2. The method of statement 1, wherein the SARS-CoV-2 virus is present at a multiplicity of infection at one or more SARS-CoV-2 virion particle per about 1000 cardiomyocyte cells; or at two or more SARS-CoV-2 virion particles per about 1000 cardiomyocyte cells; or at three or more SARS-CoV-2 virion particles per about 1000 cardiomyocyte cells; or at five or more SARS-CoV-2 virion particles per about 1000 cardiomyocyte cells; or at ten or more SARS-CoV-2 virion particles per about 1000 cardiomyocyte cells.
  • 3. The method of statement 1 or 2, wherein the SARS-CoV-2 virion particles infect cardiomyocytes, but do not infect cardiac fibroblasts, endothelial cells, or stem cells.
  • 4. The method of statement 1, 2, or 3, wherein the cardiomyocytes are generated from induced pluripotent stem cells.
  • 5. The method of statement 1-3 or 4, wherein the SARS-CoV-2 virion particles do not infect induced pluripotent stem cells.
  • 6. The method of any one of statements 1-5, wherein the cardiomyocytes are from a subject without a cardiac condition or a cardiac disease.
  • 7. The method of any one of statements 1-5, wherein the cardiomyocytes are mutant cardiomyocytes.
  • 8. The method of any one of statements 6 or 7, wherein the cardiac condition or a cardiac disease comprises a genetic mutation or a disease correlated with a genetic mutation.
  • 9. The method of any one of statements 1-7 or 8, wherein the cardiomyocytes are from a subject with a cardiac condition or a cardiac disease.
  • 10. The method of any one of statement 6-9, wherein the mutant cardiomyocytes, the cardiac condition, or the cardiac disease leads to or contributes to impairments in contractility, impairments in ability to relax (e.g., diastolic dysfunction), abnormal or improper functioning of the heart's valves, diseases of the heart muscle (e.g., cardiomyopathies), diseases such as angina pectoris, myocardial ischemia, infarction characterized by inadequate blood supply to the heart muscle, infiltrative diseases such as amyloidosis and hemochromatosis, global or regional hypertrophy (e.g., as may occur in some kinds of cardiomyopathy or systemic hypertension), abnormal communications between chambers of the heart, or a combination thereof in the subject.
  • 11. The method of any one of statement 6-10, wherein the mutant cardiomyocytes, the cardiac condition, or the cardiac disease can lead to or can contribute to a disease or dysfunction of the myocardium (heart muscle) in which a heart is abnormally enlarged, thickened and/or stiffened in a subject.
  • 12. The method of any one of statements 6-11, wherein the mutant cardiomyocytes, the cardiac condition, or the cardiac disease can lead to or can contribute to ischemic cardiomyopathy, coronary artery disease, non-ischemic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, infiltrative cardiomyopathy, congestive heart failure, myocardial infarction, cardiac ischemia, myocarditis, arrhythmia, or a combination thereof in a subject.
  • 13. The method of any one of statements 6-12, wherein the mutant cardiomyocytes, cardiac condition or a cardiac disease leads to or contributes to myocarditis, Duchenne muscular dystrophy or Emery Dreiffuss dilated cardiomyopathy in a subject.
  • 14. The method of any one of statements 1-13, comprising identifying (e.g., by Hoechst or hematoxylin staining) any of the one or more test agents that reduce cardiomyocyte enucleation compared to the control assay.
  • 15. The method of any one of statements 1-14, comprising identifying any of the one or more test agents that reduce titin protein cleavage compared to the control assay; or comprising identifying any of the one or more test agents that reduce M-band titin cleavage compared to the control assay.
  • 16. The method of any one of statements 1-15, wherein one or more of the test agents is a small molecule, an antibody, a nucleic acid, a carbohydrate, a protein, or a combination thereof.
  • 17. The method of any one of statements 1-16, wherein the one or more test agents block ACE2, inhibit cathepsin, or inhibit serine proteases.
  • 18. The method of any one of statements 1-17, further comprising manufacturing one or more of the test agents that reduce myofibrillar disruption, sarcomeric fragmentation, nuclear staining, enucleation, cardiac troponin solute levels, or a combination thereof.
  • 19. The method of any one of statements 1-18, further comprising administering to an animal one or more of the test agents that reduce myofibrillar disruption, sarcomeric fragmentation, nuclear staining, enucleation, cardiac troponin solute levels, or a combination thereof.
  • 20. One or more compounds identified by the method of any one of statements 1-19.
  • 21. The one or more compounds of statement 20 formulated into a composition.
  • 22. The one or more compounds of statement 20 or 21, comprising an ACE2 blocking agent, a cathepsin inhibitor, or a serine protease inhibitor.
  • 23. The one or more compounds of statement 20, 21 or 22, comprising an ACE2 blocking antibody, cathepsin inhibitor E-64-D, or aprotinin.
  • 24. A method comprising administering to a subject one or more of the compounds of statement 20-22 or 23.

