METHODS OF INHIBITING VIRAL REPLICATION COMPRISING THE SIGNAL PEPTIDASE COMPLEX

FIELD OF THE INVENTION

The present invention is directed to compositions targeting the signal peptidase complex and methods of use in treating and preventing flavivirus infection.

BACKGROUND OF THE INVENTION

Thus, there is a need in the art for novel antiviral therapies for the treatment of flaviviruses.

SUMMARY OF THE INVENTION

In an aspect, the disclosure provides a method to inhibit flaviviral infection, the method comprising contacting a cell with a composition comprising a compound that downregulates or inhibits the ER signal peptidase complex components SPCS1, SPCS2 and/or SPCS3.

In another aspect, the disclosure provides a method to prevent flaviviral infection in a subject, the method comprising administering to the subject a composition comprising a compound that downregulates or inhibits the ER signal peptidase complex components SPCS1, SPCS2 and/or SPCS3.

In still another aspect, the disclosure provides a method to reduce the amount of flavivirus in a subject infected with a flavivirus, the method comprising administering to the subject a composition comprising a compound that downregulates or inhibits the ER signal peptidase complex components SPCS1, SPCS2 and/or SPCS3.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E, FIG. 1F, FIG. 1G, FIG. 1H, FIG. 1I and FIG. 1J depict graphs and immunoblots showing genes required for flavivirus infection based on gene editing studies. FIG. 1A depicts WNV infection in 293T and FIG. 1B depicts WNV infection in HeLa gene-edited cells. 293T or HeLa cells were transduced with plasmids encoding sgRNA against the indicated genes (two sgRNA per gene), the Cas9 gene, and a selectable drug marker (puromycin). After three days of drug selection, cells were infected with WNV at an MOI of 5, and 12 hours later analyzed for intracellular E protein expression by flow cytometry. The results are the average of three independent experiments that were normalized to the sgRNA control. Error bars indicate standard error of the means (SEM). Statistical significance using an ANOVA with a multiple comparisons correction was as follows: 293 T cells: P<0.05: SEC61B, SERP1, SPSC1, STT3A, OST4, OSTC, EMC3, EMC5, EMC6; P<0.01: SEC63, SPSC3; HeLa cells: P<0.05: SEC61B, SEC63, OSTC, EMC2, EMC4, EMC5, EMC6; P<0.01: SERP1, SPSC1, SPSC3, STT3A. Dashed lines indicate the normalized level of WNV infection in cells transduced with an sgRNA control. FIG. 1C, FIG. 1D, FIG. 1E depict a Western blot to confirm the efficiency of gene editing for three of the genes (SEC61B, SPCS1, and SPCS3) in FIG. 1A. β-actin is included as a loading control. FIG. 1F depicts 293T cells expressing the indicated sgRNA infected with WNV (MOI of 0.01) and FIG. 1G depicts 293T cells expressing the indicated sgRNA infected with JEV (MOI of 0.1). Supernatants were titrated for infectious virus by focus-forming assay. The data is representative of three independent experiments, each performed in triplicate. Error bars indicate SEM. FIG. 1H depicts the effect of gene editing on related flavivirus JEV (MOI of 50); FIG. 1I depicts the effect of gene editing on DENV (MOI of 3); and FIG. 1J depicts the effect of gene editing on YFV (MOI of 3) infection in 293T cells. Cells were transduced with individual sgRNA against the indicated genes as described in FIG. 1A and harvested at 22 (JEV), 32 (DENV), or 38 (YFV) h after infection for processing by flow cytometry. The results are the average of three independent experiments that were normalized to the sgRNA control. Compared to the sgRNA control, for JEV and DENV, all differences were statistically significant using an ANOVA with a multiple corrections correction (P<0.01). Compared to the sgRNA control, for YFV, sgRNA against the following showed statistically significance using an ANOVA with a multiple corrections correction: (P<0.01: SEC61B, SEC63, SSR3, SPCS1, SPCS3, STT3A, OSTC).

FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D depicts graphs showing the conserved requirement for ER-associated genes in flavivirus infection in insect cells. FIG. 2A depicts Drosophila DL1 cells treated with the indicated dsRNAs and infected with WNV (Kunjin) (MOI, 4) and FIG. 2B depicts Drosophila DL1 cells treated with the indicated dsRNAs and infected with DENV-2 (MOI, 1) for 30 h, then processed for viral antigen staining by automated immunofluorescence microscopy. The percentage of infected cells was determined by automated microscopy and normalized to the control β-galactosidase siRNA. The data is expressed as the mean normalized value±SD. Statistically significant differences (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001) when compared to control siRNA (Student's t-test) are indicated. The data is pooled from four independent experiments tested in duplicate. FIG. 2C depicts cell viability analysis. DL1 cells were treated with the indicated dsRNA and 30 h later processed for cell viability. FIG. 2D depicts AAG2 cells treated with the indicated dsRNAs and infected with WNV (Kunjin) (MOI, 4).

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, FIG. 3H, FIG. 3I, FIG. 3J and FIG. 3K depict graphs, immunoblots and a schematic showing validation and mechanism of action of key genes in the flavivirus lifecycle in bulk-edited cells. FIG. 3A depicts individual sgRNA cell lines trans-complemented with cDNA expressing C-terminal FLAG-tagged versions of their respective genes and GFP or an empty vector control and GFP. Transfected cells were sorted by flow cytometry and then infected with WNV at an MOI of 5. Twelve hours later, cells were fixed, permeabilized, stained for intracellular E protein antigen, and processed by flow cytometry. The data is the average of three independent experiments performed in triplicate and reflects the percentage of WNV-infected cells in the fraction of cells expressing GFP. The indicated comparisons were statistically different (****, P<0.0001), as determined by the Mann-Whitney test. FIG. 3B depicts a Western blot of selected trans-complemented genes (e.g., SPCS1 and SPCS3) after incubating with anti-FLAG tag antibody. FIG. 3C, FIG. 3D depict the effect of sgRNA on translation and replication of a WT (FIG. 3C) and NS5 GVD polymerase mutant (FIG. 3D) WNV replicon. A cDNA launched WNV replicon with a minimal CMV promoter (eGFP-NS1-NS5) was transfected into gene-edited 293T cells. At 48 and 72 h after transfection, cells were harvested, and processed for GFP staining by flow cytometry (see FIG. 11). Statistically significant differences are indicated below the WT replicon graph as determined by ANOVA with a multiple comparisons correction. FIG. 3E depicts gene-edited cells were transfected with a WT WNV replicon as described in FIG. 3C, FIG. 3D. At 48 and 72 h after transfection, cells were harvested, stained for surface expression of NS1, and processed by flow cytometry. The data is expressed as the percentage of cells expressing NS1 compared to an isotype control MAb and is gated on GFP⁺ cells. Statistically significant differences were determined by ANOVA with a multiple comparisons correction (**, P<0.01; ****, P<0.0001). FIG. 3F depicts a schematic of the polyprotein processing strategy of flaviviruses¹³. Red and blue arrows indicate sites of cleavage by the host signalase and viral NS3 proteins, respectively. FIG. 3G, FIG. 3H depict immunoblots of control, SPCS1, and SPCS3 gene-edited 293T cells infected with WNV (MOI, 100) or mock-infected. At the indicated time points, lysates were prepared, electrophoresed and Western blotted with (FIG. 3G) anti-E (hE16) or (FIG. 3H) anti-prM-E (CR4293). Under these electrophoresis conditions, natively processed E and prM proteins migrate at ˜50 and 21 kDa, respectively. Higher molecular weight bands (E^hiand prM-E^hi) that react specifically with the E and prM-E MAbs in infected SPCS1 and SPCS3 gene-edited cells are indicated. FIG. 3I depicts prM-E transfected cells Western blotted with hE16 and FIG. 3J depicts prM-E transfected cells Western blotted with CR4293. Note, the shift of the prM-E bands to high molecular weight in cells with reduced expression of SPCS1 or SPCS3. The results are representative of three independent experiments and a loading control (β-actin) is provided immediately beneath. FIG. 3K depicts 293T cells expressing the indicated sgRNA transfected with a plasmid encoding the prM-E genes. 24 h later, supernatants were harvested and SVPs were quantitated by a capture ELISA. The results are of average several independent experiments performed in triplicate. The asterisks indicate relative SVP levels in the supernatant that are statistically different compared to control cells (****, P<0.001, ANOVA with a Dunnett's multiple comparison test).

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E and FIG. 4F depict graphs, immunoblots and an images showing the effects of SPCS1 on flavivirus protein processing and infection using a clonal SPCS1^−/− gene edited cell line. FIG. 4A depicts Western blotting to confirm the gene editing of SPSCS1 in a clonal (referred to as SPSC1^−/−) compared to a control cell line. β-actin is included as a loading control. FIG. 4B depicts SPSC1^−/− clonal cells transfected with prM-E or E expression plasmids. In both constructs, the E gene has its native signal sequence (last 25 amino acids of prM) but in the prM-E plasmid, the leader is located as an internal sequence (bottom). 48 hours after transfection, cells were subjected to Western blotting with hE16. Note, the shift of the prM-E bands to high molecular weight in SPSC1^−/− cells. The results are representative of independent experiments. FIG. 4C depicts supernatants harvested from prM-E transfected control or of SPSC1^−/− clonal cells (or untransfected cells) at 24 and 48 hours and evaluated for levels of SVP using a capture ELISA. The results are the average of two independent experiments performed in triplicate. FIG. 4D depicts WNV infection in control and SPSC1^−/− clonal cells at 72 h. Cells were infected at an MOI of 0.01 and analyzed by FFA. FIG. 4E depicts a summary of growth kinetics for WNV and FIG. 4F depicts a summary of growth kinetics for Chikungunya virus.

FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D depict a schematics and graph of the CRISPR-Cas9 screen for genes required for WNV and JEV infection. (FIG. 5A) Scheme of screen. A pooled lentivirus library containing 122,411 sgRNA (on average, 6 sgRNA per gene) was transduced into 293T-Cas9 cells at an MOI of 0.1. Ten days later, cells were left uninfected or infected with WNV or JEV (MOI of 1 and 3, respectively). After two weeks, surviving cells were harvested and the sgRNA sequences were obtained by next-generation sequencing. These results were compared to uninfected cells to determine sgRNA enrichment. The WNV screen was performed in triplicate on two independent days. The JEV screen was performed in triplicate on a single day. (FIG. 5B) Results of CRISPR-Cas9 screen for host factors required for infection with WNV in 293T cells. (FIG. 5D) Results of CRISPR-Cas9 screen for host factors required for infection with JEV in 293T cells. The x-axis is a list of all genes with sgRNA and the order was generated post-hoc to highlight genes that group together. The y-axis indicates the statistical significance of enrichment of particular genes (as reflected by sequencing of sgRNA) as compared to the uninfected population, and was determined after pooling technical and biological replicates. Filled circles represent genes identified in the virus-selected cell population. Hits were colored if they passed the statistical criteria described in the Supplementary Experimental Procedures. Significant hits were grouped by function and are colored as indicated. (FIG. 5C) Results of gene ontology (GO) enrichment biological process for genes that were enriched in the WNV screen. Enrichment analysis was performed on the 45 candidates using Panther. P values are indicated.

FIG. 6 depicts a graph showing validation of top 45 ‘hits’ from CRISPR-Cas9 screen using individual sgRNA. Lentiviruses co-expressing individual sgRNA (3 to 5 sgRNA per gene), Cas9, and puromycin were transduced into 293T cells. The gene targets were identified as the top chits' as described in the Methods and FIG. 5. After drug selection and recovery, transduced 293T cells were infected with WNV at an MOI of 5. Twelve hours later, cells were analyzed for viral E protein expression using flow cytometry. The data is the average of two independent experiments and is expressed as the percentage of cells that stained positive for WNV E protein.

FIG. 7 depicts a graph showing analysis of cell viability of gene-edited cells. WNV-infected (24 h time point) bulk CRISPR-Cas9 edited cells were evaluated for cell viability using a metabolic MTT (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results are pooled from several independent experiments performed in duplicate and data was compared to cells edited with a control sgRNA. None of the differences were statistically different compared to the control.

FIG. 8 depicts a graph showing the effect of gene editing on infection by additional RNA viruses. Lentiviruses co-expressing individual sgRNA (4 sgRNA per gene), Cas9, and puromycin were transduced into 293T cells. The 11 gene targets were identified as the top ‘hits’ as described in the text. Cells were infected with alphaviruses (SINV or CHIKV), a bunyavirus (LACV) or a rhabdovirus (VSV). Depending on the virus, cells were harvested at 12 or 24 h after infection and analyzed for intracellular viral antigen staining by flow cytometry using virus-specific monoclonal antibodies. The data is the average of two independent experiments and is expressed as relative infection (viral antigen expression) compared to the sgRNA control.

FIG. 9 depicts flow cytometry plots showing trans-complementation of sgRNA gene-edited cells with FLAG-tagged genes. (Top and middle rows) Individual sgRNA bulk gene-edited cell lines were trans-complemented with cDNA expressing C-terminal FLAG-tagged versions of their respective genes and GFP or an empty vector control and GFP. Transfected cells were analyzed by flow cytometry for expression of the FLAG-tag in the GFP⁺ cells. (Bottom row) Individual sgRNA bulk gene-edited cell lines were trans-complemented with cDNA expressing and empty vector and then stained for the FLAG-tag on the phycoerythrin channel. The data is representative of independent experiments.

FIG. 10 depicts a graph showing silencing of ER-associated SPCS2 in human U2OS cells. Human U2OS cells were transfected with either control of SPCS2 siRNAs and infected with WNV (KUN) (MOI, 1) for 18 h, DENV (MOI, 1) or SINV (MOI, 0.1) and CHIKV (MOI, 2) for 20 h, and processed for automated immunofluorescence microscopy. The percentage of infected cells was determined by automated microscopy and normalized to the control siRNA. The data is expressed as the mean normalized value±SD. Statistically significant differences (*, P<0.05; ***, P<0.001 were compared to control siRNA by a Student's t-test) are indicated. The data is pooled from at least two independent experiments tested in quadruplicate.

FIG. 11 depicts flow cytometric analysis of GFP expression in WNV replicon transfected gene-edited cells. Gene-edited bulk-selected 293T cells (sgControl, sgSTT3A, sgSPCS1, and sgSPCS3) were transfected with a cDNA launched replicon (WT or NS5 GVD polymerase dead mutant) containing a minimal CMV promoter, GFP, and the NS1 through NS5 genes of WNV. At 72 h after infection, cells were processed for GFP expression by flow cytometry. The transfection efficiency was approximately 15 to 30% (% positive cells) and the mean fluorescence intensity (MFI) of the GFP⁺ cells is indicated. In all cells tested, the GVD polymerase dead mutant expresses low levels of GFP, which reflects translation of the replicon RNA generated by the host nuclear DNA-dependent RNA polymerase. In sgControl, sgSPCS1, and sgSPCS3 (but not sgSTT3A) gene-edited cells, the WT replicon supports high levels of GFP expression, which reflects the replication activity of the NS5 RNA-dependent RNA polymerase. The results are representative of at least two independent experiments performed in triplicate, and are shown as contour plots.

FIG. 12 depicts flow cytometric analysis of surface NS1 expression in WNV replicon transfected gene-edited cells. Gene-edited bulk-selected 293T cells (sgControl, sgSTT3A, sgSPCS1, and sgSPCS3) were transfected with a cDNA launched WNV replicon (WT or NS5 GVD polymerase dead mutant) as described in FIG. 9. At 48 h after infection, cells were stained with a biotinylated anti-NS1 (9-NS1) or anti-CHIKV (CHK-152) directly to detect plasma membrane associated NS1 on the cell surface. Cells were processed by two-color flow cytometry and analyzed. The results are representative of at least two independent experiments performed in triplicate.

FIG. 13 depicts an immunoblot showing aberrant processing of WNV E protein in SPCS1 and SPCS3 gene-edited 293T cells. Note, this is an over-exposed Western blot of FIG. 3F, and is shown to highlight the accumulation of high molecular weight bands that react with anti-E protein antibody specifically in SPCS1 and SPCS3 gene-edited 293T cells. Control, SPCS1, and SPCS3 gene-edited 293T cells were infected with WNV (MOI, 100) or mock-infected for the indicated times. Lysates were prepared, boiled in SDS sample buffer, electrophoresed and Western blotted with an anti-E (hE16) MAb. Under these electrophoresis conditions, natively processed E protein migrates at ˜50 to 55 kDa, respectively. Higher molecular weight bands (E^medand E^hi) that react specifically with the E MAb in infected SPCS1 and SPCS3 gene-edited cells are present only in SPCS1 and SPCS3 gene-edited 293T cells. The data is representative of two independent experiments.

FIG. 14 depicts an immunoblot showing aberrant processing of WNV NS1 protein in SPCS1 and SPCS3 gene-edited 293T cells. Control, SPCS1, and SPCS3 gene-edited 293T cells were infected with WNV (New York 1999, MOI of 100) or mock-infected for the indicated times. Lysates were prepared, boiled in SDS sample buffer in the presence of 5% β-mercaptoethanol, electrophoresed and Western blotted with 8-NS1, a MAb that detects a linear epitope on WNV NS1¹⁴. The gel was separated into two parts (space indicated) due to the much higher signal of NS1 (48 kDa) and NS1′ (53 kDa) in the sgControl cells; thus, the top and bottom are not exposed equally. NS1′ is a C-terminal extended product of NS1 and is generated as the result of a −1 programmed ribosomal frameshift¹⁵. Note the higher molecular weight NS1 species (NS1^hi) is present only in SPCS1 and SPCS3 gene-edited cells despite the lower overall levels of NS1. Below, is a Western blot for β-actin to confirm equal loading of lysates. The results are representative of two independent experiments.

FIG. 15A and FIG. 15B depict flow cytometry plots, a graph and an immunoblot showing expression of NS1 and MHC class I in gene-edited cells. (FIG. 15A) The indicated bulk CRISPR-Cas9 edited cells were stained for surface expression of HLA-A2 class I MHC molecules using a specific mAb (W6/32) or an isotype control mAb and flow cytometry. The histograms shown are representative of two independent experiments performed in duplicate. (FIG. 15B) A summary is shown on the left of the normalized mean fluorescence intensity compared to the WT controls. A plasmid encoding WNV NS1 with a host-derived signal sequence (human CD33) was transfected into the indicated bulk CRISPR-Ca9 edited cells and 24 h later lysates were analyzed by Western blotting for NS1 (8-NS1 MAb). The results are representative of two independent experiments.

FIG. 16 depicts flow cytometry plots showing expression of complement regulators and MHC class I on the surface of SPSC1^−/− clonal cells. Control and SPSC1^−/− cells were stained for surface expression of CD55 (Decay accelerating factor (DAF), GPI-anchored), CD59 (transmembrane), HLA-A2 class I MHC molecules (transmembrane), or CD46 (Membrane cofactor protein, MCP, transmembrane) with specific primary MAbs (red and blue histograms) or isotype control MAbs (green and orange histograms) and processed flow cytometry. The histograms shown are representative of independent experiments performed in duplicate. No differences in cell surface expression between control and SPSC1^−/− cells were observed.

FIG. 17 depicts a graph showing the effect of trans-complementation of SPCS1 on WNV infection. Trans-complementation of SPCS1 restores production of WNV.

FIG. 18A, FIG. 18B and FIG. 18C depict graphs shows that the loss of expression of SPCS1 results in very little infectious flavivirus production. Virtually no infectious DENV (FIG. 18A), JEV (FIG. 18B), YFV (FIG. 18C) was recovered over time.

DETAILED DESCRIPTION OF THE INVENTION

Prior drug development efforts have been focused on defining small molecules that target flavivirus proteins including the viral protease and polymerase. Such molecules exert a rapid selective pressure that generally results in emergence of resistance due to the error prone activity of the RNA-dependent RNA polymerase. In contrast, the inventors sought out to identify host genes required for a key and conserved stage in the viral lifecycle such that inhibition of these host genes could abort flaviviral infection. Several identified genes were associated with endoplasmic reticulum (ER) functions including regulation of translocation, protein degradation, and N-linked glycosylation. Among the genes identified by the inventors, the host signal peptidase genes SPCS1 and SPCS3 were the most prominent. Reduced expression of these genes resulted in markedly lower replication of West Nile, Dengue, Japanese encephalitis, and yellow fever viruses. Remarkably, other unrelated viruses were not affected and the host cell did not show toxicity or cell injury. Accordingly, disclosed herein are compositions and methods for treating and/or preventing flaviviral infection comprising targeting ER functions, specifically, the signal peptidase complex.

