Patent Application
20170305973
The Picornaviridae family includes a variety of small, non-enveloped, icosahedral viruses with positive-strand RNA genomes1. Many picornaviruses (e.g., rhinoviruses, polioviruses, coxsackieviruses, enterovirus A71, enterovirus D68) that infect humans and cause high morbidity belong to the Enterovirus genus (EV)1. A number of these viruses have been structurally characterized by X-ray crystallography2, 3, 4, 5, establishing the general mechanisms for virus infection and for the development of effective anti-EV therapeutics. Nevertheless, rhinovirus C (RV-C), a newly discovered species among the EVs, remains enigmatic.
RV-C viruses (55 types), together with RV-A and RV-B viruses (˜100 types), are the leading cause of common colds. However, the RV-C lead to more severe respiratory infections among children than any other known rhinoviruses6. In contrast to other RV, the RV-C utilize cadherin related family member 3 (CDHR3) as a cellular receptor7. This childhood asthma susceptibility gene product is expressed in the human lower respiratory tract8. In line with this etiology, RV-Cs cause a significantly higher rate of lower respiratory tract infections in children than in adults9 and are directly associated with childhood asthma exacerbations10. Similar to influenza, RV-C infections peak in winter months. Currently, there are no vaccines or effective antiviral treatments available.
RV-C isolates have been refractory to structural characterization since their discovery in 200611 because of an inability to infect standard tissue culture (e.g., HeLa)12. Only modeled structures, based on amino acid sequence comparisons, have been available to aid biological investigations12, 13, 14. However, with recent advances in direct electron detection′ and image processing approaches16, 17 single-particle cryo-electron microscopy (cryo-EM) has now emerged as a powerful method for determining near atomic resolution (better than 4 Å) structures of macromolecular assemblies18. Cryo-EM requires only limited amount of sample without intensive purification, offering advantages over X-ray crystallography in structural studies of samples that are difficult to produce.
Picornavirus capsids are assembled from 60 copies of biological protomers, each composed of four proteins, VP1, VP2, VP3 and VP42. The three large surface polypeptides, VP1, VP2 and VP3 are folded into eight-stranded antiparallel “jelly rolls.” During the assembly process, autocatalytic cleavage of precursor VP0 into VP2 and VP4 in the presence of viral RNA results in the formation of full infectious virions19. The arrangement of jelly rolls in the virions exhibits pseudo T=3 icosahedral symmetry with an outer diameter of about 300 Å2, 3. The internal surface of the capsid is lined by the 60 copies of VP4. A surface depression or canyon2, encircling each five-fold axis, is frequently the receptor binding site for many EV20. Amino acid residues located on the outer surface of the virus but not specifically within this canyon are typically involved in forming immunogenic sites recognized by neutralizing antibodies. The canyon allows only limited access to these antibodies21. In many EV, a hydrophobic pocket within the VP1 jelly roll and situated underneath the canyon floor is occupied by a fatty-acid like molecule, or “pocket factor,”22, 23 that regulates the conformational states of the virus during cell entry24. Capsid-binding reagents that replace the pocket factor within VP1 are effective antiviral therapeutics against many EV25, but not RV-C14.
In the Examples below we report atomic resolution cryo-EM structures of the full and native empty particles (NEP) of the cell-adapted RV-C15a strain. These structures highlight novel immunogenic surfaces, a probable binding site for the glycosylated CDHR3 receptor molecule and the requirements for antiviral compound resistance. The novel immunogenic peptides identified in the work reported herein are useful targets for therapeutic antibodies and related therapeutics.
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In the Examples below Applicants report and analyze an atomic resolution structure of the rhinovirus (RV) C15a virus virion structure. Unexpectedly, in view of previous sequencing and modeling experimental efforts, a small sequence segment proximal to the carboxyl tail of the virus protein VP1 (“Virion Protein 1”) was found to have extensive surface exposure. This particular segment is not present in the sequences of RV-A and RV-B, and, therefore, the segment was not anticipated to have surface and/or immunogenic properties.