The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims.

The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

Claims

1. A method comprising: incubating one or more test agents with cardiomyocytes in the presence of SARS-CoV-2 virus; and identifying any of the one or more test agents that reduce myofibrillar disruption, sarcomeric fragmentation, nuclear staining, enucleation, cardiac troponin solute levels, or a combination thereof in the cardiomyocytes compared to a control assay comprising with cardiomyocytes in the presence of SARS-CoV-2 virus without the test agent(s).

2. The method of claim 1, wherein the SARS-CoV-2 virus is present at a multiplicity of infection of one or more SARS-CoV-2 virion particle per about 1000 cardiomyocyte cells; or of two or more SARS-CoV-2 virion particles per about 1000 cardiomyocyte cells; or of three or more SARS-CoV-2 virion particles per about 1000 cardiomyocyte cells; or of five or more SARS-CoV-2 virion particles per about 1000 cardiomyocyte cells; or of ten or more SARS-CoV-2 virion particles per about 1000 cardiomyocyte cells.

3. The method of claim 1, wherein the SARS-CoV-2 virion particles infect cardiomyocytes, but do not infect cardiac fibroblasts, endothelial cells, or stein cells.

4. The method of claim 1, wherein the cardiomyocytes are generated from induced pluripotent stem cells.

5. The method of claim 1, wherein the SARS-CoV-2 virion particles do not infect induced pluripotent stem cells.

6. The method of claim 1, wherein the cardiomyocytes are mutant cardiomyocytes.

7. The method of claim 1, wherein the cardiomyocytes are from a subject without a cardiac condition or a cardiac disease.

8. The method of claim 7, wherein the cardiac condition or a cardiac disease comprises a genetic mutation or a disease correlated with a genetic mutation.

9. The method of claim 1, wherein the cardiomyocytes comprise a mutation or genetic variation that leads to or contributes to impairments in contractility, impairments in ability to relax, diastolic dysfunction, abnormal or improper functioning of the heart's valves, cardiomyopathies, angina pectoris, myocardial ischemia, infarction, hypertension, inadequate blood supply to heart muscle, amyloidosis, hemochromatosis, global hypertrophy, regional hypertrophy, abnormal communications between heart chambers, or a combination thereof in a subject.

10. The method of claim 1, wherein the cardiomyocytes comprise a mutation or genetic variation that leads to or contributes to an abnormally enlarged, thickened heart, an abnormally stiffened heart, or a combination thereof in a subject.

11. The method of claim 1, wherein the cardiomyocytes comprise a mutation or genetic variation that leads to or contributes to ischemic cardiomyopathy, coronary artery disease, non-ischemic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, infiltrative cardiomyopathy, congestive heart failure, myocardial infarction, cardiac ischemia, myocarditis, arrhythmia, or a combination thereof in a subject.

12. The method of claim 1, wherein the cardiomyocytes comprise a mutation or genetic variation that leads to or contributes to myocarditis, Duchenne muscular dystrophy or Emery Dreiffuss dilated cardiomyopathy in a subject.

13. The method of claim 1, comprising Hoechst and/or hematoxylin staining.

14. The method of claim 1, comprising identifying one or more test agents that reduce cardiomyocyte enucleation compared to the control assay.

15. The method of claim 1, comprising identifying one or more test agents that reduce titin cleavage compared to the control assay.

16. The method of claim 1, comprising identifying any of the one or more test agents that reduce M-band titin cleavage compared to the control assay.

17. The method of claim 1, wherein one or more of the test agents is a small molecule, an antibody, a nucleic acid, a carbohydrate, a protein, or a combination thereof.

18. The method of claim 1, wherein the one or more test agents block ACE2, inhibit cathepsin, or inhibit serine proteases.

19. The method of claim 1, further comprising manufacturing one or more of the test agents that reduce myofibrillar disruption, sarcomeric fragmentation, nuclear staining, enucleation, cardiac troponin solute levels, or a combination thereof.

20. The method claim 1, further comprising administering to an animal or subject one or more of the test agents that reduce myofibrillar disruption, sarcomeric fragmentation, nuclear staining, enucleation, cardiac troponin solute levels, or a combination thereof.

21. The method of claim 1, further comprising formulating one or more test agents into a composition.

22. The method of claim 21, further comprising formulating the composition to comprise ACE2 blocking agent, a cathepsin inhibitor, or a serine protease inhibitor.

23. The method of claim 21, comprising administering the composition to an animal or subject.