Various aspects of the invention are described in more detail below.

I. Compositions

In an aspect, a composition of the invention comprises a compound that modulates ER-associated functions required for optimal flavivirus translation, polyprotein processing and replication. ER-associated functions include carbohydrate modification, translocation and ERAD. In certain embodiments, a gene involved in ER-associated translocation is selected from the group consisting of SEC63, SEC61B, SRP72, SSR1, SSR3, SPCS1, SPCS2 and SPCS3. In other embodiments, a gene involved in ER-associated carbohydrate modification is selected from the group consisting of OST4, SERP1, STT3A and OSTC. In still other embodiments, a gene involved in ER-associated protein degradation (ERAD) is selected from the group consisting of SEL1L, EMC2, EMC3 and EM6. In an embodiment, a composition of the invention comprises a compound that modulates a gene selected from the group consisting of EMC3, EMC4, EMC6, SEL1L, SEC61B, SEC63, STT3A, OSTC, SERP1, SSR3, SPCS1, and SPCS2. In another embodiment, a composition of the invention comprises a compound that modulates a gene selected from the group consisting of SEC61B, SPCS1 and SPCS3. In still another embodiment, a composition of the invention comprises a compound that modulates a gene selected from the group consisting of STT3A, SEC63, SPSC1 and SPCS3. In an embodiment, a composition of the invention comprises a compound that modulates the ER signal peptidase complex. In a specific embodiment, a composition of the invention comprises a compound that modulates the ER signal peptidase complex components SPCS1, SPCS2 and/or SPCS3. A compound that modulates ER-associated functions may be a compound that downregulates genes involved in ER-associated functions. Specifically, a compound that modulates the ER signal peptidase complex may be a compound that downregulates or inhibits SPCS1, SPCS2 and/or SPCS3. Methods to determine if a compound modulates SPCS1, SPCS2 and/or SPCS3 are known in the art. For example, SPCS1, SPCS2 and/or SPCS3 nucleic acid expression, SPCS1, SPCS2 and/or SPCS3 protein expression, or SPCS1, SPCS2 and/or SPCS3 activity may be measured as described in more detail below.

The signal peptidase complex (SPC) is a protein complex that is located in the endoplasmic reticulum membrane and cleaves the signal sequence from precursor proteins following their transport out of the cytoplasmic space. The SPC comprises signal peptidase complex subunit 1 (SPCS1, also referred to as SPC12, HSPC033, microsomal signal peptidase 12 kDa subunit and SPase 12 kDa subunit), signal peptidase complex subunit 2 (SPCS2, also referred to as SPC25, KIAA0102, microsomal signal peptidase 25 kDa subunit and SPase 25 kDa subunit) and signal peptidase complex subunit 3 (SPCS3, also referred to as SPC22, UNQ1841/PRO3567, microsomal signal peptidase 22/23 kDa subunit, SPC22/23 and SPase 22/23 kDa subunit). The SPC is a key host signalase required for efficient processing of the flavivirus polyprotein. Specifically, components of the SPC are required for proper processing of the viral prM, E and NS1 proteins.

A compound with the ability to modulate an ER-associated function in cells may potentially be used as an antiviral agent. Specifically, a compound with the ability to modulate the SPC in cells may potentially be used as an antiviral agent. Even more specifically, a compound with the ability to modulate SPCS1, SPCS2 and/or SPCS3 in cells may potentially be used as an antiviral agent. A compound with the ability to modulate SPCS1, SPCS2 and/or SPCS3 may include, without limitation, a compound, a drug, a small molecule, a peptide, a nucleic acid molecule, a protein, an antibody, a lipid, a carbohydrate, a sugar, a lipoprotein and combinations thereof. A nucleic acid molecule may be an antisense oligonucleotide, a small interfering RNA (siRNA), a ribozyme, a small nuclear RNA (snRNA), a long noncoding RNA (LncRNA), or a nucleic acid molecule which forms triple helical structures. Such compounds can be isolated from nature (e.g., isolated from organisms) or they can be produced in a laboratory (e.g., recombinantly or synthetically). Also encompassed are compounds that are combinations of natural and synthetic molecules. Methods to isolate or produce recombinant or synthetic candidate compounds are known to those skilled in the art. In certain embodiments, a compound that downregulates or inhibits SPCS1, SPCS2 and/or SPCS3 blocks enzymatic activity of SPCS1, SPCS2 and/or SPCS3. In other embodiments, a compound that downregulates or inhibits SPCS1, SPCS2 and/or SPCS3 reduces SPCS1, SPCS2 and/or SPCS3 protein expression. In still other embodiments, a compound that downregulates or inhibits SPCS1, SPCS2 and/or SPCS3 reduces SPCS1, SPCS2 and/or SPCS3 nucleic acid expression.

i. Nucleic Acid Expression

In an embodiment, SPCS1, SPCS2 and/or SPCS3 nucleic acid expression may be measured to identify a compound that downregulates or inhibits SPCS1, SPCS2 and/or SPCS3. For example, when SPCS1, SPCS2 and/or SPCS3 nucleic acid expression is decreased in the presence of a compound relative to an untreated control, the compound decreases the expression of SPCS1, SPCS2 and/or SPCS3. In a specific embodiment, SPCS1, SPCS2 and/or SPCS3 mRNA may be measured to identify a compound that decreases the expression of SPCS1, SPCS2 and/or SPCS3.

Methods for assessing an amount of nucleic acid expression in cells are well known in the art, and all suitable methods for assessing an amount of nucleic acid expression known to one of skill in the art are contemplated within the scope of the invention. The term “amount of nucleic acid expression” or “level of nucleic acid expression” as used herein refers to a measurable level of expression of the nucleic acids, such as, without limitation, the level of messenger RNA (mRNA) transcript expressed or a specific variant or other portion of the mRNA, the enzymatic or other activities of the nucleic acids, and the level of a specific metabolite. The term “nucleic acid” includes DNA and RNA and can be either double stranded or single stranded. Non-limiting examples of suitable methods to assess an amount of nucleic acid expression may include arrays, such as microarrays, PCR, such as RT-PCR (including quantitative RT-PCR), nuclease protection assays and Northern blot analyses. In a specific embodiment, determining the amount of expression of a target nucleic acid comprises, in part, measuring the level of target nucleic acid mRNA expression.

In one embodiment, the amount of nucleic acid expression may be determined by using an array, such as a microarray. Methods of using a nucleic acid microarray are well and widely known in the art. For example, a nucleic acid probe that is complementary or hybridizable to an expression product of a target gene may be used in the array. The term “hybridize” or “hybridizable” refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid. In a preferred embodiment, the hybridization is under high stringency conditions. Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. The term “probe” as used herein refers to a nucleic acid sequence that will hybridize to a nucleic acid target sequence. In one example, the probe hybridizes to an RNA product of the nucleic acid or a nucleic acid sequence complementary thereof. The length of probe depends on the hybridization conditions and the sequences of the probe and nucleic acid target sequence. In one embodiment, the probe is at least 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or more nucleotides in length.

In another embodiment, the amount of nucleic acid expression may be determined using PCR. Methods of PCR are well and widely known in the art, and may include quantitative PCR, semi-quantitative PCR, multiplex PCR, or any combination thereof. Specifically, the amount of nucleic acid expression may be determined using quantitative RT-PCR. Methods of performing quantitative RT-PCR are common in the art. In such an embodiment, the primers used for quantitative RT-PCR may comprise a forward and reverse primer for a target gene. The term “primer” as used herein refers to a nucleic acid sequence, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand is induced (e.g. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon factors, including temperature, sequences of the primer and the methods used. A primer typically contains 15-25 or more nucleotides, although it can contain less or more. The factors involved in determining the appropriate length of primer are readily known to one of ordinary skill in the art.

The amount of nucleic acid expression may be measured by measuring an entire mRNA transcript for a nucleic acid sequence, or measuring a portion of the mRNA transcript for a nucleic acid sequence. For instance, if a nucleic acid array is utilized to measure the amount of mRNA expression, the array may comprise a probe for a portion of the mRNA of the nucleic acid sequence of interest, or the array may comprise a probe for the full mRNA of the nucleic acid sequence of interest. Similarly, in a PCR reaction, the primers may be designed to amplify the entire cDNA sequence of the nucleic acid sequence of interest, or a portion of the cDNA sequence. One of skill in the art will recognize that there is more than one set of primers that may be used to amplify either the entire cDNA or a portion of the cDNA for a nucleic acid sequence of interest. Methods of designing primers are known in the art. Methods of extracting RNA from a biological sample are known in the art.

The level of expression may or may not be normalized to the level of a control nucleic acid. This allows comparisons between assays that are performed on different occasions.

SPCS1, SPCS2 and/or SPCS3 nucleic acid expression may be increased or decreased in the presence of a compound relative to an untreated control. In one embodiment, SPCS1, SPCS2 and/or SPCS3 nucleic acid expression can be compared using the ratio of the level of expression of SPCS1, SPCS2 and/or SPCS3 nucleic acid in the presence of a compound as compared with the expression level of SPCS1, SPCS2 and/or SPCS3 nucleic acid in the absence of a compound. For example, a nucleic acid is differentially expressed if the ratio of the level of expression of SPCS1, SPCS2 and/or SPCS3 nucleic acid in the presence of a compound as compared with the expression level of SPCS1, SPCS2 and/or SPCS3 nucleic acid in the absence of a compound is greater than or less than 1.0. For example, a ratio of greater than 1, 1.2, 1.5, 1.7, 2, 3, 3, 5, 10, 15, 20 or more, or a ratio less than 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.001 or less. In another embodiment, the increase or decrease in expression is measured using p-value. For instance, when using p-value, a nucleic acid is identified as being differentially expressed between a SPCS1, SPCS2 and/or SPCS3 nucleic acid in the presence of a compound and SPCS1, SPCS2 and/or SPCS3 nucleic acid in the absence of a compound when the p-value is less than 0.1, preferably less than 0.05, more preferably less than 0.01, even more preferably less than 0.005, the most preferably less than 0.001.

ii. Protein Expression

In another embodiment, SPCS1, SPCS2 and/or SPCS3 protein expression may be measured to identify a compound that downregulates or inhibits the expression of SPCS1, SPCS2 and/or SPCS3. For example, when SPCS1, SPCS2 and/or SPCS3 protein expression is decreased in the presence of a compound relative to an untreated control, the compound decreases the expression of SPCS1, SPCS2 and/or SPCS3. In a specific embodiment, SPCS1, SPCS2 and/or SPCS3 protein expression may be measured using immunoblot.

Methods for assessing an amount of protein expression are well known in the art, and all suitable methods for assessing an amount of protein expression known to one of skill in the art are contemplated within the scope of the invention. Non-limiting examples of suitable methods to assess an amount of protein expression may include epitope binding agent-based methods and mass spectrometry based methods.

In some embodiments, the method to assess an amount of protein expression is mass spectrometry. By exploiting the intrinsic properties of mass and charge, mass spectrometry (MS) can resolve and confidently identify a wide variety of complex compounds, including proteins. Traditional quantitative MS has used electrospray ionization (ESI) followed by tandem MS (MS/MS) (Chen et al., 2001; Zhong et al., 2001; Wu et al., 2000) while newer quantitative methods are being developed using matrix assisted laser desorption/ionization (MALDI) followed by time of flight (TOF) MS (Bucknall et al., 2002; Mirgorodskaya et al., 2000; Gobom et al., 2000). In accordance with the present invention, one can use mass spectrometry to look for the level of protein encoded from a target nucleic acid of the invention.

In some embodiments, the method to assess an amount of protein expression is an epitope binding agent-based method. As used herein, the term “epitope binding agent” refers to an antibody, an aptamer, a nucleic acid, an oligonucleic acid, an amino acid, a peptide, a polypeptide, a protein, a lipid, a metabolite, a small molecule, or a fragment thereof that recognizes and is capable of binding to a target gene protein. Nucleic acids may include RNA, DNA, and naturally occurring or synthetically created derivative.

As used herein, the term “antibody” generally means a polypeptide or protein that recognizes and can bind to an epitope of an antigen. An antibody, as used herein, may be a complete antibody as understood in the art, i.e., consisting of two heavy chains and two light chains, or may be any antibody-like molecule that has an antigen binding region, and includes, but is not limited to, antibody fragments such as Fab′, Fab, F(ab′)2, single domain antibodies, Fv, and single chain Fv. The term antibody also refers to a polyclonal antibody, a monoclonal antibody, a chimeric antibody and a humanized antibody. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (See, e.g. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; herein incorporated by reference in its entirety).

As used herein, the term “aptamer” refers to a polynucleotide, generally a RNA or DNA that has a useful biological activity in terms of biochemical activity, molecular recognition or binding attributes. Usually, an aptamer has a molecular activity such as binging to a target molecule at a specific epitope (region). It is generally accepted that an aptamer, which is specific in it binding to a polypeptide, may be synthesized and/or identified by in vitro evolution methods. Means for preparing and characterizing aptamers, including by in vitro evolution methods, are well known in the art (See, e.g. U.S. Pat. No. 7,939,313; herein incorporated by reference in its entirety).

In general, an epitope binding agent-based method of assessing an amount of protein expression comprises contacting a sample comprising a polypeptide with an epitope binding agent specific for the polypeptide under conditions effective to allow for formation of a complex between the epitope binding agent and the polypeptide. Epitope binding agent-based methods may occur in solution, or the epitope binding agent or sample may be immobilized on a solid surface. Non-limiting examples of suitable surfaces include microtitre plates, test tubes, beads, resins, and other polymers.

An epitope binding agent may be attached to the substrate in a wide variety of ways, as will be appreciated by those in the art. The epitope binding agent may either be synthesized first, with subsequent attachment to the substrate, or may be directly synthesized on the substrate. The substrate and the epitope binding agent may be derivatized with chemical functional groups for subsequent attachment of the two. For example, the substrate may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups. Using these functional groups, the epitope binding agent may be attached directly using the functional groups or indirectly using linkers.

The epitope binding agent may also be attached to the substrate non-covalently. For example, a biotinylated epitope binding agent may be prepared, which may bind to surfaces covalently coated with streptavidin, resulting in attachment. Alternatively, an epitope binding agent may be synthesized on the surface using techniques such as photopolymerization and photolithography. Additional methods of attaching epitope binding agents to solid surfaces and methods of synthesizing biomolecules on substrates are well known in the art, i.e. VLSIPS technology from Affymetrix (e.g., see U.S. Pat. No. 6,566,495, and Rockett and Dix, Xenobiotica 30(2):155-177, both of which are hereby incorporated by reference in their entirety).

Contacting the sample with an epitope binding agent under effective conditions for a period of time sufficient to allow formation of a complex generally involves adding the epitope binding agent composition to the sample and incubating the mixture for a period of time long enough for the epitope binding agent to bind to any antigen present. After this time, the complex will be washed and the complex may be detected by any method well known in the art. Methods of detecting the epitope binding agent-polypeptide complex are generally based on the detection of a label or marker. The term “label”, as used herein, refers to any substance attached to an epitope binding agent, or other substrate material, in which the substance is detectable by a detection method. Non-limiting examples of suitable labels include luminescent molecules, chemiluminescent molecules, fluorochromes, fluorescent quenching agents, colored molecules, radioisotopes, scintillants, biotin, avidin, stretpavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni2+, Flag tags, myc tags, heavy metals, and enzymes (including alkaline phosphatase, peroxidase, and luciferase). Methods of detecting an epitope binding agent-polypeptide complex based on the detection of a label or marker are well known in the art.

In some embodiments, an epitope binding agent-based method is an immunoassay. Immunoassays can be run in a number of different formats. Generally speaking, immunoassays can be divided into two categories: competitive immunoassays and non-competitive immunoassays. In a competitive immunoassay, an unlabeled analyte in a sample competes with labeled analyte to bind an antibody. Unbound analyte is washed away and the bound analyte is measured. In a non-competitive immunoassay, the antibody is labeled, not the analyte. Non-competitive immunoassays may use one antibody (e.g. the capture antibody is labeled) or more than one antibody (e.g. at least one capture antibody which is unlabeled and at least one “capping” or detection antibody which is labeled.) Suitable labels are described above.

In some embodiments, the epitope binding agent-based method is an ELISA. In other embodiments, the epitope binding agent-based method is a radioimmunoassay. In still other embodiments, the epitope binding agent-based method is an immunoblot or Western blot. In alternative embodiments, the epitope binding agent-based method is an array. In another embodiment, the epitope binding agent-based method is flow cytometry. In different embodiments, the epitope binding agent-based method is immunohistochemistry (IHC). IHC uses an antibody to detect and quantify antigens in intact tissue samples. The tissue samples may be fresh-frozen and/or formalin-fixed, paraffin-embedded (or plastic-embedded) tissue blocks prepared for study by IHC. Methods of preparing tissue block for study by IHC, as well as methods of performing IHC are well known in the art.

SPCS1, SPCS2 and/or SPCS3 protein expression may be increased or decreased in the presence of a compound relative to an untreated control. In one embodiment, SPCS1, SPCS2 and/or SPCS3 protein expression can be compared using the ratio of the level of expression of SPCS1, SPCS2 and/or SPCS3 protein in the presence of a compound as compared with the expression level of SPCS1, SPCS2 and/or SPCS3 protein in the absence of a compound. For example, a protein is differentially expressed if the ratio of the level of expression of SPCS1, SPCS2 and/or SPCS3 protein in the presence of a compound as compared with the expression level of SPCS1, SPCS2 and/or SPCS3 protein in the absence of a compound is greater than or less than 1.0. For example, a ratio of greater than 1, 1.2, 1.5, 1.7, 2, 3, 3, 5, 10, 15, 20 or more, or a ratio less than 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.001 or less. In another embodiment, the increase or decrease in expression is measured using p-value. For instance, when using p-value, a protein is identified as being differentially expressed between SPCS1, SPCS2 and/or SPCS3 protein in the presence of a compound and SPCS1, SPCS2 and/or SPCS3 protein in the absence of a compound when the p-value is less than 0.1, preferably less than 0.05, more preferably less than 0.01, even more preferably less than 0.005, the most preferably less than 0.001.

iii. Activity

In an embodiment, SPCS1, SPCS2 and/or SPCS3 activity may be measured to identify a compound that downregulates or inhibits SPCS1, SPCS2 and/or SPCS3. For example, processing of viral prM, E and NS1 proteins may be measured. In an embodiment, a compound that downregulates or inhibits SPCS1, SPCS2 and/or SPCS3 may reduce the amount of E protein present during viral infection and/or increase the molecular weight of E protein detected following viral infection. In another embodiment, a compound that downregulates or inhibits SPCS1, SPCS2 and/or SPCS3 may reduce the amount of prM-E protein present during viral infection and/or increase the molecular weight of prM-E protein detected following viral infection. In still another embodiment, a compound that downregulates or inhibits SPCS1, SPCS2 and/or SPCS3 may reduce the amount of NS1 protein present during viral infection and/or increase the molecular weight of NS1 protein detected following viral infection. In a different embodiment, a compound that downregulates or inhibits SPCS1, SPCS2 and/or SPCS3 may reduce the level of secreted viral particles (SVPs) following viral infection.

(a) Components of the Composition

The present disclosure also provides pharmaceutical compositions. The pharmaceutical composition comprises a compound that modulates ER-associated functions, as an active ingredient(s), and at least one pharmaceutically acceptable excipient, carrier or diluent. Further, a composition of the invention may contain binders, fillers, pH modifying agents, disintegrants, dispersants, lubricants, taste-masking agents, flavoring agents, preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. The amount and types of excipients utilized to form pharmaceutical compositions may be selected according to known principles of pharmaceutical science.