Using their knowledge of this new putative epitope, Applicants produced two synthetic peptides. One peptide included the new potentially immunogenic segment of the 15 residue C15 VP1 sequence and the other included a previously identified, possibly immunogenic 13 residue site nearby on the virion surface that was contributed by the viral VP2 capsid protein. These peptides were each inoculated into five mice. When Applicants tested the resulting murine polyclonal sera in Western assays, the VP1 peptide, but not the VP2 peptide, was found to have elicited an immune response in all five animals.
When these sera were tested in micro-neutralization assays, three of the five sera elicited to the predicted VP1 protein were able to neutralize the virus itself. None of the mice immunized with the VP2 peptide were reactive with virus, or produced neutralizing sera.
Therefore, Applicants have defined the reactive immunogenicity of an RV-C15 virus VP1 segment that can elicit protective, neutralizing antibodies. This segment is unique in structure and sequence (i.e. defining the dominant immunology) to the RV-C species of viruses. Knowledge of this segment allows Applicants to predict a corresponding segment in other members of the RV-C class. Peptide or virus reagents which elicit antibodies to this segment are likely vaccine and antiviral targets.
In one embodiment, the present invention is a composition comprising the immunogenic peptides described below. In a preferred embodiment, the invention is a peptide comprising a Rhinovirus-C structural protein 1 (VP1) rhinovirus immunogen peptide, wherein the peptide does not comprise flanking sequence with which the amino acids are contiguous in a naturally occurring rhinovirus. By “Rhinovirus C immunogen peptide” we mean to include the peptide as defined in RV-C15 and all other known and unknown strains of Rhinovirus C.
The Examples below define the immunogen peptide of RV-C15 as amino acids 252-266 (SEQ ID NO: 1 [YKPNSSGPDEHVLKD]) of the VP1 protein (see also
b
c
ce
c
a
YKPNSSGPDEHVLKD
bNCBI GenBank entry encoding this sequence.
cVP1 protein sequences on the N-terminus and COOH terminus of the structurally analogous VP1 immunogenic sequence are shown for context. Neither of the context flanking sequences is required for the external VP1 loop to exhibit immunogenicity when encoded in the virus capsid, proper. Maximum analogous length is 15 amino acids (aa); minimum length is 8 aa, average/mode length is 12 aa.
dThese sequences are identical. HM236966 is potentially misclassified in the literature as a type 29. It should be a type 20.
dAlphabetical list of the 59 structurally analogous VP1 sequence segments in this table: FKSKENENILVP (SEQ ID NO: 57), FKTVHEGKNILKD (SEQ ID NO: 106), IAKPNSGGLLEQ (SEQ ID NO: 30), IKDGEQGMLRQ (SEQ ID NO: 123), IPKTGSTTSELEQ (SEQ ID NO: 75), IPVEGGSGGLRE (SEQ ID NO: 117), IRDGDQGMLKQ (SEQ ID NO: 86), KEENESEQNLKD (SEQ ID NO: 44), KIKVEATKELEQ (SEQ ID NO: 102), KIKVETTKELEQ (SEQ ID NO: 104), KKKVPTDPNNHELTK (SEQ ID NO: 48), KPETEGSNILIQ (SEQ ID NO: 23), KPKTKGSNELEP (SEQ ID NO: 15), KPKTSGSTELEP (SEQ ID NO: 8), KQKPDSGGQVEP (SEQ ID NO: 3), KRKQPTNPDNHELTK (SEQ ID NO: 80), LPKEGSNDLTE (SEQ ID NO: 127), NVKDQGTKALEQ (SEQ ID NO: 52), REVNETTTDLTK (SEQ ID NO: 110), RKLADNTLKV (SEQ ID NO: 63), RKVNETTTDLTK (SEQ ID NO: 108), RKVNSTSHDLTK (SEQ ID NO: 125), RKVRDDSHDLEK (SEQ ID NO: 67), RKVSDDDSTLTR (SEQ ID NO: 25), RRVNPNSEDSTLTR (SEQ ID NO: 13), RRVYPDSADST (SEQ ID NO: 88), RRVYPDSADSTLTK (SEQ ID NO: 90), SVKSGTLNDLEQ (SEQ ID NO: 50), TKADESGNLKE (SEQ ID NO: 61), TKNASNENVLEE (SEQ ID NO: 121), TKVNDTQVLKE (SEQ ID NO: 46), TNKGSTTELEE (SEQ ID NO: 6), TNKGTTTELEE (SEQ ID NO: 114), TPTGASDGTLKE (SEQ ID NO: 21), TRKNNTSELEE (SEQ ID NO: 94), TRTGENNGTLEE (SEQ ID NO: 