In one embodiment, the excipient may be a diluent. The diluent may be compressible (i.e., plastically deformable) or abrasively brittle. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., acetate and butyrate mixed esters), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, corn starch, phosphated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol, xylitol, maltodextrin, and trehalose. Non-limiting examples of suitable abrasively brittle diluents include dibasic calcium phosphate (anhydrous or dihydrate), calcium phosphate tribasic, calcium carbonate, and magnesium carbonate.

In another embodiment, the excipient may be a binder. Suitable binders include, but are not limited to, starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, and combinations thereof.

In another embodiment, the excipient may be a filler. Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone. By way of non-limiting example, the filler may be calcium sulfate, both di- and tri-basic, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starches, lactose, sucrose, mannitol, or sorbitol.

In still another embodiment, the excipient may be a buffering agent. Representative examples of suitable buffering agents include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline).

In various embodiments, the excipient may be a pH modifier. By way of non-limiting example, the pH modifying agent may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

In a further embodiment, the excipient may be a disintegrant. The disintegrant may be non-effervescent or effervescent. Suitable examples of non-effervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, and tragacanth. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

In yet another embodiment, the excipient may be a dispersant or dispersing enhancing agent. Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.

In another alternate embodiment, the excipient may be a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelators such as EDTA or EGTA; and antimicrobials, such as parabens, chlorobutanol, or phenol.

In a further embodiment, the excipient may be a lubricant. Non-limiting examples of suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate or stearic acid.

In yet another embodiment, the excipient may be a taste-masking agent. Taste-masking materials include cellulose ethers; polyethylene glycols; polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers; monoglycerides or triglycerides; acrylic polymers; mixtures of acrylic polymers with cellulose ethers; cellulose acetate phthalate; and combinations thereof.

In an alternate embodiment, the excipient may be a flavoring agent. Flavoring agents may be chosen from synthetic flavor oils and flavoring aromatics and/or natural oils, extracts from plants, leaves, flowers, fruits, and combinations thereof.

In still a further embodiment, the excipient may be a coloring agent. Suitable color additives include, but are not limited to, food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drug and cosmetic colors (Ext. D&C).

The weight fraction of the excipient or combination of excipients in the composition may be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the composition.

The composition can be formulated into various dosage forms and administered by a number of different means that will deliver a therapeutically effective amount of the active ingredient. Such compositions can be administered orally (e.g. inhalation), parenterally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, or intrasternal injection, or infusion techniques. Formulation of drugs is discussed in, for example, Gennaro, A. R., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (18^thed, 1995), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980). In a specific embodiment, a composition may be a food supplement or a composition may be a cosmetic.

Solid dosage forms for oral administration include capsules, tablets, caplets, pills, powders, pellets, and granules. In such solid dosage forms, the active ingredient is ordinarily combined with one or more pharmaceutically acceptable excipients, examples of which are detailed above. Oral preparations may also be administered as aqueous suspensions, elixirs, or syrups. For these, the active ingredient may be combined with various sweetening or flavoring agents, coloring agents, and, if so desired, emulsifying and/or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular, intra-articular and intraperitoneal), the preparation may be an aqueous or an oil-based solution. Aqueous solutions may include a sterile diluent such as water, saline solution, a pharmaceutically acceptable polyol such as glycerol, propylene glycol, or other synthetic solvents; an antibacterial and/or antifungal agent such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and the like; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as etheylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and/or an agent for the adjustment of tonicity such as sodium chloride, dextrose, or a polyalcohol such as mannitol or sorbitol. The pH of the aqueous solution may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Oil-based solutions or suspensions may further comprise sesame, peanut, olive oil, or mineral oil. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

For topical (e.g., transdermal or transmucosal) administration, penetrants appropriate to the barrier to be permeated are generally included in the preparation. Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. In some embodiments, the pharmaceutical composition is applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes. Transmucosal administration may be accomplished through the use of nasal sprays, aerosol sprays, tablets, or suppositories, and transdermal administration may be via ointments, salves, gels, patches, or creams as generally known in the art.

In certain embodiments, a composition a compound that modulates ER-associated functions is encapsulated in a suitable vehicle to either aid in the delivery of the compound to target cells, to increase the stability of the composition, or to minimize potential toxicity of the composition. As will be appreciated by a skilled artisan, a variety of vehicles are suitable for delivering a composition of the present invention. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems. Methods of incorporating compositions into delivery vehicles are known in the art.

In one alternative embodiment, a liposome delivery vehicle may be utilized. Liposomes, depending upon the embodiment, are suitable for delivery a compound that modulates ER-associated functions in view of their structural and chemical properties. Generally speaking, liposomes are spherical vesicles with a phospholipid bilayer membrane. The lipid bilayer of a liposome may fuse with other bilayers (e.g., the cell membrane), thus delivering the contents of the liposome to cells. In this manner, a compound that modulates ER-associated functions may be selectively delivered to a cell by encapsulation in a liposome that fuses with the targeted cell's membrane.

Liposomes may be comprised of a variety of different types of phosolipids having varying hydrocarbon chain lengths. Phospholipids generally comprise two fatty acids linked through glycerol phosphate to one of a variety of polar groups. Suitable phospholids include phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fatty acid chains comprising the phospholipids may range from about 6 to about 26 carbon atoms in length, and the lipid chains may be saturated or unsaturated. Suitable fatty acid chains include (common name presented in parentheses) n-dodecanoate (laurate), n-tretradecanoate (myristate), n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate (arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate), cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate), cis,cis-9,12-octadecandienoate (linoleate), all cis-9, 12, 15-octadecatrienoate (linolenate), and all cis-5,8,11,14-eicosatetraenoate (arachidonate). The two fatty acid chains of a phospholipid may be identical or different. Acceptable phospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS, distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl, oleoyl PS, palmitoyl, linolenyl PS, and the like.

The phospholipids may come from any natural source, and, as such, may comprise a mixture of phospholipids. For example, egg yolk is rich in PC, PG, and PE, soy beans contains PC, PE, PI, and PA, and animal brain or spinal cord is enriched in PS. Phospholipids may come from synthetic sources too. Mixtures of phospholipids having a varied ratio of individual phospholipids may be used. Mixtures of different phospholipids may result in liposome compositions having advantageous activity or stability of activity properties. The above mentioned phospholipids may be mixed, in optimal ratios with cationic lipids, such as N-(1-(2,3-dioleolyoxy)propyl)-N,N,N-trimethyl ammonium chloride, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate, 3,3′-deheptyloxacarbocyanine iodide, 1,1′-dedodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate, 1,1′-dioleyl-3,3,3′,3′-tetramethylindo carbocyanine methanesulfonate, N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or 1,1,-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate.

Liposomes may optionally comprise sphingolipids, in which spingosine is the structural counterpart of glycerol and one of the one fatty acids of a phosphoglyceride, or cholesterol, a major component of animal cell membranes. Liposomes may optionally contain pegylated lipids, which are lipids covalently linked to polymers of polyethylene glycol (PEG). PEGs may range in size from about 500 to about 10,000 daltons.

Liposomes may further comprise a suitable solvent. The solvent may be an organic solvent or an inorganic solvent. Suitable solvents include, but are not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone, N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide, tetrahydrofuran, or combinations thereof.

Liposomes carrying a compound that modulates ER-associated functions (i.e., having at least one methionine compound) may be prepared by any known method of preparing liposomes for drug delivery, such as, for example, detailed in U.S. Pat. Nos. 4,241,046, 4,394,448, 4,529,561, 4,755,388, 4,828,837, 4,925,661, 4,954,345, 4,957,735, 5,043,164, 5,064,655, 5,077,211 and 5,264,618, the disclosures of which are hereby incorporated by reference in their entirety. For example, liposomes may be prepared by sonicating lipids in an aqueous solution, solvent injection, lipid hydration, reverse evaporation, or freeze drying by repeated freezing and thawing. In a preferred embodiment the liposomes are formed by sonication. The liposomes may be multilamellar, which have many layers like an onion, or unilamellar. The liposomes may be large or small. Continued high-shear sonication tends to form smaller unilamellar liposomes.

As would be apparent to one of ordinary skill, all of the parameters that govern liposome formation may be varied. These parameters include, but are not limited to, temperature, pH, concentration of methionine compound, concentration and composition of lipid, concentration of multivalent cations, rate of mixing, presence of and concentration of solvent.

In another embodiment, a composition of the invention may be delivered to a cell as a microemulsion. Microemulsions are generally clear, thermodynamically stable solutions comprising an aqueous solution, a surfactant, and “oil.” The “oil” in this case, is the supercritical fluid phase. The surfactant rests at the oil-water interface. Any of a variety of surfactants are suitable for use in microemulsion formulations including those described herein or otherwise known in the art. The aqueous microdomains suitable for use in the invention generally will have characteristic structural dimensions from about 5 nm to about 100 nm. Aggregates of this size are poor scatterers of visible light and hence, these solutions are optically clear. As will be appreciated by a skilled artisan, microemulsions can and will have a multitude of different microscopic structures including sphere, rod, or disc shaped aggregates. In one embodiment, the structure may be micelles, which are the simplest microemulsion structures that are generally spherical or cylindrical objects. Micelles are like drops of oil in water, and reverse micelles are like drops of water in oil. In an alternative embodiment, the microemulsion structure is the lamellae. It comprises consecutive layers of water and oil separated by layers of surfactant. The “oil” of microemulsions optimally comprises phospholipids. Any of the phospholipids detailed above for liposomes are suitable for embodiments directed to microemulsions. A compound that modulates ER-associated functions may be encapsulated in a microemulsion by any method generally known in the art.

In yet another embodiment, a compound that modulates ER-associated functions may be delivered in a dendritic macromolecule, or a dendrimer. Generally speaking, a dendrimer is a branched tree-like molecule, in which each branch is an interlinked chain of molecules that divides into two new branches (molecules) after a certain length. This branching continues until the branches (molecules) become so densely packed that the canopy forms a globe. Generally, the properties of dendrimers are determined by the functional groups at their surface. For example, hydrophilic end groups, such as carboxyl groups, would typically make a water-soluble dendrimer. Alternatively, phospholipids may be incorporated in the surface of a dendrimer to facilitate absorption across the skin. Any of the phospholipids detailed for use in liposome embodiments are suitable for use in dendrimer embodiments. Any method generally known in the art may be utilized to make dendrimers and to encapsulate compositions of the invention therein. For example, dendrimers may be produced by an iterative sequence of reaction steps, in which each additional iteration leads to a higher order dendrimer. Consequently, they have a regular, highly branched 3D structure, with nearly uniform size and shape. Furthermore, the final size of a dendrimer is typically controlled by the number of iterative steps used during synthesis. A variety of dendrimer sizes are suitable for use in the invention. Generally, the size of dendrimers may range from about 1 nm to about 100 nm.

II. Methods

In an aspect, the present invention encompasses a method to inhibit flaviviral infection. The method comprises contacting a cell with a composition comprising a compound that modulates ER-associated functions required for optimal flavivirus translation, polyprotein processing and replication. In an embodiment, the composition comprises a compound that downregulates or inhibits the ER signal peptidase complex. In another embodiment, the composition comprises a compound that downregulates or inhibits the ER signal peptidase complex components SPCS1, SPCS2 and/or SPCS3. In a specific embodiment, the composition comprises a compound that downregulates or inhibits SPCS1. In another specific embodiment, the flaviviral infection is due to a flavivirus selected from the group consisting of West Nile virus, Dengue virus, Japanese encephalitis virus or yellow fever virus. Since a composition of the present invention is useful for inhibiting infection by a flavivirus, a composition of the invention may be used to protect a subject from flaviviral infection. As used herein, the term “protect” refers to prophylactic as well as therapeutic use. Thus, one embodiment of the present invention is a method to prevent flaviviral infection in a subject by administering a composition comprising a compound that downregulates or inhibits the ER signal peptidase complex components SPCS1, SPCS2 and/or SPCS3.

In another aspect, the present invention encompasses a method to reduce the amount of flavivirus in a subject infected with a flavivirus. The method comprises administering a composition comprising a compound that modulates ER-associated functions required for optimal flavivirus translation, polyprotein processing and replication. In an embodiment, the composition comprises a compound that downregulates or inhibits the ER signal peptidase complex. In another embodiment, the composition comprises a compound that downregulates or inhibits the ER signal peptidase complex components SPCS1, SPCS2 and/or SPCS3. In a specific embodiment, the composition comprises a compound that downregulates or inhibits SPCS1. In another specific embodiment, the flaviviral infection is due to a flavivirus selected from the group consisting of West Nile virus, Dengue virus, Japanese encephalitis virus or yellow fever virus.

In still another aspect, the present invention encompasses a method to protect a subject from flavivirus infection. The method comprises administering to the subject a composition comprising a compound that modulates ER-associated functions required for optimal flavivirus translation, polyprotein processing and replication. In an embodiment, the composition comprises a compound that downregulates or inhibits the ER signal peptidase complex. In another embodiment, the composition comprises a compound that downregulates or inhibits the ER signal peptidase complex components SPCS1, SPCS2 and/or SPCS3. In a specific embodiment, the composition comprises a compound that downregulates or inhibits SPCS1. In another specific embodiment, the flaviviral infection is due to a flavivirus selected from the group consisting of West Nile virus, Dengue virus, Japanese encephalitis virus or yellow fever virus.

As used herein, the terms “viral infection”, “viral infectivity”, “infection by a virus”, “viral propagation”, and the like, refer to the ability of a virus to carry out all steps in the viral life cycle, resulting in the production of infectious particles. Such a life cycle comprises a variety of steps including, for example, attachment, uncoating, transcription, translation, protein processing, replication of nucleic acid molecules, assembly of viral particles, intracellular transport of viral particles, budding, release and the like. Other steps may also be included depending on the virus.

As used herein, the terms “inhibit viral infection”, “inhibit infection by a virus”, “inhibit viral infectivity”, “inhibit viral propagation”, and the like, refer to decreasing the amount of virus present in an infected cell or subject relative to the amount of virus present in a cell or subject that has not been contacted with or treated with the disclosed methods or compounds. Also encompassed is the ability to prevent viral infection. Inhibition of viral infection can be effected in a patient infected with a flavivirus, or it can be effected in cells in culture (e.g., tissue culture). It should be appreciated that the terms amount and concentration can be used interchangeably. An amount of virus can also be referred to as a titer. It is also understood by those of skill in the art that the amount of virus can refer to the total number of viral particles, or it can refer to the number of viral particles that are infectious, i.e. capable of carrying out the viral life cycle, including the ability to effect another cycle of infectious particle formation. For example, in a given population of virus particles, some or all of the particles may be unable to carry out a specific step in its life cycle (e.g., attachment or entry) due to a deficiency in a molecule needed to perform that step. While the number of particles in the population may be large, the number of infectious particles could be small to none. Thus the amount of virus determined by counting virus particles may differ from that determined by measuring functional virus in, for example, a plaque assay. Accordingly methods of the present invention can affect the total number of viral particles produced, as well as the number of infectious viral particles produced. Appropriate methods of determining the amount of virus are understood by those skilled in the art and include, but are not limited to, directly counting virus particles, titering virus in cell culture e.g., plaque assay), measuring the amount of viral protein(s), measuring the amount of viral nucleic acids, or measuring the amount of a reporter protein, e.g., luciferase, GFP.

Inhibition of viral infection can result in a partial reduction in the amount of virus, or it can result in complete elimination of virus from a cell or subject or in prevention of viral infection. In one embodiment of the present invention, the amount of virus is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In another embodiment, the amount of virus is reduced by a factor of at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, or at least 10,000. In one embodiment the viral infection is completely inhibited (i.e., there are no infectious particles).

As used herein, the term “contacting” refers to bringing the compound and the cell into proximity so that the compound is capable of interacting with a gene involved in ER-associated function, or more specifically, SPCS1, SPCS2 and/or SPCS3. Such contacting can be achieved by introducing the compound to the cell when the cell is in a tissue culture environment, or it can be achieved when the cell is present in a subject. Consequently contacting the compound with the infected cell can be achieved through introducing the compound into a subject, for example, through an oral medication, an injection or other route of administration. The compound can interact with and remain on outside of the cell, or it can enter the cell and interact with a gene involved in ER-associated function, or more specifically, SPCS1, SPCS2 and/or SPCS3 within the cell.

The composition is described in Section I, the subject and administration are described in more detail below.

(a) Subject

A method of the invention may be used to treat or prevent flaviviral infection in a subject that is a human, a livestock animal, a companion animal, a lab animal, or a zoological animal. In one embodiment, the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas. In yet another embodiment, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In certain embodiments, the animal is a laboratory animal. Non-limiting examples of a laboratory animal may include rodents, canines, felines, and non-human primates. In other embodiments, the animal is a rodent. Non-limiting examples of rodents may include mice, rats, guinea pigs, etc. In a specific embodiment, the subject is a human.

Given that many flaviviruses are arthropod-transmitted, in some embodiments, a subject may be an arthropod. Arthropods include insects, arachnids, myriapods, and crustaceans. In an embodiment, the arthropod is an insect. In a specific embodiment, the insect is a mosquito. In an exemplary embodiment, the insect is Drosophila.

(b) Administration

In certain aspects, a therapeutically effective amount of a composition of the invention may be administered to a subject. Administration is performed using standard effective techniques, including peripherally (i.e. not by administration into the central nervous system) or locally to the central nervous system. Peripheral administration includes but is not limited to oral, inhalation, intravenous, intraperitoneal, intra-articular, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. Local administration, including directly into the central nervous system (CNS) includes but is not limited to via a lumbar, intraventricular or intraparenchymal catheter or using a surgically implanted controlled release formulation. The route of administration may be dictated by the disease or condition to be treated. It is within the skill of one in the art, to determine the route of administration based on the disease or condition to be treated.

Pharmaceutical compositions for effective administration are deliberately designed to be appropriate for the selected mode of administration, and pharmaceutically acceptable excipients such as compatible dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents and the like are used as appropriate. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition, incorporated herein by reference in its entirety, provides a compendium of formulation techniques as are generally known to practitioners. It may be particularly useful to alter the solubility characteristics of the peptides useful in this discovery, making them more lipophilic, for example, by encapsulating them in liposomes or by blocking polar groups.

Effective peripheral systemic delivery by intravenous or intraperitoneal or subcutaneous injection is a preferred method of administration to a living patient. Suitable vehicles for such injections are straightforward. In addition, however, administration may also be effected through the mucosal membranes by means of nasal aerosols or suppositories. Suitable formulations for such modes of administration are well known and typically include surfactants that facilitate cross-membrane transfer. Such surfactants are often derived from steroids or are cationic lipids, such as N-[1-(2,3-dioleoyl)propyl]-N,N,N-trimethyl ammonium chloride (DOTMA) or various compounds such as cholesterol hemisuccinate, phosphatidyl glycerols and the like.

For therapeutic applications, a therapeutically effective amount of a composition of the invention is administered to a subject. A “therapeutically effective amount” is an amount of the therapeutic composition sufficient to produce a measurable response (e.g., a reduction in infection, reduction in viral particles, reduction in symptoms associated with viral infection). Actual dosage levels of active ingredients in a therapeutic composition of the invention can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject. The selected dosage level will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, the flavivirus, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art of medicine.

The timing of administration of the treatment relative to the disease itself and duration of treatment will be determined by the circumstances surrounding the case. Treatment could begin in a hospital or clinic itself, or at a later time after discharge from the hospital or after being seen in an outpatient clinic.

Duration of treatment could range from a single dose administered on a one-time basis to a life-long course of therapeutic treatments. The duration of treatment can and will vary depending on the subject and the disease or disorder to be treated. For example, the duration of treatment may be for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. Or, the duration of treatment may be for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks. Alternatively, the duration of treatment may be for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months. In still another embodiment, the duration of treatment may be for 1 year, 2 years, 3 years, 4 years, 5 years, or greater than 5 years. It is also contemplated that administration may be frequent for a period of time and then administration may be spaced out for a period of time. For example, duration of treatment may be 5 days, then no treatment for 9 days, then treatment for 5 days.

The frequency of dosing may be once, twice, three times or more daily or once, twice, three times or more per week or per month, or as needed as to effectively treat the symptoms or disease. In certain embodiments, the frequency of dosing may be once, twice or three times daily. For example, a dose may be administered every 24 hours, every 12 hours, or every 8 hours. In other embodiments, the frequency of dosing may be once, twice or three times weekly. For example, a dose may be administered every 2 days, every 3 days or every 4 days. In a different embodiment, the frequency of dosing may be one, twice, three or four times monthly. For example, a dose may be administered every 1 week, every 2 weeks, every 3 weeks or every 4 weeks.

A compound of the present invention, or a composition thereof, may be administered alone or in combination with one or more other pharmaceutical agents, including other compounds of the present invention.

Although the foregoing methods appear the most convenient and most appropriate and effective for administration of a composition of the invention, by suitable adaptation, other effective techniques for administration, such as intraventricular administration, transdermal administration and oral administration may be employed provided proper formulation is utilized herein.

In addition, it may be desirable to employ controlled release formulations using biodegradable films and matrices, or osmotic mini-pumps, or delivery systems based on dextran beads, alginate, or collagen.

Typical dosage levels can be determined and optimized using standard clinical techniques and will be dependent on the mode of administration.

EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1. CRISPR/Cas9 Screen Identifies an Endoplasmic Reticulum-Associated Signal Peptidase Complex Required for Infectivity of Multiple Flaviviruses

West Nile virus (WNV) is a mosquito-transmitted flavivirus that infects humans and other vertebrate animals and is closely related to several other pathogens (e.g., Dengue (DENV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses) that cause global disease¹. Despite almost 400 million flavivirus infections annually, there is no specific antiviral therapy for this group of viruses. We reasoned that an improved understanding of the host factors required for efficient infection might identify genes that could be targeted pharmacologically to control infection of multiple members of the viral genus. Although genome-wide siRNA screens have been performed with WNV and other flaviviruses in different laboratories^2-4, the results have varied.

To identify genes required for infection and to overcome off-target effects associated with RNA silencing-based screens, we performed genome-wide CRISPR/Cas9 gene-editing screens in human 293T cells with WNV and JEV. The CRISPR/Cas9 system uses small guide RNAs (sgRNA) that facilitate sequence-dependent insertion or deletion of nucleotides, which enables functional knockout of both alleles in diploid mammalian cells^5,8. We designed an inhibition of cytopathic effect screen to identify genes that were required for WNV (strain New York 2000) or JEV (strain 14-14-2) infection in human 293T cells expressing the Cas9 RNA-guided DNA endonuclease (FIG. 5A). We transduced 293T-Cas9 cells with a commercial library of 122,411 sgRNA targeting 19,050 genes; sgRNA were packaged into lentiviruses, pooled to create a master library, and transduced at a low multiplicity of infection to limit the number of sgRNAs in each cell. Lentivirus transduced cells were then either infected with WNV or JEV or left untreated, followed by culture for 14 days. In the absence of library lentivirus transduction, no cells in virus-infected cultures survived. Colonies of lentivirus-transduced 293T-Cas9 cells surviving WNV or JEV infection were expanded and pooled separately, and sgRNA were amplified by PCR, subjected to next-generation sequencing, and compared to the library generated from the uninfected cells cultured in parallel. For additional validation and comparison, we performed the screens with two technical replicates or on separate days.

Based on analysis of the uninfected cell library, the sgRNA coverage was ˜93% of human genes. In cells surviving WNV infection, on average, we obtained ˜100 sgRNA reads that showed ˜10- or greater fold enrichment (Table 1) in the surviving cell population. Prioritization of gene ‘hits’ was based on sequencing data showing multiple different sgRNA per gene, the number of sequencing reads per gene, the enrichment of a given sgRNA compared to the uninfected cell library, and the reproducibility across the technical and biological repeats. Based on these criteria, 45 genes (Table 2) were selected as candidates for validation. Gene ontology enrichment analysis suggested that the majority of these were involved in endoplasmic reticulum (ER)-associated functions including carbohydrate modification (OST4, OSTC, STT3A, and SERP1), translocation (SEC63, SEC61B, SSR1, SSR3, SPSC1, and SPSC3), and protein degradation (ERAD: SEL1L, EMC3, and EMC6) (FIG. 5B, FIG. 5C). Genes also were identified in the heparan sulfate biosynthesis pathway (EXT2, SLC36B2, HS3ST5, HST3ST3A1), which was not unanticipated given the role of heparans in enhancing cellular attachment of flaviviruses^7,8. JEV infection of lentivirus-transduced 293T-Cas9 cells also identified several of the same ER-associated gene ‘hits’ (e.g., STT3A, SEC63, SEC61B, EMC3, and EMC6) (FIG. 5D), suggesting that conserved host pathways are required by multiple flavivirus family members for optimal infectivity.

To validate the top 45 genes that emerged from computational analysis, 293T cells were transduced with a vector expressing Cas9, puromycin, and one of five different sgRNAs for each gene (Table 3). Four days after drug selection, bulk cells were infected with WNV at an MOI of 5, and 12 hours later infectivity was assessed by flow cytometry by staining for intracellular E protein expression. Notably, 12 genes (EMC3, EMC4, EMC6, SEL1L, SEC61B, SEC63, STT3A, OSTC, SERP1, SSR3, SPCS1, and SPCS2) were validated by this assay, with reduced infection observed in 293T cells expressing at least 2 different sgRNA against the same gene (FIG. 1A, FIG. 6). Importantly, sgRNA expression did not decrease WNV infection because of cellular toxicity, as cell viability was equivalent in the presence or absence of sgRNA at baseline (data not shown) or 24 h after infection (FIG. 7). Additional validation in a second cell line, human HeLa cells, with the two sgRNAs used to validate studies in 293T cells showed that editing of many of the 12 ‘hits’ also reduced WNV infection (FIG. 1B). We determined the knockout efficiency of our sgRNA in bulk-transduced cells by Western blotting for the selected genes (SEC61B, SPCS1, SPCS3) that we could obtain a validated antibody. These results confirmed that cells transduced with specific sgRNAs led to substantive decreases in protein expression (FIG. 1C, FIG. 1D, FIG. 1E).

We extended our studies to a multi-step growth assay in bulk gene-edited 293T cells with a subset of our validated genes; we selected STT3A, SEC63, SPSC1, or SPCS3 for further analysis because of their phenotypes in both 293T and HeLa cells after infection with WNV. Gene editing of STT3A, SEC63, SPSC1, or SPCS3 resulted in a 50 to 1,000-fold reduction in WNV yield at different time points after infection (FIG. 1F). Since the magnitude of the phenotype was large in this multi-step growth kinetic assay, these genes may have important roles in viral replication or cell-to-cell spread, which would be less apparent in single cycle infections, which were used in the primary screen.

We next tested the role of the genes validated from the WNV screen against other globally relevant flaviviruses, including JEV, DENV serotype 2 (DENV-2), or YFV (FIG. 1H, FIG. 1I, FIG. 1J). Expression of 7 of the validated sgRNA also reduced infection of closely (JEV, ˜85% amino acid identity) and distantly (DENV and YFV, ˜45% amino acid identity) related flaviviruses. Of note, the magnitude of reduction of infection using the flow cytometric assay was greatest for DENV-2. Similar to the results with WNV, editing of STT3A, SEC63, SPSC1, or SPCS3 resulted in markedly (up to 1,000-fold) reduced JEV yield (FIG. 1G). An important role for these ER-associated genes was relatively specific to flaviviruses, as we observed less or no impact of gene editing on infection by unrelated positive or negative sense RNA viruses including alphaviruses, bunyaviruses, and rhabdoviruses (FIG. 8). One exception was genes modifying carbohydrate processing (STT3A and OSTC), which when edited, showed reduced infection of Sindbis and vesicular stomatitis viruses.

Given that many flaviviruses are arthropod-transmitted, we evaluated the roles of the gene orthologs in Drosophila insect cells using WNV and DENV-2. We tested 11 genes and found that silencing of Drosophila orthologs in the same ER-associated pathways of carbohydrate modification (dCG1518 [STT3A]), translocation and processing (dSEC63, dSEC61b, dSPCS2, dSRP72, dCG5885 [SSR3]), and protein degradation (ERAD: dCG17556 [EMC2] and dCG6750 [EMC3]) resulted in an loss of infection by WNV and DENV-2 (FIG. 2A, FIG. 2B); similar to the results seen in mammalian cells, the magnitude of the effects were larger for DENV compared to WNV. Importantly, silencing of these genes in insect cells did not affect viability (FIG. 2C). An analogous reduction in WNV infection was observed in AAG2 Aedes aegypti mosquito cells after gene silencing of SEC63, SRP72, and SPSC2 (FIG. 2D). Altogether, several of the validated genes that were required for efficient flavivirus infectivity in human cells had analogous impact on infection in insect cells.

Although the observation of reduced infection of flaviviruses with multiple sgRNAs lessened the possibility of off-target gene editing effects, we validated our findings using trans-complementation with four ER-associated genes that regulate ER translocation (SEC61B, SPCS1, and SPCS3) or carbohydrate modification (STT3A) (FIG. 3A). Transfection of gene edited CRISPR cells with tagged versions of the wild-type (WT) alleles, which was confirmed by Western blotting (FIG. 3B, FIG. 9), resulted in enhanced WNV infection compared to control vector-transfected cells. Since we identified two of the three components of the Signal Peptide Processing Complex (SPSC1 and SPSC3) in the genome-wide CRISPR screen, we also tested whether SPSC2 was required for flavivirus infection using siRNAs. Indeed, depletion of SPSC2 in human U2OS cells led to reduced infection of WNV and DENV yet had no impact on alphavirus (CHIKV or SINV) infection (FIG. 10). However, we were unable to validate a SPCS2 gene-edited 293T cell despite attempts with multiple different sgRNA.

We next evaluated the stage in the viral lifecycle that was affected by loss of expression of several of the ER-associated genes that we validated. To determine whether genes were required for efficient translation and/or replication, we utilized WT and NS5 RNA-dependent RNA polymerase loss-of-function mutant (NS5 GDD→GVD⁹) WNV replicons (FIG. 3C, FIG. 3D). Transfection of cells with this cDNA-launched GFP-expressing replicon (GFP-NS1→NS5) results in the production of relatively low levels of viral RNA from an enhancer-less minimal CMV promoter that is independent of viral RNA replication, as measured by GFP expression (compare WT and GVD, FIG. 11). Viral RNA replication results in a significant increase in GFP-expression with time that depends on a functional RNA-dependent RNA polymerase, allowing a comparative measure of viral RNA replication. Accordingly, in control sgRNA cells, the mutant (NS5 GDD→GVD) replicon was translated but did not replicate and accordingly, the GFP signal remained dim over time (FIG. 3C, FIG. 3D), whereas the fluorescence intensity of GFP in control sgRNA cells transfected with the WT replicon increased over time. In STT3A gene-edited cells, GFP signal at 48 h and 72 h was diminished markedly compared to the control sgRNA cell, although no difference was seen with the non-replicating mutant GVD replicon. This suggests that STT3A is required for efficient replication but not translation. The results with the SPCS1 and SPCS3, however, were distinct. Despite the marked defect in viral yield by multi-step growth analysis (see FIG. 1F) in SPCS1 and SPCS3 gene-edited cells, near wild-type levels of GFP accumulation were observed after transfection of the WNV replicon. Analogous phenotypes were observed when we analyzed surface or intracellular levels of NS1 after transfection of WT replicons in the different gene-edited cells (FIG. 3E, FIG. 12).

SPCS1 and SPCS3 have annotated functions as components of a signal peptidase complex^10,11, and SPCS1 reportedly is required for hepatitis C virus (HCV) assembly¹². Because a deficiency of SPCS1 and SPCS3 resulted in substantially reduced WNV and JEV yield while only modestly impacting replication of the WNV replicon, we speculated that the SPCS complex was a key host signalase required for efficient processing of the flavivirus polyprotein¹³. Flavivirus structural, NS1, and NS4B proteins require cleavage by unknown host signal peptidase(s), whereas the remaining non-structural proteins are cleaved in cis by the viral NS2B-NS3 protease (FIG. 3F and text missing or illegible when filed ^14,15). To assess the role of the SPCS complex in polyprotein processing, gene-edited 293T cells were infected with WNV at a high multiplicity of infection. Lysates were prepared at different time points and subjected to Western blotting to monitor expression of the viral proteins. Studies with an anti-E protein antibody (hE16¹⁶) (FIG. 3G) revealed that less E protein was present in SPCS1 and SPCS3 gene-edited cells than the control cells at 12 h after WNV infection. By 24 h after infection, higher molecular weight aberrant bands reacted with the anti-E protein antibody, and this was more apparent in an over-exposed blot (FIG. 13). Of note, the pattern of high molecular weight bands that reacted with anti-E antibody were overlapping but not identical in SPSC1 and SPSC3 gene-edited cells, suggesting that the absence of an individual subunit could impact the efficiency of cleavage of a given target site in the WNV genome. Higher molecular weight yet non-identical bands also were apparent in the SPCS1 and SPCS3 gene-edited cells after blotting with an antibody (CR4293¹⁷) that binds to a shared determinant on prM and E (FIG. 3H). Consistent with these findings, less NS1 accumulated in WNV-infected cells that were gene-edited for SPCS1 or SCPC3, and a high molecular weight (˜100 kDa) band was apparent after blotting under highly denaturing conditions (boiled in SDS plus 5% β-mercaptoethanol) (FIG. 14). Collectively, this data suggests that components of the SPCS complex are required for proper processing of the viral prM, E, and NS1 proteins.

To isolate the effects of the SPCS complex on prM and E protein processing we used a plasmid encoding only the WNV prM-E structural genes, which upon translation and processing can produce secreted subviral particles (SVPs)¹⁸. We transfected this WNV prM-E plasmid into bulk gene-edited cell lines and assessed intracellular and extracellular production of prM and E proteins. Western blotting of cell lysates for E protein (˜55 kDa) showed both reduced levels and a higher molecular weight band (˜80 kDa) in cells deficient in SPCS1 or SPCS3 (FIG. 3I). These data suggest a defect in E processing from prM. This finding was corroborated by Western blotting with the CR4293 antibody, which showed reduced levels of prM and E and the emergence of the high molecular weight 80 kDa band in the SPCS1 and SPCS3 gene-edited cells (FIG. 3J). To confirm that the defect observed impacted particle assembly and release, we measured levels of secreted SVPs in the supernatant at 24 h after prM-E transfection. Substantive reductions were apparent in cells deficient in SPCS1 or SPCS3 (FIG. 3K). This effect of SPCS1 and SPCS3 on flavivirus protein processing was not global in nature, as we observed normal levels of endogenous HLA-A2 class I MHC antigen were present on the surface of these cells (FIG. 15A, FIG. 15B, left) and no impact on processing of a genetically engineered form of NS1 that depends on the endogenous CD33 leader sequence for processing (FIG. 15B, right).

As bulk-selected cells might still retain one WT allele, we transduced sgRNA against SPCS1 or SPCS3 and selected clonal lines after limiting dilution cloning. SPCS3^−/− clones were not obtained despite several attempts, suggesting this gene may be essential. Several SPCS1^−/− clonal lines emerged and one was chosen for functional analysis after confirming both alleles contained non-sense mutations and/or deletions and the SPCS1 protein was absent (FIG. 4A). Transfection experiments with the single prM-E plasmid followed by Western blotting with anti-E MAb showed that loss of expression of SPSC1 was associated with almost complete loss of E protein expression, with a residual uncleaved prM-E band present (FIG. 4B, lanes 1-2). Consistent with this data, SVPs were not detected in the supernatant of SPCS1^−/− cells at 24 or 48 h after transfection, compared to high levels observed in control cells (FIG. 4C). Remarkably, infectious WNV and DENV failed to accumulate in the supernatant of SPCS1^−/− cells even 72 h after infection, with at least a 10,000-fold reduction in titer observed (FIG. 4D, FIG. 4E). The SPSC1^−/− cell line did not have general defects in processing of host proteins destined for the secretory pathway, as wild type levels of the complement regulator Membrane cofactor protein (MCP; CD46) were observed by Western blotting and cell surface expression of Decay accelerating factor (DAF; CD55), CD59, CD46, and class I MHC molecules anchored by glycophosphatidylinositol (GPI) or transmembrane anchors was unaffected (FIG. 16). Indeed, relatively smaller (5-fold) effects on yield in the supernatant of CHIKV, an unrelated alphavirus were observed in SPCS1^−/− cells (FIG. 4F).

In yeast, there exist parallel signal recognition targeting pathways, with specificity conferred by differences in the hydrophobic core of signal sequences^19,20. Given the specific reduction in processing and secretion of flavivirus structural proteins in SPCS1^−/− cells, we speculated that SPSC1 uniquely facilitated recognition of the hydrophobic character and/or internal leader sequence of flavivirus structural proteins. To test this hypothesis, we compared E protein expression when E was transfected as part of the prM-E plasmid or as a separate plasmid, with both E genes downstream of their native signal sequence (FIG. 4B, bottom). Whereas transfection of the prM-E in a single plasmid with an internal leader peptide did not result in efficient processing of the E protein, transfection of the E gene alone resulted in protein expression that was normal in size, albeit at slightly lower levels than observed in control cells (FIG. 4B, lanes 3-4).

Our screen preferentially identified genes with several ER-associated functions (carbohydrate modification, translocation, and ERAD) required for optimal flavivirus translation, polyprotein processing, and replication. Two of our top gene hits, the ER signal peptidase components SPCS1 and SPCS3 were required for flavivirus polyprotein processing as evidenced by a reduction in cleavage and accumulation of prM-E in the form of subviral particles; these latter experiments suggest that this complex is one of the previously unidentified host signalases required for viral polyprotein cleavage. Although the SPCS1 and SPCS3 were largely dispensable for flavivirus RNA replication, remarkably their loss did not impact surface expression or processing of host proteins including class I MHC molecules or complement regulatory factors, the processing of a recombinant flavivirus NS1 protein containing a heterologous human CD33 signal sequence, or alphavirus structural proteins or infectivity. This specificity suggests that the SPCS complex in mammalian and likely insect cells may represent one of several host signal peptidases that can promote cleavage of signal peptides for entry into the ER lumen, each with unique target site preference. Alternatively, SPCS1, SPSC2, and SPCS3 confer substrate specificity to a larger signal peptidase complex, and these proteins preferentially recognize flavivirus cleavage sites.

In separate interactome analysis, we observed that SPSC2 can bind to WNV NS2B (S. Cherry, unpublished results). Given that SPCS1 and SPCS3 were required for efficient cleavage of prM-E, and that flavivirus NS2B-3 has been reported to modulate the activity of the host signal peptidase cleavage at the C-prM junction²¹, flavivirus non-structural proteins (e.g., NS2B-3) might modulate the target specificity of the SPCS1/SPCS3 enzyme complex to facilitate additional cleavage events of the viral polyprotein.

A subset of our ER-related genes were identified in a prior RNAi screen with WNV in Drosophila cells⁴, and dSec61B was identified in an RNAi screen with DENV³. Virtually all of the human genes identified in our CRISPR screen involved in ER biology with insect orthologs also were required for optimal infection by two different flaviviruses, WNV and DENV, in insect cells. This suggests that flaviviruses utilize highly conserved host pathways in invertebrate and vertebrate cells to facilitate infection in multiple species. This is relevant because many flaviviruses (e.g., WNV) are highly promiscuous and can replicate in insects, birds, and many mammalian species. The ER is a particularly important site in the flavivirus lifecycle as its membranes support viral translation, polyprotein processing, replication, virion morphogenesis and carbohydrate modification of structural proteins. Thus, the identification of gene targets, especially those with enzymatic functions (e.g., signal peptidases) that are required for efficient flavivirus infection across phylogeny provide intriguing new candidates for pharmacological manipulation.

References for Example 1

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2 Krishnan, M. N. et al. RNA interference screen for human genes associated with West Nile virus infection. Nature 455, 242-245 (2008).

3 Sessions, O. M. et al. Discovery of insect and human dengue virus host factors. Nature 458, 1047-1050 (2009).

4 Yasunaga, A. et al. Genome-Wide RNAi Screen Identifies Broadly-Acting Host Factors That Inhibit Arbovirus Infection. PLoS Pathog 10, e1003914, doi:10.1371/journal.ppat.1003914 PPATHOGENS-D-13-01089 [pii] (2014).