84), TTAPEGGGLLKE (SEQ ID NO: 33), VIKKGTTSDLEQ (SEQ ID NO: 17), VIKTGTTTGELEQ (SEQ ID NO: 92), VKKPDDTTGLLIQ (SEQ ID NO: 11), VKKPGDDTGLLIQ (SEQ ID NO: 119), VNIGETKELTE (SEQ ID NO: 82), VPDSNETDGLKE (SEQ ID NO: 40), VPIEGGSGNLKE (SEQ ID NO: 78), VPTQANDGTLEE (SEQ ID NO: 112), YKADDNTSTLTD (SEQ ID NO: 54), YKADENSPLKD (SEQ ID NO: 96), YKANDTSPLEDV (SEQ ID NO: 71), YKAPDATPQQLES (SEQ ID NO: 19), YKAPGNAQELKD (SEQ ID NO: 73), YKEEESEQNLKD (SEQ ID NO: 42), YKEKDASEDTLKS (SEQ ID NO: 65), YKKEDTTLEDRHFI (SEQ ID NO: 100), YKKGDALE (SEQ ID NO: 98), YKPDGEGHALTD (SEQ ID NO: 59), YKPNADEHILED (SEQ ID NO: 38), YKPNSSGPDEHVLKD (SEQ ID NO: 1), YKPNSSGPDQHVLED (SEQ ID NO: 36), YRTGTEGNYTLKN (SEQ ID NO: 27)
In one preferred version of the present invention, a nucleic acid encoding the peptide is part of an expression vector. In one version, the vector comprises a peptide operably linked to a transcriptional regulatory element wherein the peptide encodes the epitope described above. Preferable expression vectors include those listed in the GenScript web site: https://www.jpt.com/products/peptide-conjugates-klh-bsa/.
In another version, the present invention is a cell comprising the vector or peptide described above. In another version, the cell expresses the protein. In another version, the present invention is the peptide expressed by the cell.
The present invention includes a vaccine comprising the rhinovirus immunogen peptide described above, preferably including a pharmaceutically acceptable carrier. Preferably, the vaccine comprises an adjuvant.
In one embodiment, the vaccine is a peptide vaccine. Peptide vaccines are useful in eliciting an immunogenic response but are sometimes found to not stimulate cells in exactly the same way as a traditional vaccine. For example, a peptide vaccine may not cause a thymus cell, or T-cell, to react as much as other vaccines. To combat this, the peptide vaccine can be bound to a carrier protein or peptide to improve cell interaction. A suitable carrier will present the epitope peptide in a way that improves the immunogenicity of the peptide and allows the enhanced production of antibodies against the peptide. One of skill in the art would understand the construction of a peptide vaccine. Good information on peptides and their administration as vaccines (or to raise antibodies), comes from the GenScript web site: https://www.jpt.com/products/peptide-conjugates-klh-bsa/.
In one version, the present invention is a pharmaceutical composition comprising the immunogen peptide described above, preferably combined with pharmaceutical carriers.
In another version, the present invention is an antibody or antibody binding fragment thereof which binds to the epitope comprising the immunogen peptide described above. Preferably, the antibody is neutralizing against rhinovirus C.
Preferably, the antibody inhibits rhinovirus infection in a subject, such as a human or animal subject. By “inhibits,” we mean that infection is decreased or inhibited or that the rate of infection is reduced. One may wish to give the pharmaceutical preparation of the present invention in a prophylactic manner.
In a preferred embodiment, the antibody ameliorates symptoms of rhinovirus C infection in a subject, wherein the antibody is administered to a subject after infection with the rhinovirus.
Monoclonal antibody therapies (immunotherapy) are now quite common in cancer treatment. For example, common monoclonal antibody therapies for managing colon cancer are Bevacizumab (Avastin), Cetuximab (Erbitux), and Panitumumab (Vectibix). There are many more examples. The process is described on the ACS web site at http://www.cancer.org/treatment/treatmentsandsideeffects/treatmenttypes/immunotherapy/immu notherapy-monoclonal-antibodies.