5 Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823, doi:10.1126/science.1231143 science.1231143 [pii] (2013).

6 Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471, doi:10.7554/eLife.00471. 00471 [pii] (2013).

7 Chen, Y. et al. Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nat Med 3, 866-871 (1997).

8 Lee, E., Hall, R. A. & Lobigs, M. Common E protein determinants for attenuation of glycosaminoglycan-binding variants of Japanese encephalitis and West Nile viruses. J Virol 78, 8271-8280 (2004).

9 Khromykh, A. A., Kenney, M. T. & Westaway, E. G. trans-Complementation of flavivirus RNA polymerase gene NS5 by using Kunjin virus replicon-expressing BHK cells. J Virol 72, 7270-7279 (1998).

10 Evans, E. A., Gilmore, R. & Blobel, G. Purification of microsomal signal peptidase as a complex. Proc Natl Acad Sci USA 83, 581-585 (1986).

11 Meyer, H. A. & Hartmann, E. The yeast SPC22/23 homolog Spc3p is essential for signal peptidase activity. J Biol Chem 272, 13159-13164 (1997).

12 Suzuki, R. et al. Signal peptidase complex subunit 1 participates in the assembly of hepatitis C virus through an interaction with E2 and NS2. PLoS Pathog 9, e1003589, doi:10.1371/journal.ppat.1003589 PPATHOGENS-D-13-00314 [pii] (2013).

13 Lindenbach, B. D., Murray, C. L., Thiel, H. J. & Rice, C. M. in Fields Virology Vol. 1 (eds D. M. Knipe & P. M. Howley) 712-746 (Lippincott Williams & Wilkins, 2013).

14 Chambers, T. J., Grakoui, A. & Rice, C. M. Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites. J Virol 65, 6042-6050 (1991).

15 Falgout, B., Pethel, M., Zhang, Y. M. & Lai, C. J. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol 65, 2467-2475 (1991).

16 Oliphant, T. et al. Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nature Medicine 11, 522-530 (2005).

17 Throsby, M. et al. Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus. J Virol 80, 6982-6992 (2006).

18 Schalich, J. et al. Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions. J Virol 70, 4549-4557 (1996).

19 Hann, B. C. & Walter, P. The signal recognition particle in S. cerevisiae. Cell 67, 131-144, doi:0092-8674(91)90577-L [pii] (1991).

20 Ng, D. T., Brown, J. D. & Walter, P. Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J Cell Biol 134, 269-278 (1996).

21 Stocks, C. E. & Lobigs, M. Signal peptidase cleavage at the flavivirus C-prM junction: dependence on the viral NS2B-3 protease for efficient processing requires determinants in C, the signal peptide, and prM. J Virol 72, 2141-2149 (1998).

22 Ma, H. et al. A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death. Cell Rep, doi:S2211-1247(15)00675-0 [pii]. 10.1016/j.celrep.2015.06.049 (2015).

Methods for Example 1
Cells and Viruses

Vero, BHK21, HeLa, U205, and 293T cells were cultured at 37° C. in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS). C6/36 Aedes albopictus cells were cultured at 28° C. in L15 supplemented with 10% FBS and 25 mM HEPES pH 7.3. Drosophila DL1 cells were cultured at 28° C. in Schneiders' medium supplemented with 10% FBS as described¹. The following viruses were used in screening and validation studies: WNV (New York 2000), WNV (Kunjin), JEV (14-14-2), DENV-2 (16681 and New Guinea C strains), YFV (17D), LACV (original strain), VSV (Indiana), and SINV (Toto). All viruses were propagated in Vero or C6/36 cells and titrated by standard plaque or focus-forming assays².

sgRNA Library and Screen.

A pooled library encompassing 122,411 different sgRNA against 19,050 human genes was derived by the Zheng laboratory³and obtained from a commercial source (Addgene). The library was packaged using a lentivirus expression system. 293T cells were transfected using Fugene®HD (Promega). Forty-eight hours after transfection, supernatants were harvested, clarified by centrifugation (300 g×5 min), filtered, and aliquotted for storage at −80° C.

For the screen, we generated 293T-Cas9 cells by transfecting the lentiCas9-Blast plasmid (Addgene #52962) using Fugene®HD transfection reagent and blasticidin selection. These 293T-Cas9 cells (5×10⁷) were infected with lentiviruses encoding individual sgRNA at a multiplicity of infection (MOI) of 0.1. Two days later, after extensive washing, transduced cells were infected with WNV or JEV at an MOI of 1 and then incubated for 14 days. In parallel, untransduced 293T-Cas9 cells were infected to ensure virus-induced infection and cell death. The experiments were performed parallel as either duplicate or triplicate technical replicates, and for WNV the screen was repeated in an independent biological experiment.

Genomic DNA was extracted from the cells that survived WNV or JEV infection, and sgRNA sequences were amplified. The amplified product was subjected to next generation sequencing using an Illumina Hi-Seq 2500 platform, and the sgRNA sequences against specific genes were recovered after removal of the tag sequences.

Gene Validation.

Bioinformatic analysis was used to determine the sgRNA sequences that were enriched in the cells that survived WNV or JEV infection. This was achieved using a program, and accounted for the number of sequencing reads per gene, and the enrichment of a given sgRNA compared to the uninfected cell library, which was prepared in parallel. A further cut-off of candidate genes was made manually and reflected the reproducibility across the different technical and biological repeats. From this, we identified 45 top ‘hits’. These candidate genes were tested for validation by designing 4 to 5 independent sgRNA per gene as oligonucleotides and cloning them into the pLentiCRISPR v2 (Addgene plasmid 52961) per the manufacturer's instructions. A control sgRNA was designed. Plasmids were transfected into 293T or HeLa cells using Lipofectamine 2000 (Life Technologies) and puromycin was added one day later. Three days later, puromycin was removed, and cells were allowed to recover for three additional days prior to infection with different viruses.

For flow cytometric analyses, gene-edited 293T or HeLa cells were infected with WNV (MOI, 5), JEV (MOI, 50), DENV-2 (MOI, 3), YFV (MOI, 3), CHIKV, SINV, LACV, or VSV and analyzed 12 or 24 hours later depending on the individual virus. Cells were fixed with 1% paraformaldehyde (PFA, Electron Microscopy Sciences) diluted in PBS for 20 min at room temperature and permeabilized with Perm buffer (HBSS (Invitrogen), 10 mM HEPES, 0.1% (w/v) saponin (Sigma), and 0.025% NaN₃(Sigma)) for 10 min at room temperature. Cells then were rinsed one additional time with Perm buffer. Cells (5×10⁴) were transferred to a U-bottom plate and incubated for 1 h at 4° C. with 1 mg/ml of the following virus-specific or isotype control mouse antibodies. After washing, cells were incubated with an Alexa Fluor 647-conjugated goat anti-mouse or anti-human IgG (Invitrogen) for 1 h at 4° C. Cells were fixed in 1% PFA in PBS, processed on a FACS Array (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Validation also was performed by an infectious virus yield assay. Gene-edited 293T cells were infected with WNV or JEV (MOI, 0.01). Supernatants were harvested at specific times after infection and focus-forming assays were performed in 96-well plates as described previously⁴. Following infection, cell monolayers were overlaid with 100 ml per well of medium (1×DMEM, 4% FBS) containing 1% carboxymethylcellulose, and incubated for 16 to 18 hours at 37° C. with 5% CO₂. Cells were then fixed by adding 100 ml per well of 1% paraformaldehyde directly onto the overlay at room temperature for 40 minutes. Cells were washed twice with PBS, permeabilized (in 1×PBS, 0.1% saponin, and 0.1% BSA) for 20 minutes, and incubated with cross-reactive antibodies specific for WNV or JEV (mouse WNV E18⁵) E glycoprotein for 1 h at room temperature. After rinsing cells twice, cells were incubated with species-specific HRP-conjugated secondary antibodies (Sigma). After further washing, foci were developed by incubating in 50 ml/well of TrueBlue peroxidase substrate (KPL) for 10 min at room temperature, after which time cells were washed twice in water. Well images were captured using Immuno Capture software (Cell Technology Ltd.), and foci counted using BioSpot software (Cell Technology Ltd.).

Insect Cell Infections.

dsRNAs were generated as described⁶. To silence genes using RNAi, insect cells were passaged into serum-free media containing dsRNAs targeting the indicated genes. Cells were serum-starved for one hour, after which complete media was added and cells were incubated for 3 days. Cells were infected with WNV (Kunjin strain) at an MOI of 4 or DENV-2 (NGC strain) at an MOI of 1 for 30 h and then processed for microscopy with automated image analysis as described⁷.

siRNA Treatments in Human Cells.

Human U2OS cells were transfected with siRNAs against either control or SPCS2 for three days and infected with WNV (KUN) (MOI, 1) for 18 h, or SINV (MOI, 0.1) and CHIKV (MOI, 2) for 20 h, and processed for microscopy with automated image analysis as described⁷.

Gene Ontology Enrichment Analysis.

Enrichment analysis was performed on the 45 top candidates that were identified by CRISPR-Cas9 screening using Panther.

Replicon Transfection and Analysis.

The construction of WT and NS5 polymerase mutant (GDD→GVD) WNV replicons (lineage I, strain New York 1999) was based on a previously described cDNA launched molecular done system⁸. The backbone of this strategy, a plasmid containing a truncated WNV genome under the control of a CMV promoter (pWNV-backbone); was designed to be complemented via ligation of a structural gene DNA fragment; transfection of pWNV-backbone alone does not result in production of a self-replicating RNA molecule. Using overlap extension PCR and unique restriction endonuclease sites, pWNV-backbone was modified by the introduction of a fragment downstream of the CMV promoter encoding [5′UTR-cylization sequence of capsid-FMDV2a protease-signal sequence of E-NS1] to complement the [NS2→NS5-3′UTR] already present in the pWNV-backbone plasmid, generating the replicon plasmid pWNVI-rep. The reporter gene GFP then was cloned upstream of the FMDV2a protease sequence via a unique Mlul site to generate pWNVI-rep-GFP. The construction and organization of this WNVI replicon is analogous to a previously described lineage II WNV replicon (pWNVIIrep-GFP)⁹. Finally, QuikChange mutagenesis (Agilent Technologies) was used to delete the enhancer portion of the CMV immediate early enhancer/promoter, generating pWNVI-minCMV-rep-GFP, and to generate the GDD→GVD NS5 polymerase variant. Although the CMV enhancer/promoter combination commonly found in cloning vectors results in robust and constitutive expression, inclusion of only the minimal CMV promoter (no enhancer) results in low level expression¹⁰. As such, direct transfection of pWNVI-minCMV-rep-GFP results in a low GFP signal, which reflects translation of the RNA generated by DNA-dependent RNA translation. RNA polymerase-dependent replication of the WT (but not GVD mutant) replicon results in higher production of GFP over time. The eGFP is bracketed by the FMDV2a autocleavage site, and does not rely on host or viral proteases for processing. WT and NS5 GVD variants of pWNVI-minCMV-rep-GFP (200 ng) were transfected into 10 controller gene-edited 293T cells (96 well plates) using Lipofectamine 2000. At various times after transfection, cells were harvested, cooled to 4° C., stained sequentially with a biotinylated anti-9NS1¹¹(or biotin anti-chikungunya virus negative control MAb) and Alexa 647 conjugated streptavidin. In some samples, cells were fixed with 4% paraformaldehyde in PBS (10 min, room temperature) and permeabilized with 0.1% saponin (w/v). Cells were processed for two-color flow cytometry using a FACSScan (Becton Dickinson).

prM-E or NS1 Plasmid Transfection.

CRISPR-Cas9 293T cells were transfected with a pWN-AB plasmid expressing prM and E genes from the New York 1999 WNV strain¹²or an expression plasmid encoding the signal sequence of human CD33 linked to the full length WNV NS1 (gift of M. Edeling and D. Fremont, St Louis, Mo.) using FuGENE HD (Roche). Supernatants containing prM-E subviral particles (SVPs) were collected 24 and 48 h after transfection, filtered through a 0.2-μm filter, and stored aliquoted at −80° C. For the capture ELISA, Nunc MaxiSorp polystyrene 96-well plates were coated overnight at 4° C. with mouse E60 mAb⁵(5 μg/ml) in a pH 9.3 carbonate buffer. Plates were washed three times in enzyme-linked immunosorbent assay (ELISA) wash buffer (PBS with 0.02% Tween 20) and blocked for 1 h at 37° C. with ELISA block buffer (PBS, 2% bovine serum albumin, and 0.02% Tween 20). Supernatants from prM-E plasmid transfected cells were captured on plates coated with E60 for 90 min at room temperature (RT). Subsequently, plates were rinsed five times in wash buffer and then incubated with humanized anti-WNV E16 (1 μg/ml in block buffer) in triplicate for 1 h at RT. Plates were washed five times and then incubated with pre-absorbed biotinylated goat anti-human IgG antibody (1 mg/ml; Jackson Laboratories) for 1 h at RT in blocking buffer. Plates were washed again five times and then sequentially incubated with 2 μg/ml of horseradish peroxidase-conjugated streptavidin (Vector Laboratories) and tetramethylbenzidine substrate (Dako). The reaction was stopped with the addition of 2 N H₂SO₄to the medium, and emission (450 nm) was read using an iMark microplate reader (Bio-Rad).

Western Blotting.

CRISPR-Cas9 gene-edited 293T cells (10⁶) were lysed directly in 50 ml 5×SDS sample buffer. After heating samples (95° C., 5 min), 10 ml of the preparation was electrophoresed (10% SDS-PAGE) and proteins were transferred to nylon membranes using an iBlot2 Dry Blotting System (Life Technologies). Membranes were blocked with 5% non-fat dry powdered mile and then probed with antibodies. For studies with prM-E and NS1 transfected cells, membranes were probed with anti-E (human WNV E16), anti-NS1 (mouse 8-NS1), and anti-prM (human CR4293¹³), and the relevant secondary antibodies.

293T Cell Viability Assay.

A Vybrant MTT cell viability assay (Life Technologies) was used according to the manufacturer's instructions. Briefly, 10 ml of 12 mM MTT (4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) was added to 10⁵293 T cells (different gene-edited lines, with or without WNV infection) in 100 ml of phenol-red free medium. Cells were incubated for 4h at 37° C., at which time medium was removed and formazan crystals solubilized in 100 ml of DMSO were added for 10 min at 37° C. Liquid was analyzed for absorbance at 540 nm using a Synergy H1 Hybrid Plate Reader (Biotek).

HLA-A2 Surface Protein Expression.

Surface expression of HLA-A2 class I MHC molecules was evaluated using W6/32 (BioLegend), a mouse mAb that recognizes a common determinant on HLA-A, -B, and -C molecules. W6/32 (10 mg/ml) was incubated at 4° C. with individual CRISPR-Cas9 gene-edited cell lines. After incubation with an Alexa Fluor-488 conjugated goat anti-mouse secondary antibody, cells were processed by flow cytometry on a BD FACSArray (Becton Dickinson), and data was processed with FlowJo software (Tree Star, Inc).

Statistical Analysis.

Statistical significance was assigned when P values were <0.05 using GraphPad Prism Version 5.04 (La Jolla, Calif.). Viral antigen staining after expression of sgRNA was analyzed using a one-way ANOVA adjusting for repeated measures with a Dunnett's multiple comparison test or with a Mann-Whitney test depending on the number of comparison groups. Analysis of levels of E protein in the supernatant from CRISPR-Cas9 gene edited cells was analyzed by a one-way ANOVA. Analysis of siRNA in insect and human cells was performed using a Student's T-test.

REFERENCES FOR THE METHODS

1 Rose, P. P. et al. Natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts. Cell Host Microbe 10, 97-104, doi:10.1016/j.chom.2011.06.009 S1931-3128(11)00218-6 [pii] (2011).

2 Brien, J. D., Lazear, H. M. & Diamond, M. S. Propagation, quantification, detection, and storage of West Nile virus. Curr Protoc Microbiol 31, 15D 13 11-15D 13 18, doi:10.1002/9780471729259.mc15d03s31 (2013).

3 Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11, 783-784, doi:10.1038/nmeth.3047. nmeth.3047 [pii] (2014).

4 Fuchs, A., Pinto, A. K., Schwaeble, W. J. & Diamond, M. S. The lectin pathway of complement activation contributes to protection from West Nile virus infection. Virology 412, 101-109, doi:S0042-6822(11)00008-0 [pii]. 10.1016/j.viro1.2011.01.003 (2011).

5 Oliphant, T. et al. Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein. J Virol 80, 12149-12159 (2006).

6 Boutros, M. et al. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303, 832-835, doi:10.1126/science.1091266. 303/5659/832 [pii] (2004).

7 Hackett, B. A. et al. RNASEK is required for internalization of diverse acid-dependent viruses. Proc Natl Acad Sci USA 112, 7797-7802, doi:10.1073/pnas.1424098112. 1424098112 [pii] (2015).

8 Lin, T. Y. et al. A novel approach for the rapid mutagenesis and directed evolution of the structural genes of west nile virus. J Virol 86, 3501-3512, doi:JVI.06435-11 [pii]. 10.1128/JVI.06435-11 (2012).

9 Pierson, T. C. et al. A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus. Virology 346, 53-65 (2006).

10 Mishin, V. P., Cominelli, F. & Yamshchikov, V. F. A ‘minimal’ approach in design of flavivirus infectious DNA. Virus Res 81, 113-123, doi:S0168170201003719 [pii] (2001).

11 Chung, K. M. et al. Antibodies against West Nile virus non-structural (NS)-1 protein prevent lethal infection through Fc gamma receptor-dependent and independent mechanisms. J Virol 80, 1340-1351 (2006).

12 Vogt, M. R. et al. Human Monoclonal Antibodies Induced by Natural Infection Against West Nile Virus Neutralize at a Post-Attachment Step. J Virol 83, 6494-6507 (2009).

13 Throsby, M. et al. Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus. J Virol 80, 6982-6992 (2006).

14 Youn, S., Cho, H., Fremont, D. H. & Diamond, M. S. A short N-terminal peptide motif on flavivirus nonstructural protein NS1 modulates cellular targeting and immune recognition. J Virol 84, 9516-9532 (2010).

15 Melian, E. B. et al. NS1′ of flaviviruses in the Japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness. J Virol 84, 1641-1647 (2010).