In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody.
In a preferred version of the present invention, the monoclonal antibody recognizes VP1 of rhinovirus C, wherein the epitope that binds or is recognized by said antibody is within SEQ ID NO:1 or the other immunogen peptides listed in Table 1.
The present invention is also a hybridoma cell line that produces a monoclonal antibody as described above.
In another embodiment, the present invention is an immunogenic composition comprising an isolated preparation of empty Rhinovirus C particles, as described below in the Examples. In a preferred embodiment, the preparation is useful as a vaccine and contains no live virus. In another embodiment, the isolated preparation is combined with an element selected from the group consisting of pharmaceutical carriers and adjuvants.
In one embodiment, the present invention is a method of eliciting an immune response against rhinovirus C in a subject. Typically, the method comprises administering to the subject the immunogen peptide or a vaccine as described above.
A sufficient immune response can typically be measured as follows: Blood serum from an inoculated individual can be tested as per
In one version of the invention, one may wish to isolate and use the empty RV-C particles described below as an immunogenic composition.
In another embodiment, the present invention is a method of eliciting neutralizing antibodies against rhinovirus C, the method comprising the steps of providing to a subject an effective amount of an immunogenic composition comprising the immunogen peptide vaccine as described above.
In one version of the invention, one may wish to isolate and use the empty RV-C particles described below as an immunogenic composition.
Production of RV-C15a Viruses.
Recently, a recombinant RV-C15 virus12, adapted for tissue culture growth by serial passage in HeLa-E8 cells7 (a transduced HeLa cell line expressing CDHR3) led to new protocols for enhanced virus yields. The derivative, RV-C15a, represents a cell-adapted, uncloned population. The consensus sequence of this population differs in the capsid region from that of RV-C15, primarily by a single, high-frequency, nucleotide polymorphism. The substitution converts residue 1125 from Thr to Lys. (Numbering convention adds 1000 to VP1 residues, 2000 to VP2 residues, 3000 to VP3 residues, and 4000 to VP4 residues.) In the present work, an RV-C15a sample, purified only by sucrose cushion sedimentation, was used for cryo-EM structure analysis. To achieve an optimal number of particles per micrograph, data collection was carried out at a low magnification. Specifically, movies of frozen RV-C15a particles within a thin layer of vitreous ice were recorded at a nominal magnification of 14,000× using a Gatan K2 Summit direct electron detector. However, the trade-off was a low signal-to-noise ratio and a high anisotropic magnification distortion compared to what would be the case were high magnifications used for data collection. The primary data were collected in less than one week.
Biochemical Characterization of Two Forms of Particles.
Cryo-EM micrographs of RV-C15a showed the presence of two major forms of particles. One form lacked density at their centers and another form had density at their centers (
Cryo-EM Structure Determination.
Images of full and empty particles were separated by reference-free 2D classification using the program Relion16. A “truly independent” procedure of 3D reconstruction was employed to avoid overfitting to noise17. Essentially, initial model calculations, low resolution refinements and high resolution refinements were performed independently for each of the two half-data subsets. Parameters of anisotropic magnification distortion, a major resolution limiting factor for large assemblies (e.g., viruses), were estimated using powder diffraction patterns of polycrystalline gold particles26. The resultant parameters were used in the program jspr17 for correcting anisotropic magnification distortion on individual particles. Refinements of particle center, orientation, defocus, astigmatism, scale, and beam tilt resulted in icosahedral reconstructions of 8,973 full particles and 3,614 empty particles at 2.8 Å and 3.2 Å resolution, respectively (
RV-C15a has a Spiky Structure.
The structure of the RV-C15a full particle has 60 dominant spike-like protrusions, or “fingers,” on the outer surface of the virion (
Because of relatively large deletions (21-35 residues) in parts of the VP1 BC, DE and HI loops, the RV-C15a structure lacks a protruding “plateau” around each of the 5-fold vertices, a characteristic feature of other EV (
A Sequence-Conserved Depression could Bind Glycosylated CDHR3.
Sialic acid is the glycan moiety recognized by EV-D68 when it interacts with its cellular receptor24. Superposition of EV-D68 structure complexed with sialic acid (
The VP1 Hydrophobic Pocket is Unsuitable for Antiviral Capsid Binding Agents.
Unlike many EV structures the hydrophobic pocket within the VP1 jelly roll fold, where a pocket factor is typically bound4, 5, 22, is collapsed in RV-C15a (
Comparison of the Full and Empty Particle Structures.
RV-C15a full and empty particles differed mainly in regions on the inner surfaces of their capsid shells (
The cryo-EM structure of RV-C15a showed a collapsed hydrophobic pocket in VP1 that is filled with multiple bulky residues that inhibit the entrance of compounds which inhibit other EV by binding into the VP1 pocket. This is reminiscent of the collapsed pockets of non-EV picornaviruses, such as foot-and-mouth disease virus (FMDV, genus Aphthovirus)31 and Mengovirus (genus Cardiovirus)32. Those pockets are similarly occupied by multiple bulky, hydrophobic side chains and are unable to serve as drug targets.
Inclusion of large hydrophobic residues into the VP1 pocket of RV-Cs and incorporation of a fatty-acid like pocket factor into the VP1 pocket of many other EVs produce similar hydrophobic effects that favor the folding of the VP1 “jelly roll” β barrel. On one hand large hydrophobic residues (e.g., Phe, Trp, Met) are more effective than small residues (e.g., Val, Ala) at reducing solvent accessible surface areas in the pocket. Likewise, a pocket factor with a long hydrophobic tail (more than eight carbon atoms) plays a crucial role in expelling water out of the VP1 pocket of many EVs that contain multiple small residues. On the other hand, the presence of large hydrophobic side chains or a pocket factor in a VP1 pocket keeps the two sheets (one with β strands C, H, E, F and the other with β strands B, I, D, G) in a favorable distance for forming a β barrel and offers hydrophobic interactions that stabilize the “jelly roll” fold.
The large interior rearrangements characterizing the full and the native empty particles of RV-C15a, contrast with the conserved external surfaces. Both particle types have the same diameter, and display the same finger protrusions, truncated 5-fold vertices, and putative glycan binding regions. Possibly the RV-C use these native empty particles as immunogenic molecular decoys during infections, or they are merely byproducts of the assembly process.
The atomic structures of an RV-C virus, as reported here, show novel molecular targets for designing anti-RV-C therapeutics or effective vaccines by utilizing the non-infectious empty particles. These possibilities have clinical relevance since many RV-C, including RV-C15, are associated with severe, hospitalization-category infections in young children, especially those with asthma, and can also lead to significant adult respiratory problems, including chronic obstructive pulmonary disease.
Growth and Purification of RV-C15a.
RV-C15a (adapted) is a virus preparation derived by serial passage (13×) of recombinant C15 virus12, in HeLa-E8 cells, a lentivirus-transduced line expressing the full-length human CDHR3 gene (Tyr529) linked to a GFP reporter sequence7. To produce viruses for structure determination, HeLa-E8 cells were infected with RV-C15a at 34° C. At 40 h post infection after complete cytopathic effect (CPE) was observed, infected cells (2.4×108) were supplemented with HEPES (to 50 mM, pH 7.2) and then subject to multiple freeze-thaw cycles (3×). Clarified supernatants were treated with RNAse A, then concentrated by pelleting through 30% sucrose, before resuspension and being assayed for titer7, 33. The procedure gave ˜1.2×1010 PFU/PFUe (by plaque assay/qRT-PCR using HeLa-E8 cells) equivalent to ˜125 μg RNA-containing, infectious particles, assuming a particle-to-PFU ratio of ˜200.
Characterization of Two Forms of RV-C15a Particles.
A sample of RV-C15a, as prepared for structure determination, was sedimented through a 10-40% sucrose gradient (SW41 rotor, 24,600 rpm for 3 h at 4° C.). Fractions (1 ml) were collected (from the top) and then probed for VP2/VP0 content by Western blot analyses using anti-RV-C15-VP2 mouse monoclonal antibody, clone #517 (kindly provided by MedImmune Inc., Gaithersberg Md.). The fractions were also tested for infectivity according to CPE34, and for RNA content by qRT-PCR7.
Cryo-Electron Microscopy.