TABLE 1

sgRNA showing enrichment

Screen 1—Technical repeat 1
Screen 2—Technical repeat 2
Screen 2

Un-
WNV
Fold-

Un-
WNV
Fold-

Un-
WNV
Fold-

gene
infected
infected
enrich
gene
infected
infected
enrich
gene
infected
infected
enrich

SPCS3
174
1424744
8188.18
SPCS3
235
2542792
10820.39
SEC63
158
406252
2571.22

RAP1GAP
57
377951
6630.72
ZYX
104
199532
1918.58
MARK3
244
559773
2294.15

SEC63
200
601326
3006.63
PARVG
41
54507
1329.44
SEL1L
237
211140
890.89

SLC35G5
18
32150
1786.11
SEC63
264
321061
1216.14
EMC4
378
259120
685.50

SEC61A1
36
34519
958.86
RAPSN
18
13253
736.28
SPCS1
114
52000
456.14

OR6T1
88
84108
955.77
SEC63
280
205583
734.23
RPL23A
65
25561
393.25

RRAS
255
192540
755.06
SLIT2
14
9254
661.00
CYP11B2
108
42208
390.81

RAB2B
105
74975
714.05
CYB5A
159
61636
387.65
SEL1L
195
68199
349.74

CCSER2
22
8368
380.36
TPTE2
33
12402
375.82
KLRK1
280
93843
335.15

SMAD4
276
90740
328.77
CYP1A2
34
11091
326.21
SERP1
98
23999
244.89

OSTC
221
72308
327.19
OR52E2
218
70413
323.00
FIG4
7
1644
234.86

ATOH7
81
19372
239.16
FBXL20
243
76780
315.97
FAM212A
281
60908
216.75

SPCS3
67
13809
206.10
SEC61B
58
17688
304.97
PCP2
50
10099
201.98

LRRC37A3
348
69652
200.15
AP1S3
150
40256
268.37
SLC26A9
116
22944
197.79

OST4
143
20481
143.22
CPSF3L
42
9501
226.21
CTSD
382
74112
194.01

FEZ2
56
7261
129.66
hsa-mir-4421
16
3084
192.75
ESPN
155
29154
188.09

SEC63
98
11879
121.21
STT3A
149
28383
190.49
USB1
308
49168
159.64

DAPL1
169
18351
108.59
OSTC
270
49888
184.77
FAM151B
481
72317
150.35

TBPL1
266
28757
108.11
ZKSCAN4
256
46519
181.71
TRERF1
296
43508
146.99

CHCHD7
116
12437
107.22
HSPA13
84
12914
153.74
PCDH9
263
37138
141.21

CLEC2D
119
12143
102.04
SEC61B
242
34312
141.79
FBXL20
196
27535
140.48

CHMP1B
84
7678
91.40
PCDHGA6
15
2099
139.93
GKN2
129
15933
123.51

PRH2
283
25498
90.10
ALDH16A1
51
7024
137.73
GORAB
502
60958
121.43

hsa-mir-6775
23
2024
88.00
ASB16
170
21989
129.35
UNKL
469
53570
114.22

SSR3
241
20343
84.41
KCTD13
40
5089
127.23
HSPA13
120
12706
105.88

SEC61B
70
5835
83.36
HIST1H2BI
146
16571
113.50
STT3A
275
27910
101.49

SEC63
297
23086
77.73
SCLT1
95
8745
92.05
ASPSCR1
8
763
95.38

EMC6
95
7224
76.04
ATG4D
95
8718
91.77
NCOA7
46
4190
91.09

GGTLC1
94
6416
68.26
APBB3
130
10341
79.55
C12orf50
220
16684
75.84

RBMX2
76
4410
58.03
NCAPH2
40
3006
75.15
KCP
26
1873
72.04

C11orf65
339
17813
52.55
ZSCAN23
154
10946
71.08
DNAJC25
199
13734
69.02

MORN2
714
37202
52.10
SEC63
109
7492
68.73
STT3A
205
13819
67.41

TMEM232
347
13916
40.10
DDIT4
139
8975
64.57
CD63
155
9623
62.08

TAAR8
270
10202
37.79
FAM111A
173
11151
64.46
RGMB
767
40968
53.41

GZMM
355
13053
36.77
C1orf110
226
14339
63.45
ADAMTS15
165
7303
44.26

PPP1R13B
132
4596
34.82
DNAJB1
242
14930
61.69
SSR3
476
20802
43.70

IL26
184
6211
33.76
KIDINS220
170
10456
61.51
PASK
75
3030
40.40

SERP1
216
6723
31.13
DCTN5
153
9297
60.76
USP7
180
7110
39.50

DNAJB14
118
3593
30.45
STT3A
96
5807
60.49
IFT20
240
8557
35.65

NDST1
156
4545
29.13
CYP17A1
45
2667
59.27
PNP
361
11816
32.73

hsa-mir-802
511
14197
27.78
hsa-mir-4442
9
513
57.00
PHF10
210
6679
31.80

RER1
206
5723
27.78
TMEM168
361
20449
56.65
RAB39A
258
7507
29.10

CES4A
108
2981
27.60
SERP1
204
10941
53.63
TRIM21
277
7957
28.73

COA5
68
1761
25.90
MED26
37
1943
52.51
SYT10
46
1278
27.78

SEC61B
225
5128
22.79
ABCC9
18
902
50.11
GGT1
74
2000
27.03

PANX2
136
2880
21.18
TBK1
95
4706
49.54
DDR2
235
6309
26.85

RASA1
120
2527
21.06
OR5L2
184
8853
48.11
ANKIB1
130
3145
24.19

MOGS
364
7590
20.85
AP4S1
136
6491
47.73
ESF1
62
1457
23.50

EXO1
148
2931
19.80
FNBP4
57
2587
45.39
NT5C2
86
1960
22.79

ANTXR2
148
2852
19.27
ATP6AP1L
103
4605
44.71
BCAS1
142
3200
22.54

VPS4B
49
935
19.08
TRIM72
26
1143
43.96
BOP1
76
1711
22.51

hsa-mir-18a
582
11000
18.90
THRB
143
6250
43.71
ZNF75D
165
3601
21.82

NPFF
85
1480
17.41
hsa-mir-548as
41
1766
43.07
UBE2J2
196
4157
21.21

hsa-mir-4647
147
2495
16.97
SYNE2
189
7808
41.31
MGLL
19
395
20.79

MMGT1
154
2460
15.97
MKL2
570
23450
41.14
PRDM13
110
2278
20.71

RGMA
149
2339
15.70
hsa-mir-506
550
22568
41.03
NASP
93
1760
18.92

PLCL1
185
2878
15.56
XIRP1
179
7183
40.13
AP2B1
382
7205
18.86

GRIP1
19
295
15.53
PTK2
714
28185
39.47
SSX1
262
4750
18.13

EMC2
29
424
14.62
VPRBP
94
3640
38.72
OR2T2
575
10281
17.88

TG
83
1107
13.34
SHC1
632
23483
37.16
TECPR1
76
1345
17.70

TRAF5
186
2201
11.83
hsa-mir-4666b
126
4538
36.02
HIST1H2AK
141
2444
17.33

ASB5
371
4082
11.00
hsa-mir-5696
186
6527
35.09
BEND5
592
10144
17.14

SERP1
155
1682
10.85
UPK1B
20
677
33.85
EMC3
203
3365
16.58

FES
278
3012
10.83
DKKL1
41
1382
33.71
ARL6IP5
33
507
15.36

USP20
3
30
10.00
DDC
152
5116
33.66
TSPYL5
85
1215
14.29

HPRT1
104
967
9.30
KRTAP13-1
39
1288
33.03
OR10W1
71
1007
14.18

MUM1L1
362
3204
8.85
ME2
74
2408
32.54
FAM120A
222
2919
13.15

hsa-mir-132
168
1415
8.42
BHMT2
247
7889
31.94
DGKK
89
1115
12.53

hsa-mir-4738
37
310
8.38
hsa-mir-758
183
5636
30.80
MRPL52
223
2783
12.48

CC2D2B
158
1312
8.30
GLRB
231
7077
30.64
SFMBT2
283
3516
12.42

SYNDIG1L
117
956
8.17
TMEM170B
29
855
29.48
MCCD1
656
7964
12.14

MMGT1
41
315
7.68
MTM1
200
5857
29.29
OR4D2
175
2112
12.07

DYNC1LI2
118
899
7.62
SLC35B4
195
5710
29.28
RPS25
427
5084
11.91

FAM32A
704
4936
7.01
PQLC1
293
8496
29.00
SCARF1
593
6977
11.77

FAM73B
71
487
6.86
RLN1
205
5884
28.70
SCCPDH
402
4717
11.73

STT3A
186
1227
6.60
KIAA1239
135
3837
28.42
KLF3
234
2726
11.65

PVRL3
164
1077
6.57
PPP1CC
122
3463
28.39
ZC2HC1A
567
6394
11.28

EREG
310
2004
6.46
OR13H1
208
5891
28.32
KRTAP5-6
538
5769
10.72

SCGB2B2
126
811
6.44
FHIT
320
8861
27.69
ALDOB
144
1500
10.42

NPFFR2
125
767
6.14
GPR63
38
1032
27.16
EMC4
320
3262
10.19

FAM209B
267
1637
6.13
MC3R
308
7941
25.78
HNRNPF
100
1000
10.00

SLC10A5
73
437
5.99
CDH13
47
1188
25.28
FAM210A
70
666
9.51

BRD3
276
1605
5.82
HBG1
184
4633
25.18
GDPD1
171
1613
9.43

GUCA1C
134
774
5.78
BEGAIN
109
2724
24.99
CCR5
156
1437
9.21

TMEM134
29
167
5.76
OST4
162
3981
24.57
C20orf85
20
182
9.10

hsa-mir-644a
105
603
5.74
RBM25
287
7018
24.45
SMARCB1
242
2162
8.93

LY6H
135
770
5.70
DEF6
105
2555
24.33
KLHL34
241
2061
8.55

MESDC1
19
104
5.47
STXBP5L
7
169
24.14
PQLC2
367
3061
8.34

DHRS13
225
1210
5.38
hsa-mir-4723
113
2657
23.51
LPCAT2
240
1925
8.02

hsa-mir-521-2
222
1146
5.16
ZNF264
95
2221
23.38
ZNF347
112
892
7.96

ASB9
109
532
4.88
ANO10
176
3831
21.77
NABP1
132
1043
7.90

PREX1
171
805
4.71
SVEP1
182
3539
19.45
GPR25
83
646
7.78

MFSD3
308
1433
4.65
ETS1
94
1827
19.44
FRS2
786
5987
7.62

ENPP5
103
455
4.42
KLHL7
14
268
19.14
RSPO2
294
2209
7.51

ANXA2
517
2263
4.38
LOC100130451
28
533
19.04
ARL5A
180
1349
7.49

PRRT3
66
282
4.27
AKAP5
295
5604
19.00
TCAIM
287
2120
7.39

EIF1
112
471
4.21
BUB3
45
843
18.73
GBP4
396
2893
7.31

C12orf75
34
142
4.18
hsa-mir-1-1
81
1517
18.73
SEC63
115
827
7.19

AFTPH
68
280
4.12
NAA25
156
2896
18.56
PRAF2
96
688
7.17

POC1B
466
1900
4.08
BEAN1
81
1502
18.54
CROCC
208
1412
6.79

hsa-mir-4780
272
1067
3.92
SULT4A1
77
1411
18.32
RBX1
206
1393
6.76

SEC61B
214
837
3.91
LEAP2
119
2179
18.31
HDLBP
130
878
6.75

AR
210
809
3.85
C17orf72
94
1677
17.84
ADAMTS14
276
1825
6.61

CAV2
173
655
3.79
MS4A6E
375
6333
16.89
EXT2
129
820
6.36

PLEK2
89
328
3.69
PTOV1
523
8787
16.80
CCDC178
658
4137
6.29

PRELID2
75
274
3.65
FBXO33
62
1019
16.44
PPRC1
303
1886
6.22

NCKIPSD
177
646
3.65
OTUD6B
300
4832
16.11
FBXL14
87
536
6.16

FCRLA
293
1013
3.46
HIST1H2AB
48
767
15.98
VSIG10L
117
716
6.12

CASP8
175
594
3.39
GUCY2F
368
5713
15.52
DMXL2
327
1989
6.08

SAYSD1
201
680
3.38
C20orf201
40
613
15.33
BCAR3
133
808
6.08

ABCA10
124
415
3.35
DIO3
250
3783
15.13
PSMG3
400
2414
6.04

LYPLA2
148
495
3.34
PIGL
487
7186
14.76
PEX11B
296
1773
5.99

PHEX
306
1002
3.27
DNAJC24
322
4650
14.44
TCERG1
267
1587
5.94

ZNF564
264
855
3.24
CLDND1
79
1135
14.37
C9orf41
224
1319
5.89

NOA1
51
161
3.16
MMGT1
63
905
14.37
USH1G
209
1228
5.88

SBDS
143
447
3.13
OSTC
178
2556
14.36
SFT2D3
161
940
5.84

LRRC40
126
386
3.06
STAB2
185
2594
14.02
MYO3B
183
1067
5.83

BRI3BP
122
373
3.06
CLVS2
46
644
14.00
MEIG1
585
3388
5.79

MPND
767
2338
3.05
GALNT1
29
404
13.93
PDCD6
119
680
5.71

RLF
317
961
3.03
SAMD9
462
6398
13.85
BTN2A2
560
3158
5.64

hsa-mir-3166
233
699
3.00
INS
200
2740
13.70
NVL
70
389
5.56

TMEM203
203
594
2.93
HIST1H2AA
49
667
13.61
C2orf40
85
468
5.51

C4orf48
144
413
2.87
SERP1
157
2133
13.59
ADIPOR2
262
1430
5.46

CNOT4
178
509
2.86
P2RY14
83
1100
13.25
WBSCR28
232
1254
5.41

THAP8
178
506
2.84
VIM
195
2534
12.99
AKAP13
411
2217
5.39

ERMP1
272
770
2.83
CTPS1
103
1331
12.92
ARF1
184
989
5.38

MCCD1
152
428
2.82
CST3
32
412
12.88
HIST1H2BL
183
954
5.21

HSPA13
103
285
2.77
SLC25A14
37
474
12.81
LCN6
221
1146
5.19

hsa-mir-5696
334
867
2.60
ZFP36L1
216
2761
12.78
FGR
116
594
5.12

hsa-mir-1289-2
101
254
2.51
SLU7
186
2375
12.77
EIF4A3
69
352
5.10

ELANE
356
879
2.47
GYPB
394
5029
12.76
NT5DC3
271
1372
5.06

FAM210A
192
466
2.43
PSORS1C1
438
5585
12.75
MYEOV2
94
474
5.04

HSPA13
137
331
2.42
STX8
64
810
12.66
RBP5
28
139
4.96

BRIP1
456
1099
2.41
STT3A
135
1704
12.62
LIN7A
484
2357
4.87

FAM181B
284
683
2.40
RAP1GAP2
99
1247
12.60
DOK1
388
1886
4.86

XKRX
165
394
2.39
GREM2
200
2508
12.54
RUNDC3B
76
364
4.79

MRPS7
43
100
2.33
FGFR2
187
2330
12.46
NLRP2
95
442
4.65

FBP1
100
229
2.29
SEC11A
474
5892
12.43
SLC25A42
237
1101
4.65

SDPR
618
1414
2.29
hsa-mir-1976
176
2159
12.27
NARF
589
2680
4.55

PTPRU
320
682
2.13
SNRNP200
97
1189
12.26
ASCC2
353
1604
4.54

STT3A
127
266
2.09
hsa-mir-4683
297
3554
11.97
EIF4ENIF1
314
1416
4.51

OR2G6
233
479
2.06
RAB37
128
1527
11.93
EMC3
490
2188
4.47

YLPM1
132
269
2.04
NTRK3
61
727
11.92
GNG8
275
1212
4.41

SPCS1
14
28
2.00
SHROOM2
271
3198
11.80
MCM8
185
812
4.39

CLCC1
38
75
1.97
ATF2
214
2480
11.59
AKIP1
132
577
4.37

RPL5
14
27
1.93
RGS22
106
1227
11.58
FAM72D
396
1676
4.23

CLCC1
286
547
1.91
LSMEM2
84
957
11.39
ABCC4
215
896
4.17

MARVELD2
44
84
1.91
KLHL28
56
626
11.18
HTR1E
242
1998
7.06

CD163L1
399
761
1.91
ZNF620
67
747
11.15
HIF3A
114
468
4.11

APCS
112
213
1.90
AK9
126
1386
11.00
FBXO9
565
2309
4.09

hsa-mir-323a
476
874
1.84
MICALL1
128
1407
10.99
FUT2
610
2399
3.93

ACSM4
158
284
1.80
SENP5
227
2486
10.95
HIST2H2BF
273
1058
3.88

GPSM3
190
340
1.79
SHQ1
82
865
10.55
TMEM253
42
162
3.86

PRR14L
302
535
1.77
C4orf21
322
3348
10.40
INPP5B
249
957
3.84

XYLT1
161
284
1.76
POLR2A
666
6877
10.33
C16orf97
485
1824
3.76

ALDH1A2
119
209
1.76
DFNB31
354
3619
10.22
AK8
99
369
3.73

hsa-mir-4804
126
217
1.72
BEX5
9
90
10.00
MAP9
229
848
3.70

hsa-mir-9-3
124
211
1.70
AMOTL2
260
2588
9.95
MICU3
117
432
3.69

hsa-mir-137
20
34
1.70
hsa-mir-103a-1
284
2773
9.76
CINP
225
829
3.68

SERPINI2
86
146
1.70
MS4A4A
97
918
9.46
CYP2B6
174
633
3.64

BAG5
225
366
1.63
SP5
357
3342
9.36
CCDC179
448
1625
3.63

GSC
153
248
1.62
hsa-mir-4295
57
532
9.33
TMEM200B
64
227
3.55

KIF2B
48
76
1.58
hsa-mir-146b
172
1587
9.23
CLOCK
143
503
3.52

SAMD5
67
105
1.57
NFIB
134
1233
9.20
SNAPC1
111
388
3.50

E2F6
206
314
1.52
MAN1B1
78
717
9.19
DCHS1
226
788
3.49

ZNF18
313
471
1.50
hsa-mir-4440
48
440
9.17
HAP1
108
373
3.45

LIMK2
448
665
1.48
hsa-mir-196a-2
285
2603
9.13
PDE11A
468
1612
3.44

ACAD10
77
114
1.48
DTX3
439
3966
9.03
PNO1
164
558
3.40

GIF
385
557
1.45
NEGR1
78
698
8.95
CLEC11A
414
1408
3.40

FAM20B
121
175
1.45
SEC62
178
1573
8.84
RAB11FIP5
276
935
3.39

C22orf24
181
258
1.43
RD3
236
2084
8.83
GSE1
154
506
3.29

C1orf168
170
242
1.42
AP1B1
141
1243
8.82
GCC1
133
436
3.28

PSG8
217
306
1.41
GGT6
64
558
8.72
RABEP2
127
407
3.20

TECRL
105
147
1.40
hsa-mir-1825
93
805
8.66
TMEM14A
254
805
3.17

EMC6
95
133
1.40
SMARCE1
183
1569
8.57
EPAS1
328
1028
3.13

hsa-mir-759
96
133
1.39
METTL20
174
1485
8.53
SPINK4
692
2125
3.07

PLA2G10
283
385
1.36
EFCAB3
292
2490
8.53
STAU2
328
1007
3.07

hsa-mir-140
272
370
1.36
SET
72
611
8.49
PDE10A
144
441
3.06

ZFYVE16
234
316
1.35
NDUFAF1
116
974
8.40
TMEM100
303
902
2.98

XDH
89
119
1.34
OR5J2
187
1565
8.37
IFRD2
127
375
2.95

SENP8
61
81
1.33
ZCCHC10
165
1380
8.36
SHC1
150
440
2.93

INSIG2
255
338
1.33
HAUS2
230
1921
8.35
FADS3
42
123
2.93

S100A11
156
204
1.31
BRWD3
125
1035
8.28
APCDD1L
401
1167
2.91

CDKN2C
452
583
1.29
PLCH1
119
985
8.28
WFDC6
88
253
2.88

PXDC1
324
415
1.28
EP400
149
1224
8.21
C1QL1
277
795
2.87

VWA3B
209
266
1.27
FYN
285
2340
8.21
EPHA3
154
428
2.78

TTC18
111
138
1.24
hsa-mir-6131
171
1387
8.11
SLITRK4
305
847
2.78

NTM
873
1080
1.24
SMIM17
117
942
8.05
BRD3
228
626
2.75

SYVN1
192
226
1.18
FRMD1
98
781
7.97
GPIHBP1
76
206
2.71

PHTF2
215
253
1.18
IRF7
154
1221
7.93
MRPL2
122
327
2.68

GLIPR1L2
97
112
1.15
NCMAP
123
974
7.92
COX19
154
411
2.67

SDSL
69
79
1.14
hsa-mir-539
73
577
7.90
OAS3
143
381
2.66

TIMP4
113
127
1.12
LYN
253
1988
7.86
STT3A
127
326
2.57

EMC3
80
89
1.11
MBNL3
91
708
7.78
KDM5D
877
2220
2.53

IL19
117
129
1.10
FFAR1
126
969
7.69
TMCO4
261
642
2.46

GDF1
148
163
1.10
TBCEL
347
2662
7.67
PFKM
156
381
2.44

ZNF653
249
274
1.10
DST
350
2670
7.63
ACIN1
178
433
2.43

PRSS45
54
59
1.09
TSSK6
135
1019
7.55
SMTN
152
369
2.43

EMC6
141
153
1.09
PCGF1
142
1070
7.54
HTR4
206
492
2.39

ZNF670
331
354
1.07
AK3
534
3996
7.48
CHIC2
262
624
2.38

FUCA2
385
410
1.06
VEZT
122
894
7.33
ZCCHC24
16
38
2.38

ADAP1
109
113
1.04
ADAM20
51
363
7.12
CTSV
71
168
2.37

OTUD4
67
69
1.03
ANK2
389
2738
7.04
SLC20A1
279
643
2.30

KCTD16
468
481
1.03
RAB30
114
801
7.03
ABCC1
285
654
2.29

WNT5B
208
212
1.02
TMC7
411
2865
6.97
SLC8A2
219
500
2.28

FXYD3
82
82
1.00
OR4D5
28
194
6.93
DGKZ
378
863
2.28

SRP72
61
61
1.00
STAT4
241
1658
6.88
HOXD8
225
507
2.25

LYNX1
192
185
0.96
CAMSAP1
269
1842
6.85
HAS2
141
316
2.24

MTMR2
394
379
0.96
TDRD7
292
1993
6.83
CCDC8
283
630
2.23

hsa-mir-891b
365
348
0.95
hsa-mir-500a
454
3074
6.77
KIF25
289
638
2.21

MED10
77
72
0.94
CDRT1
86
581
6.76
TRANK1
557
1223
2.20

BPIFB4
88
82
0.93
RNF112
233
1565
6.72
LDLRAD4
590
1277
2.16

ARHGAP18
169
157
0.93
COPS5
348
2336
6.71
ALB
153
330
2.16

GABRG3
156
144
0.92
NOC4L
83
539
6.49
CARNS1
110
236
2.15

hsa-mir-302e
184
168
0.91
ATL3
267
1690
6.33
ZIC2
331
705
2.13

RHEB
211
189
0.90
ZNF844
107
676
6.32
RBM25
98
207
2.11

SLC28A3
425
378
0.89
CEACAM21
184
1158
6.29
NMNAT2
263
542
2.06

DCTD
188
166
0.88
RAPGEF4
88
545
6.19
OR51M1
259
532
2.05

GSTT1
126
110
0.87
DCST2
126
776
6.16
DENND3
143
291
2.03

INPP4A
228
196
0.86
EHBP1
137
840
6.13
C9orf57
176
357
2.03

KIF4B
60
50
0.83
INPP5F
91
551
6.05
CCL26
201
407
2.02

KRTAP5-5
243
200
0.82
hsa-mir-1260a
140
833
5.95
COX6C
234
470
2.01

GLG1
176
144
0.82
EPHX3
283
1669
5.90
ITFG3
180
358
1.99

hsa-mir-521-1
436
356
0.82
ZNF488
115
674
5.86
PAX5
262
520
1.98

TMEM151B
304
243
0.80
HSPA12B
88
515
5.85
COQ10A
190
377
1.98

HBD
389
310
0.80
TRIM69
119
687
5.77
GPR161
293
581
1.98

ART1
87
68
0.78
SLITRK6
318
1820
5.72
KIAA0930
213
414
1.94

DHX35
31
24
0.77
CAPNS1
220
1249
5.68
MAP2K5
402
779
1.94

hsa-mir-4803
75
58
0.77
hsa-mir-31
115
643
5.59
C11orf65
218
422
1.94

DENND3
121
92
0.76
HBM
264
1475
5.59
GFRA4
804
1550
1.93

CYP2F1
175
132
0.75
WDR96
70
390
5.57
PPA1
142
273
1.92

ELP4
290
217
0.75
SEMA4A
65
362
5.57
LOC100287177
243
465
1.91

GTF2B
223
166
0.74
hsa-mir-891b
427
2376
5.56
EML6
223
423
1.90

TNFAIP8L2
166
123
0.74
MAGEA1
100
552
5.52
FAM214B
241
447
1.85

YPEL4
403
293
0.73
CCDC25
403
2201
5.46
ACSS1
189
344
1.82

HES7
29
21
0.72
SLC30A10
147
796
5.41
PVRL3
195
349
1.79

EOMES
46
33
0.72
ZC3H4
126
672
5.33
PCDHA13
146
261
1.79

GNPDA1
380
266
0.70
OR56B1
132
698
5.29
CHM
56
100
1.79

BCHE
48
33
0.69
hsa-mir-7515
7
37
5.29
TADA2A
68
121
1.78

ASPM
382
260
0.68
TRAF1
149
787
5.28
VPS37D
157
279
1.78

GPR68
73
49
0.67
ERVFRD-1
255
1346
5.28
LTA4H
526
934
1.78

TMEM72
229
153
0.67
CD1D
171
877
5.13
OR51E1
307
545
1.78

EMC3
696
450
0.65
C1orf50
12
61
5.08
DTHD1
88
156
1.77

ZYX
129
83
0.64
ERVV-1
65
329
5.06
BLVRB
214
374
1.75

XPO4
266
167
0.63
SLC26A6
185
934
5.05
KLB
285
493
1.73

OR1G1
74
46
0.62
SOD3
198
989
4.99
CNTROB
327
564
1.72

RAVER1
274
170
0.62
PER1
253
1236
4.89
PFN4
449
766
1.71

SETD9
274
170
0.62
UBL7
240
1170
4.88
ST8SIA6
500
845
1.69

TMEM165
420
259
0.62
C17orf85
222
1070
4.82
WNK3
241
405
1.68

TBATA
69
42
0.61
PLCXD3
166
798
4.81
ZNF543
234
387
1.65

DENND5B
267
162
0.61
FAM109A
228
1095
4.80
SH3GL1
293
482
1.65

ERLEC1
237
141
0.59
DCST1
151
721
4.77
CPXM2
748
1228
1.64

SPTA1
341
202
0.59
SERPINC1
236
1124
4.76
ZNF706
523
850
1.63

CHIC2
491
290
0.59
SOS2
140
664
4.74
SFRP2
93
148
1.59

INTS2
78
46
0.59
WBSCR16
229
1083
4.73
COX7A1
335
532
1.59

OR5L1
212
125
0.59
C10orf25
121
572
4.73
NR3C1
225
356
1.58

NAT10
232
136
0.59
SEC24B
479
2262
4.72
RCBTB2
201
318
1.58

FAM19A4
95
53
0.56
ARHGAP8
199
930
4.67
TNRC6C
84
132
1.57

GLIS3
85
47
0.55
TRPV6
154
715
4.64
LACTB2
331
514
1.55

TNNI2
152
84
0.55
EFHC2
197
914
4.64
NUCB1
667
1035
1.55

TTBK2
241
132
0.55
CPSF6
95
428
4.51
YIPF4
200
308
1.54

KRT8
165
90
0.55
CCNL1
16
71
4.44
HSPA12B
41
63
1.54

hsa-mir-1302-5
410
223
0.54
hsa-mir-4675
193
855
4.43
OTOP2
117
179
1.53

PRKCG
70
38
0.54
TTC16
138
600
4.35
OR8D1
142
217
1.53

GCG
405
218
0.54
UBE2G2
376
1616
4.30
GLI4
207
310
1.50

HIST1H3F
73
39
0.53
IDO1
190
795
4.18
ZNF490
147
220
1.50

hsa-mir-578
86
45
0.52
PCMTD1
346
1446
4.18
AUP1
59
88
1.49

FNDC3B
267
138
0.52
AUP1
259
1080
4.17
PDE7B
1039
1542
1.48

CDKN2D
327
169
0.52
EPOR
61
253
4.15
MAN1B1
29
43
1.48

hsa-mir-96
273
140
0.51
WDFY2
237
982
4.14
OR2T4
253
374
1.48

DDA1
181
92
0.51
NUP107
199
824
4.14
PITPNC1
143
211
1.48

FAM217A
28
14
0.50
SLC39A2
201
832
4.14
CCDC47
120
177
1.48

CCDC167
8
4
0.50
CTIF
157
637
4.06
C1orf112
222
327
1.47

CNBD1
155
77
0.50
CRISP2
30
121
4.03
FUCA1
797
1169
1.47

FAM180A
222
110
0.50
OR5P3
648
2605
4.02
ALDH4A1
172
245
1.42

STEAP4
145
69
0.48
C1QL3
155
621
4.01
ODF3L2
224
315
1.41

CCR6
358
169
0.47
LAMA1
299
1195
4.00
IFNGR2
566
794
1.40

OLIG2
114
53
0.46
hsa-mir-548ai
111
442
3.98
CHCHD4
233
325
1.39

VAPA
91
42
0.46
SPANXF1
896
3526
3.94
SLC35D1
236
329
1.39

C15orf62
128
59
0.46
BRWD1
107
419
3.92
FCRLB
184
252
1.37

ZBTB5
204
94
0.46
hsa-mir-4660
130
505
3.88
HIVEP2
46
63
1.37

AMOT
163
75
0.46
GK2
244
943
3.86
TTC29
336
455
1.35

EMC2
64
29
0.45
WWC3
186
709
3.81
FAM26D
89
119
1.34

PARVG
31
14
0.45
TNFAIP8
73
277
3.79
PKN1
122
163
1.34

hsa-mir-130b
60
27
0.45
RASSF9
159
603
3.79
DEDD2
231
308
1.33

CCDC106
147
66
0.45
PCDHB14
126
474
3.76
C12orf54
879
1164
1.32

DCAF4L2
148
66
0.45
ABLIM3
355
1333
3.75
RBBP6
38
50
1.32

hsa-mir-2681
275
122
0.44
FAM71F2
520
1936
3.72
P2RY12
121
157
1.30

IL22RA1
46
20
0.43
HDAC10
136
505
3.71
HBB
169
219
1.30

KIAA1217
148
64
0.43
POU5F2
36
130
3.61
NADSYN1
62
80
1.29

hsa-mir-4286
132
57
0.43
DOCK2
677
2388
3.53
TACO1
273
345
1.26

RBMX
106
44
0.42
TPR
78
275
3.53
TYW1
587
738
1.26

LUZP2
122
50
0.41
TAS2R39
151
529
3.50
CTSF
105
131
1.25

NXPH4
108
44
0.41
C15orf39
120
417
3.48
JRKL
206
256
1.24

KIAA0907
370
149
0.40
TAS2R46
696
2409
3.46
BTBD16
100
124
1.24

SLC4A5
243
97
0.40
ITGA7
341
1176
3.45
PDE8A
192
238
1.24

ZNF93
254
100
0.39
C4orf26
175
594
3.39
XPO7
682
844
1.24

AP4M1
379
149
0.39
hsa-mir-6836
84
285
3.39
ZNF560
19
23
1.21

GCSAML
51
20
0.39
SYT15
55
186
3.38
CBLN4
232
277
1.19

VASH2
291
111
0.38
hsa-mir-1208
124
413
3.33
POP7
53
63
1.19

HAP1
250
94
0.38
hsa-mir-4692
70
233
3.33
MPZL3
95
112
1.18

TRNT1
104
39
0.38
hsa-mir-4792
43
143
3.33
SDR9C7
175
206
1.18

hsa-mir-378f
235
88
0.37
OR51Q1
31
103
3.32
OR14J1
501
587
1.17

SOX30
206
77
0.37
HGF
284
939
3.31
ZNF407
120
140
1.17

GUSB
163
60
0.37
OTUB1
376
1232
3.28
TMEM194B
65
75
1.15

MAPRE3
300
110
0.37
UBFD1
282
914
3.24
OR10R2
549
633
1.15

STAG3
267
97
0.36
SOSTDC1
235
727
3.09
SHISA2
300
345
1.15

R3HDM2
205
74
0.36
UBL4A
692
2139
3.09
CENPC1
327
374
1.14

KRTAP9-6
347
124
0.36
DEXI
327
1009
3.09
ZNF529
154
174
1.13

hsa-mir-2861
79
28
0.35
FLVCR1
45
138
3.07
KRT6B
177
199
1.12

SNRPN
289
102
0.35
HMGB2
185
560
3.03
FAM151B
280
314
1.12

LEO1
153
54
0.35
hsa-mir-3187
383
1151
3.01
HPDL
203
226
1.11

IMPDH2
88
31
0.35
COL25A1
326
978
3.00
LSM3
54
60
1.11

hsa-mir-200b
216
76
0.35
SNX7
101
303
3.00
CSF2
224
248
1.11

HLA-DRB5
172
60
0.35
GPR61
55
165
3.00
PCDH11Y
182
201
1.10

hsa-mir-556
73
25
0.34
PSME1
167
499
2.99
UTS2
195
215
1.10

SCML2
280
95
0.34
GZMB
403
1204
2.99
NFATC2
427
470
1.10

GABRA3
118
40
0.34
FAM3D
66
196
2.97
ADCYAP1R1
191
210
1.10

IL9
210
70
0.33
ZNF329
148
439
2.97
SLAMF9
119
130
1.09

VSNL1
46
15
0.33
EXOC7
52
151
2.90
SETD7
273
297
1.09

GNAL
120
39
0.33
SGK3
123
355
2.89
MXRA7
585
636
1.09

TXN2
238
77
0.32
hsa-mir-8057
34
98
2.88
SLC14A1
627
677
1.08

C5orf20
598
192
0.32
MNS1
53
152
2.87
MSC
293
313
1.07

RSAD2
202
63
0.31
MANBA
339
972
2.87
LRRC29
91
97
1.07

ALOXE3
209
65
0.31
PLEKHO2
133
377
2.83
TEK
92
98
1.07

FAM24A
164
51
0.31
MLL5
157
445
2.83
SOST
71
72
1.01

SYPL1
342
106
0.31
OR10X1
513
1449
2.82
SCGB3A2
443
446
1.01

RBP5
459
141
0.31
RILPL2
45
127
2.82
TAS2R3
641
643
1.00

ARSB
212
65
0.31
BBS10
44
124
2.82
SLC24A4
434
435
1.00

TNFSF12
92
28
0.30
WFDC8
258
726
2.81
MYOZ3
94
94
1.00

hsa-mir-4733
211
64
0.30
MRPL46
146
409
2.80
APLNR
5
5
1.00

EFCAB4B
63
19
0.30
ZNF597
584
1633
2.80
CASP3
750
749
1.00

hsa-mir-107
355
105
0.30
AREG
166
463
2.79
FAM204A
76
75
0.99

FZD1
44
13
0.30
FSCB
272
758
2.79
NUCKS1
478
468
0.98

RILPL1
184
54
0.29
CDKN1B
269
741
2.75
PSAP
270
263
0.97

COG4
103
30
0.29
RAP1GAP
43
118
2.74
BARHL1
152
147
0.97

SLC24A2
171
49
0.29
KCNN3
131
357
2.73
RESP18
449
430
0.96

OR5H15
161
46
0.29
AEBP2
139
377
2.71
MMGT1
298
285
0.96

ZFP69B
146
41
0.28
VSX1
561
1516
2.70
CCDC74A
438
414
0.95

IBTK
364
102
0.28
ERP29
219
590
2.69
DUSP8
88
83
0.94

CXCR1
204
56
0.27
OMD
322
865
2.69
PPP2R5E
193
181
0.94

hsa-mir-1182
51
14
0.27
AICDA
151
404
2.68
ORC5
484
445
0.92

TMEM213
128
35
0.27
ANTXR1
18
48
2.67
CCDC88C
147
135
0.92

PRSS8
549
150
0.27
KPNA2
112
298
2.66
CD19
356
321
0.90

MAP2K7
112
30
0.27
PKP4
78
205
2.63
FAM175B
182
164
0.90

hsa-mir-492
273
73
0.27
TUFM
247
643
2.60
ZSWIM2
421
375
0.89

FLVCR2
185
49
0.26
GJA3
303
788
2.60
GLIPR2
166
147
0.89

FSCN2
68
18
0.26
FBX018
235
611
2.60
ERAL1
224
198
0.88

HCK
87
23
0.26
TMIGD2
88
228
2.59
CLCN7
445
390
0.88

CLLU1OS
250
66
0.26
USP16
361
935
2.59
COL4A6
102
89
0.87

BCKDHA
163
43
0.26
HP1BP3
169
435
2.57
PRPH2
112
97
0.87

ZNF770
133
35
0.26
SRMS
168
431
2.57
CTSV
261
226
0.87

POLR1E
76
20
0.26
C17orf112
132
337
2.55
PCNX
94
81
0.86

SLC8A1
88
23
0.26
TSFM
11
28
2.55
FOPNL
114
98
0.86

DCAF6
174
45
0.26
NQO1
288
733
2.55
ZNF581
139
119
0.86

KIAA1024L
396
102
0.26
TRAPPC5
162
412
2.54
CD3G
468
399
0.85

hsa-mir-6778
231
59
0.26
DENR
29
73
2.52
EMC6
161
137
0.85

FGL2
149
38
0.26
hsa-mir-1277
308
774
2.51
INADL
92
78
0.85

EBP
564
142
0.25
CDCA7L
222
553
2.49
TIE1
332
281
0.85

FAM153A
864
217
0.25
SLC2A14
110
273
2.48
CCDC134
457
385
0.84

METAP1D
52
13
0.25
hsa-mir-645
102
253
2.48
NAA35
139
117
0.84

PRB3
279
69
0.25
TSHR
196
486
2.48
RWDD2B
131
110
0.84

UBAC2
267
65
0.24
EMC6
175
433
2.47
RNF215
710
596
0.84

PCDHA9
153
37
0.24
COMTD1
434
1063
2.45
ZIC2
143
120
0.84

ADRM1
397
96
0.24
RAP1GAP
273
667
2.44
ZNF79
1043
864
0.83

SVOPL
406
98
0.24
CD300LF
199
482
2.42
SNX16
290
239
0.82

MTERFD3
531
128
0.24
MPPED1
186
447
2.40
MSX2
78
64
0.82

MAP9
241
57
0.24
FAM47B
40
96
2.40
GPBP1
122
99
0.81

FMR1NB
102
24
0.24
SMARCA2
147
352
2.39
MLXIP
163
132
0.81

ABHD14B
64
15
0.23
ADH5
190
454
2.39
MINOS1
227
183
0.81

TAS2R19
444
104
0.23
SLC10A7
522
1238
2.37
CCDC13
313
251
0.80

PPA1
284
66
0.23
ZFP91
127
298
2.35
SRPK3
349
279
0.80

BRD2
194
45
0.23
LOC100506388
441
1033
2.34
SST
357
285
0.80

ENTPD7
441
101
0.23
KIF15
199
466
2.34
ZNF786
550
435
0.79

hsa-mir-2116
206
47
0.23
FBX048
538
1256
2.33
EEF1B2
42
33
0.79

UBXN11
197
44
0.22
PTGIS
160
373
2.33
MMP21
606
472
0.78

hsa-mir-758
701
156
0.22
PSG5
381
886
2.33
ARHGEF5
163
126
0.77

hsa-mir-1289-1
207
46
0.22
FN3KRP
245
565
2.31
CD33
163
126
0.77

RHEBL1
9
2
0.22
SPINT2
107
244
2.28
ZNF491
245
189
0.77

OR5H2
221
49
0.22
MTUS2
194
442
2.28
C2orf83
165
127
0.77

HLA-F
193
42
0.22
ADAT1
27
61
2.26
YIPF7
502
380
0.76

PDIA6
92
20
0.22
WWC2
273
614
2.25
HTR2B
415