Aliquots of 2.8 μL of purified RV-C15a sample were applied onto glow-discharged holey carbon EM grids (400 mesh, Ted Pella Inc.). Grids were blotted for ˜8 s at a relative humidity of 80% and then plunge-frozen in liquid ethane cooled down by liquid nitrogen using a Cryoplunge 3 system (Gatan). Movies of frozen RV-C15a particles embedded in vitreous ice were collected at liquid nitrogen temperature using a Titan Krios transmission electron microscope (FEI) operated at 300 kV and equipped with a Gatan K2 Summit direct electron detector (3838×3710 in physical pixels). All the movies were automatically recorded in super resolution mode using Leginon35 at a nominal magnification of 14,000× and with a defocus range of 0.7-3.5 μm. This resulted in a super resolution pixel size of 1.04 Å/pixel. The dose rate was approximately 8e−/pixel/s. For each movie, the total electron dose was about 25.7 e−/Å2 that was fractionated into 70 frames with an exposure time of 200 ms per frame.
Image Processing.
Cryo-EM data were collected of the RV-15a particles. A total of 2979 movies were subjected to whole-frame motion correction using a modified version of MOTIONCORR15 as modified by Wen Jiang (Purdue University). This process was integrated into the Appion data processing pipeline36. Aligned frames were subsequently summed to obtain individual micrographs. Micrographs that had ice contamination or severe drift were discarded. The remaining micrographs were used for estimating of the contrast transfer function (CTF) parameters using CTFFIND337. A total of 24,882 particles were selected initially semi-automatically using e2boxer.py in the EMAN2 package38 and later automatically using DoG picker39. Individual particle images were boxed, extracted and subjected to reference-free 2D classification into 156 classes using the program Relion16. Some of these classes were clearly composed of full particles, some of empty particles and some were just junk. This yielded 13,390 full particles and 5,324 empty particles. Particles were re-boxed and re-extracted from the micrographs using jspr17. CTF parameters of the particles from each micrograph were estimated using fitctf2.py40.
Images of the full particles were divided into two half data subsets. A “truly independent” 3D reconstruction strategy, using the program jspr17, was applied to each of the two subsets assuming icosahedral symmetry. For each subset, eightfold binned particle images (squares of 8×8 original pixels separated by 1.04 Å were averaged to represent one pixel with a spacing of 8.32 Å) were used to compute eight initial 3D reconstructions by assigning random initial angles to each of 150 particle images. Refinements were performed by searching for the best orientation and particle center of each particle image relative to the eight current 3D reconstructions. Three of these structures were selected for further refinement with all the available particle images in the half subset. After multiple iterations these structures converged to roughly the same reconstruction. One of these structures was randomly chosen to extend the refinement with 4-binned, then 2-binned data and finally unbinned data. At this point, anisotropic magnification distortion remained the major resolution limiting factor. Ten images of polycrystalline gold particles were taken at a nominal magnification of 14,000× in super resolution mode. Fourier transform of these gold particle images gave powder diffraction like patterns that were used to estimate parameters of anisotropic magnification distortion26. The estimated degree of distortion and angle were 2.87% and 31.3°, respectively. These parameters were then employed to correct anisotropic magnification distortion for individual particle images using jspr17. Subsequent refinement of particle center, orientation, defocus, astigmatism, scale and beam tilt using jspr led to the final optimal reconstructions in terms of resolution. Fourier Shell Correlation (FSC) between the two subsets was used to monitor convergence. The same procedures were used for determining the 3D structure of the empty particles. A 2.79 Å resolution map of the full particle was reconstructed using 8,973 particles, and a 3.16 Å resolution map of the empty particle was reconstructed using 3,614 particles. The map resolution was determined based on the FSC between the two half maps (masked with a soft mask) independently calculated using the two half data subsets following the 0.143 cut-off criterion27, 41. To further validate the map resolution, phase randomized (beyond 5 Å) data were refined using the same procedures as were used for the original data that were not phase randomized. A “true FSC” curve′ was calculated using the FSC curve based on the original data and the FSC curve based on the phase randomized data. The full and empty particle maps were sharpened′ using a B factor of −108.4 Å2 and −122.2 Å2, respectively.
Model Building and Refinement.
For the full particle structure, a predicted atomic structure of the RV-C1513 (including coordinates for a protomer, VP1-VP4) was manually fitted into a region of the final EM map that corresponds to one protomer of the capsid using Chimera′. Atomic positions were refined using Phenix44 in real space to maximize the correlation coefficient between the final EM map and a map calculated based on the coordinates. Model statistics including bond lengths, bond angles and all-atom clash, rotamer statistics, and Ramachandran plot statistics were monitored. This was followed by model rebuilding with the program Coot45. The combination of real space refinement in Phenix and model rebuilding in Coot were repeated multiple times to achieve an optimized fit between the coordinates and the final EM map. At this point, the coordinates fit well into the densities by visual inspection.
Next, a mask, which included all grid points within a radius of 5 Å around each atom, was employed to cut out densities from the final EM map using the CCP446 program suite. The resultant segment of the final EM map was placed into a pseudo crystallographic unit cell (P1 space group) and was back transformed into pseudo structure factors (including both amplitudes and phases.) The coordinates were then subjected to refinement of individual B factors, atom positions and occupancy against the pseudo structure factors using standard reciprocal space refinement procedures in Phenix44. R factors were monitored during the refinement cycles. Only the coordinates were refined, whereas the map was kept constant. Subsequently, the coordinates were refined in real space against the final EM map by applying non-crystallographic symmetry (60-fold) constraints using Phenix44. Validation of the final coordinates was based on the criteria of MolProbity47. The full particle atomic model (excluding VP4 and the VP1 N-terminal residues 1101-1160) was used as a starting atomic model for model building and refinement of the empty particle structure.
As a further validation of the EM map resolution and of the fitting between the atomic model and the final EM map, a density map was calculated based on the atomic model specifying a resolution of 2.79 Å for the full particle structure and 3.16 Å for the empty particle structure. An FSC was computed between the resultant density map and the final EM map. The resolution determined using 0.5 FSC as a cut-off was 2.85 Å (full particle) and 3.24 Å (empty particle). Oligomers were generated using VIPERdb48. Figures were made using Chimera43 and Pymol (https://www.pymol.org/).
Example 2 refers to
Referring to
Peptide sequences chosen for peptide antibody analysis in the RVC15a VP1 and VP2 proteins are highlighted in
Referring to
Our conclusion is that serum from animals: 1520, 1526, 1647 and 1649 (shown in red in
aPixel size in super resolution mode. The physical pixel size is 2.08 Å/pixel.
bResolution was determined by the Fourier shell correlation between two half-maps using 0.143 as a cut-off value.
cReal space correlation coefficient (around atoms) between the final EM map and a density map calculated based on the coordinates.
dBased on the criteria of Molprobity (44)
1096
Phe
Leu
Leu
Ile
Pro
Tyr
Thr
1106
Phe
Phe
Leu
Val
Leu
Leu
Leu
1114
Phe
Phe
Phe
Phe
Phe
Phe
Phe
1178
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
1180
Met
Met
Cys
Trp
Asn
His
Val
1198
Ile
Thr
Ile
Ala
Ile
Ala
Ile
1246
Tyr
His
Gly
Ala
Lys
—
Ala
1118
Val
Ile
Tyr
Phe
Leu
Leu
Ile
1120
Ile
Met
Ile
Phe
Phe
Phe
Ile
1130
Ile
Tyr
Ala
Tyr
Ile
Ile
Ala
1132
Phe
Tyr
Tyr
Phe
Tyr
Tyr
Phe
1154
Pro
Ala
Pro
Pro
Pro
Pro
Ala
1156
Val
Val
Val
Val
Val
Ile
Val
1167
Phe
Phe
Phe
Val
Met
Ile
Met
1207
Leu
Leu
Met
Phe
Leu
Leu
Leu
3024
Ile
Ala
Ala
Ile
Ala
Ala
Val
aIn analogy with EV that have a pocket factor bound in the VP1 pocket, RV-C15a residues that are close to the head, the middle part, and the end of the pocket factor are colored bold, white, and italics, respectively.
bResidues are numbered based on RV-C15a numbering.
This application claims benefit of U.S. Patent Application 62/326,327 filed Apr. 22, 2016, which is incorporated herein by reference.
This invention was made with government support under AI104317 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62326327 | Apr 2016 | US |