314
0.76

LZTFL1
69
15
0.22
hsa-mir-23c
145
326
2.25
GTF3C5
273
206
0.75

FHL3
65
14
0.22
UEVLD
291
654
2.25
ATP8A2
524
395
0.75

hsa-mir-200c
451
97
0.22
hsa-mir-5697
251
563
2.24
CCL14
539
403
0.75

DAAM2
280
60
0.21
ABCB6
186
416
2.24
CCDC108
414
309
0.75

ZC3HAV1
196
42
0.21
PYDC1
192
428
2.23
GDPD5
332
247
0.74

EXT2
99
21
0.21
AKT2
89
198
2.22
TEX15
233
173
0.74

CLRN3
302
64
0.21
WIPF1
499
1106
2.22
TENC1
186
138
0.74

ABCC2
90
19
0.21
THUMPD1
275
609
2.21
CNOT7
364
270
0.74

SSTR3
133
28
0.21
GSN
441
972
2.20
SNAPC5
173
128
0.74

DPP9
281
59
0.21
C8A
412
907
2.20
C5orf49
259
191
0.74

ALKBH 7
447
93
0.21
CEACAM7
138
302
2.19
ITGB1BP2
387
285
0.74

OR52L1
267
55
0.21
EPHA1
70
153
2.19
B4GALT2
269
198
0.74

LRIT1
122
25
0.20
OR52E2
18
39
2.17
ACOT13
303
223
0.74

MXI1
124
25
0.20
TEX101
184
398
2.16
HABP2
331
242
0.73

C11orf82
273
55
0.20
RAD23B
186
402
2.16
ACSM2B
556
405
0.73

hsa-mir-4421
5
1
0.20
SSR3
252
535
2.12
CNTN4
327
237
0.72

EPC1
431
86
0.20
CCDC144NL
225
475
2.11
CELSR3
112
81
0.72

NDRG4
393
78
0.20
hsa-mir-4779
169
355
2.10
AP5M1
341
246
0.72

KLHL7
162
32
0.20
VARS2
88
184
2.09
SH3TC2
185
133
0.72

METAP1
291
57
0.20
CBX7
254
529
2.08
AIM1
213
153
0.72

ATOH7
97
19
0.20
ATP6AP1
466
967
2.08
INSR
340
244
0.72

HMP19
128
25
0.20
LSMEM1
151
313
2.07
CXADR
194
139
0.72

C6orf70
134
26
0.19
FCHO2
48
99
2.06
EAPP
305
218
0.71

PCDHB14
182
35
0.19
PDYN
143
292
2.04
SLC35B2
313
223
0.71

ZNF695
236
45
0.19
HEATR3
77
157
2.04
C5orf15
152
108
0.71

FAM114A2
273
52
0.19
THRA
26
53
2.04
KDM4D
114
81
0.71

FGG
428
81
0.19
ENPP3
149
303
2.03
ZNF835
270
191
0.71

RPP40
106
20
0.19
STC2
184
374
2.03
ZNF408
149
105
0.70

PYCARD
203
38
0.19
ERLEC1
221
445
2.01
BTBD10
267
188
0.70

ZBTB42
391
73
0.19
CDS1
327
657
2.01
PAPOLA
27
19
0.70

hsa-mir-4276
162
30
0.19
SYVN1
238
478
2.01
ZNF586
157
110
0.70

CCL4
348
64
0.18
hsa-mir-6824
463
929
2.01
SPATA4
353
247
0.70

NUDT2
490
89
0.18
PRR22
67
132
1.97
DDOST
457
318
0.70

FAM21B
281
51
0.18
ACSL6
132
260
1.97
TMEM159
495
343
0.69

OR6N1
238
43
0.18
TMEM100
63
124
1.97
PRMT5
314
217
0.69

SH3YL1
195
35
0.18
ANAPC2
154
303
1.97
OR4C13
157
107
0.68

LOC100288524
301
54
0.18
ALOX12B
241
471
1.95
P4HA3
781
532
0.68

FLT3
552
99
0.18
hsa-mir-20b
509
993
1.95
NLGN3
456
308
0.68

CFLAR
147
26
0.18
NEIL2
411
793
1.93
C9orf91
351
237
0.68

PTPRC
221
39
0.18
OR6K2
228
439
1.93
C15orf62
273
182
0.67

TPTE2
17
3
0.18
OR5L1
277
533
1.92
TM4SF18
244
161
0.66

DAPL1
159
28
0.18
PTPRB
118
227
1.92
C1orf100
75
49
0.65

HYLS1
239
42
0.18
hsa-mir-4674
150
288
1.92
KIF20A
679
440
0.65

SPACA4
97
17
0.18
NR2C2AP
37
71
1.92
PELI1
252
162
0.64

GPATCH2
556
97
0.17
CRYM
17
32
1.88
CNST
223
143
0.64

ZNF77
310
54
0.17
CD40LG
168
316
1.88
ABLIM1
508
325
0.64

VAMP2
219
38
0.17
SLC2A3
72
135
1.88
LY86
157
100
0.64

C1orf174
173
30
0.17
STX19
253
473
1.87
DKK1
138
87
0.63

TSSK6
243
42
0.17
CYP19A1
183
342
1.87
SLTM
73
46
0.63

ANKRD28
369
63
0.17
PSG6
325
607
1.87
IL11
154
97
0.63

CLRN2
223
38
0.17
PLIN5
124
231
1.86
GBE1
54
34
0.63

ZC2HC1A
94
16
0.17
GPR161
113
208
1.84
IFIT3
310
195
0.63

TGFBR1
316
53
0.17
EXT1
219
403
1.84
DIAPH3
197
123
0.62

STK31
373
62
0.17
ANO1
236
434
1.84
NAA15
527
328
0.62

CD163
121
20
0.17
hsa-mir-377
86
158
1.84
SNRNP25
305
189
0.62

SAGE1
294
48
0.16
AFP
226
415
1.84
PGLYRP3
206
126
0.61

ITFG1
184
30
0.16
CTDSP1
218
396
1.82
INTS12
347
211
0.61

SLC37A2
351
57
0.16
AQP10
291
528
1.81
RFK
160
97
0.61

GCSAM
111
18
0.16
NKX1-2
75
136
1.81
EXOC3
88
52
0.59

CACNG1
205
33
0.16
MVD
118
213
1.81
CERKL
275
162
0.59

AZI2
201
32
0.16
TMPRSS11D
71
128
1.80
ABHD12
461
271
0.59

CYP1A2
45
7
0.16
C16orf72
227
409
1.80
SLC22A6
223
131
0.59

IGDCC4
52
8
0.15
KIAA0907
424
759
1.79
GNG10
175
102
0.58

AMER3
13
2
0.15
HDAC5
376
673
1.79
TMED7-
534
311
0.58

TICAM2

FAM110D
438
67
0.15
VIP
78
139
1.78
RNASE4
358
207
0.58

DTNB
113
17
0.15
KCNB2
150
267
1.78
NRAP
410
236
0.58

hsa-mir-513c
88
13
0.15
MLXIP
45
80
1.78
C4BPB
42
24
0.57

RAD51
285
42
0.15
APOBEC3F
210
373
1.78
RAB26
302
172
0.57

P2RY1
163
24
0.15
PYGM
308
547
1.78
HIST1H2AG
390
222
0.57

ANKRD20A1
34
5
0.15
ARRDC2
113
200
1.77
GTF2H2D
60
34
0.57

ZCCHC14
48
7
0.15
hsa-mir-4769
56
99
1.77
IPP
825
466
0.56

hsa-mir-551a
174
25
0.14
KCNC4
302
527
1.75
RARRES3
514
290
0.56

hsa-mir-4678
42
6
0.14
HNRNPH1
298
519
1.74
SRR
695
391
0.56

C10orf90
220
31
0.14
KIF1B
89
154
1.73
SDR42E1
136
75
0.55

PCDHB10
57
8
0.14
LRP2
321
555
1.73
KRT3
565
311
0.55

MYL12B
646
90
0.14
VN1R5
253
437
1.73
ZNF555
255
140
0.55

CLEC17A
133
18
0.14
SLPI
220
380
1.73
BAIAP2
596
327
0.55

KLHL3
215
29
0.13
FAM71C
143
247
1.73
TANC1
483
265
0.55

PPP5C
157
21
0.13
OR4Q3
43
74
1.72
GFOD2
174
95
0.55

RAPSN
15
2
0.13
SOWAHC
235
404
1.72
ZNF101
194
105
0.54

PINX1
219
29
0.13
SPINK14
7
12
1.71
ENG
320
173
0.54

ESRP2
144
19
0.13
BIRC5
114
195
1.71
OR5H2
288
155
0.54

CYB5A
147
19
0.13
RABL3
230
389
1.69
WISP1
164
88
0.54

VCX2
217
28
0.13
CCR6
314
527
1.68
LYPD1
235
126
0.54

OST4
527
67
0.13
OSCAR
93
155
1.67
NFKB1
162
86
0.53

SGPP1
269
34
0.13
CLCN5
83
138
1.66
PLCL2
276
146
0.53

KLHL2
319
40
0.13
KIF13A
126
209
1.66
BHLHE41
82
43
0.52

PLA2G10
639
80
0.13
MX1
315
518
1.64
TNFSF4
284
148
0.52

SERP1
104
13
0.13
NR1H4
213
350
1.64
DPCR1
142
74
0.52

DENR
8
1
0.13
TRPC6
230
373
1.62
CA12
224
116
0.52

PDIK1L
211
26
0.12
ZNF57
359
577
1.61
RERE
141
73
0.52

PEBP1
180
22
0.12
TREM1
217
347
1.60
BCAN
114
59
0.52

MED7
304
37
0.12
RGP1
402
640
1.59
KRT12
177
91
0.51

HTN3
189
23
0.12
FAM115C
277
439
1.58
BCL2L13
283
143
0.51

UBE2Q1
222
27
0.12
COX7A2
110
174
1.58
CTCFL
321
162
0.50

KRTAP10-4
141
17
0.12
CAPRIN1
597
938
1.57
LYPD4
298
149
0.50

XCR1
83
10
0.12
EIF5B
72
113
1.57
DOHH
54
27
0.50

ARPP21
167
20
0.12
ZNF488
220
342
1.55
CCDC166
22
11
0.50

NAALAD2
59
7
0.12
ZNF318
150
233
1.55
MDH2
247
123
0.50

TIRAP
287
34
0.12
CCDC24
96
149
1.55
RTTN
181
90
0.50

ZNF883
423
50
0.12
ARHGAP22
127
197
1.55
CHCHD7
212
105
0.50

CMTM2
128
15
0.12
hsa-mir-299
274
424
1.55
HMX2
85
42
0.49

EHF
293
34
0.12
PLA2G10
689
1066
1.55
UBE2D4
334
165
0.49

KLHL9
181
21
0.12
hsa-mir-196b
151
232
1.54
KLRG1
189
93
0.49

TRAPPC8
315
36
0.11
CXCR4
156
239
1.53
ANAPC7
316
155
0.49

SEMA6A
168
19
0.11
hsa-mir-5687
115
176
1.53
SF3B14
351
171
0.49

VWA5B2
142
16
0.11
LARGE
168
257
1.53
KSR2
187
91
0.49

LOC440563
45
5
0.11
ARIH2
294
449
1.53
NIPSNAP3B
101
49
0.49

hsa-mir-154
9
1
0.11
B4GALT2
105
160
1.52
ABCF2
385
186
0.48

TERT
226
25
0.11
NEURL4
156
235
1.51
UXS1
118
57
0.48

CLEC4D
182
20
0.11
OR6V1
342
515
1.51
MAGEA12
292
141
0.48

hsa-mir-628
110
12
0.11
hsa-mir-4654
186
280
1.51
SLC4A1AP
460
221
0.48

PKHD1LI
230
25
0.11
APOL2
93
140
1.51
SH3YL1
465
223
0.48

hsa-mir-217
92
10
0.11
TMSB10
194
292
1.51
PLA2G4B
484
232
0.48

ADAM17
378
41
0.11
NRL
196
294
1.50
SASH3
84
40
0.48

TCTEX1D4
93
10
0.11
TLR2
269
403
1.50
CLN8
259
123
0.47

TWISTNB
298
32
0.11
RASA1
121
181
1.50
ACTN4
57
27
0.47

UBAP2L
149
16
0.11
IFNA10
610
912
1.50
TMEM191C
146
69
0.47

OR10G4
112
12
0.11
TTC7A
116
173
1.49
IMPAD1
345
163
0.47

hsa-mir-4494
131
14
0.11
FOXD3
72
107
1.49
GALNT13
575
269
0.47

UBE2V1
113
12
0.11
C1QL1
373
552
1.48
ELL2
620
289
0.47

hsa-mir-5584
396
42
0.11
ITGA10
174
256
1.47
LRRC8E
251
116
0.46

FBXL20
265
28
0.11
SIGLEC7
62
91
1.47
TBC1D29
362
166
0.46

hsa-mir-383
771
81
0.11
SLC3A1
340
499
1.47
CHRNA4
132
60
0.45

CLCC1
182
19
0.10
hsa-mir-8068
75
110
1.47
ALDH1L1
22
10
0.45

GTF2A1
154
16
0.10
NEUROD4
176
257
1.46
MIP
214
97
0.45

DNAJC3
799
81
0.10
ZNF346
59
86
1.46
FAM162A
759
343
0.45

MMP28
168
17
0.10
TMEM186
326
475
1.46
KRTCAP2
175
79
0.45

PSMD13
277
28
0.10
CXCR5
125
182
1.46
NTAN1
100
45
0.45

CBFB
357
36
0.10
STOML2
121
176
1.45
CYYR1
147
66
0.45

AP4E1
130
13
0.10
hsa-mir-6797
33
48
1.45
EMC6
171
76
0.44

CALB2
10
1
0.10
CPEB2
565
821
1.45
STOM
363
161
0.44

METTL17
171
17
0.10
TOP2B
274
398
1.45
BCL2L11
428
189
0.44

TPPP
550
54
0.10
REEP4
125
181
1.45
BDNF
222
98
0.44

LOC154872
369
36
0.10
GYLTL1B
102
146
1.43
LRRC14
114
50
0.44

SEMA6C
72
7
0.10
HMP19
91
130
1.43
IL12B
57
25
0.44

NAP1L5
319
31
0.10
ABR
165
235
1.42
MYB
504
221
0.44

DNAJC2
228
22
0.10
L1CAM
148
210
1.42
PATE1
211
92
0.44

RB1
685
66
0.10
ZNF286B
78
110
1.41
CDH20
186
80
0.43

CISD1
461
44
0.10
RBM6
98
138
1.41
NWD1
119
51
0.43

SRPK2
42
4
0.10
CROCC
56
78
1.39
PDXP
7
3
0.43

HNRNPF
21
2
0.10
AGPAT6
90
125
1.39
NLRP1
150
64
0.43

KIAA1430
200
19
0.10
KRTAP27-1
50
69
1.38
PHLDA3
214
90
0.42

MANEAL
443
42
0.09
PRLHR
241
332
1.38
OR5D16
239
100
0.42

ARHGEF1
285
27
0.09
hsa-mir-5089
134
184
1.37
SYNDIG1L
787
326
0.41

hsa-mir-183
454
43
0.09
FAM129C
207
284
1.37
DHRS7C
237
98
0.41

RASGEF1B
286
27
0.09
COPS7B
297
405
1.36
CES1
199
82
0.41

ZMYND10
435
41
0.09
SPCS2
22
30
1.36
NDRG2
261
107
0.41

LHX5
340
32
0.09
SPATA19
127
173
1.36
BRS3
244
100
0.41

PTPRE
812
76
0.09
ATRN
377
512
1.36
TCF19
159
65
0.41

CHMP4A
294
27
0.09
AQPEP
263
356
1.35
IQCB1
306
125
0.41

MIS18BP1
98
9
0.09
EEF1G
51
69
1.35
SETD1A
642
262
0.41

RASSF8
98
9
0.09
TOPAZ1
179
240
1.34
DDX27
302
123
0.41

SOX13
297
27
0.09
AXIN1
77
103
1.34
RBM44
106
43
0.41

ABCC9
11
1
0.09
TMEM199
113
151
1.34
C3orf72
227
92
0.41

ARHGEF12
11
1
0.09
TAZ
143
191
1.34
CDC25C
385
156
0.41

C12orf60
264
23
0.09
BEST4
332
442
1.33
NCR1
358
145
0.41

TMPRSS6
138
12
0.09
GPR112
160
213
1.33
MAP1LC3B2
159
64
0.40

RNF185
461
40
0.09
BROX
483
641
1.33
MYO1G
110
44
0.40

ORC2
58
5
0.09
MAPKBP1
178
236
1.33
FRS3
78
31
0.40

SEMA3C
199
17
0.09
YIPF4
203
269
1.33
IGJ
172
68
0.40

LPPR5
285
24
0.08
hsa-mir-7703
31
41
1.32
NFKBID
363
143
0.39

MNT
264
22
0.08
FAM110C
47
62
1.32
NANOG
216
85
0.39

ARSJ
48
4
0.08
SPHAR
29
38
1.31
DNASE2B
102
40
0.39

OR56B4
327
27
0.08
BRD4
614
804
1.31
ZFYVE26
82
32
0.39

CEP250
73
6
0.08
C1orf194
145
189
1.30
SYCE3
510
199
0.39

NYX
225
18
0.08
VWA5B2
290
377
1.30
ZNF664-
217
84
0.39

FAM101A

hsa-mir-132
100
8
0.08
CERKL
246
319
1.30
CEBPG
223
86
0.39

R3HCC1L
113
9
0.08
SOST
54
70
1.30
KDM4E
236
91
0.39

NT5C1B
165
13
0.08
SPG20
350
453
1.29
AVPR2
205
79
0.39

CCDC160
128
10
0.08
ULBP3
131
169
1.29
NSMAF
330
127
0.38

ERLIN2
90
7
0.08
MAGEB10
45
57
1.27
SPINK6
169
65
0.38

ACAN
309
24
0.08
GAB1
166
210
1.27
ZNF22
241
92
0.38

BICD2
362
28
0.08
NEK5
267
337
1.26
CLEC7A
160
61
0.38

ASB1
569
44
0.08
C2orf44
313
395
1.26
PTPRO
193
73
0.38

FAM3D
273
21
0.08
HMGA2
463
583
1.26
CES5A
766
289
0.38

NFKBIL1
26
2
0.08
PTTG2
255
321
1.26
FHOD1
345
130
0.38

SPINK14
13
1
0.08
ALCAM
209
263
1.26
ATF3
316
119
0.38

MBD5
378
29
0.08
RHOBTB3
194
244
1.26
SPTBN4
170
64
0.38

DNAJC16
170
13
0.08
ACSF2
156
195
1.25
SPCS1
75
28
0.37

TMEM123
158
12
0.08
FAM47A
20
25
1.25
SPINK14
418
156
0.37

GNAS
79
6
0.08
LPCAT2
90
112
1.24
EGLN2
201
75
0.37

DEFB118
358
27
0.08
CEP44
64
79
1.23
HILPDA
183
68
0.37

SGCZ
347
26
0.07
hsa-mir-376a-2
505
620
1.23
CTLA4
253
94
0.37

PCDP1
107
8
0.07
TMEM114
314
385
1.23
OR8B4
27
10
0.37

C9orf64
228
17
0.07
LRRC28
99
121
1.22
FAM21C
130
48
0.37

hsa-mir-3671
121
9
0.07
hsa-mir-1273g
470
573
1.22
ARMC2
271
100
0.37

MKX
175
13
0.07
ASIP
52
63
1.21
ADCY6
125
46
0.37

LILRB3
245
18
0.07
OVCH2
53
64
1.21
PLCG1
226
83
0.37

INSM1
123
9
0.07
LPIN3
58
70
1.21
BTAF1
131
48
0.37

CCDC106
411
30
0.07
hsa-mir-4657
29
35
1.21
HDAC4
202
74
0.37

PGM1
274
20
0.07
LSP1
103
124
1.20
SOX15
706
258
0.37

TRIML1
96
7
0.07
CXCL5
208
250
1.20
ZNF202
451
164
0.36

RAB18
55
4
0.07
RBP4
224
269
1.20
PSMB11
942
342
0.36

hsa-mir-29b-2
252
18
0.07
MFGE8
85
102
1.20
TCHHL1
711
257
0.36

RAB37
84
6
0.07
TAS2R30
36
43
1.19
FZD7
183
66
0.36

SLC37A1
70
5
0.07
CSTF2T
350
417
1.19
GP5
379
136
0.36

FAM19A1
323
23
0.07
NABP1
144
169
1.17
THNSL2
306
109
0.36

SPAG7
127
9
0.07
BST1
410
480
1.17
PRG3
287
102
0.36

STUB1
113
8
0.07
RAB3IP
102
119
1.17
FCN3
197
70
0.36

LST1
229
16
0.07
GPBP1L1
139
161
1.16
RAB1A
364
129
0.35

ATP5G1
276
19
0.07
SON
121
140
1.16
ZGLP1
398
141
0.35

MID2
204
14
0.07
HN1L
720
832
1.16
HMGCLL1
211
74
0.35

OR52E2
219
15
0.07
EAF2
45
52
1.16
CCDC67
341
119
0.35

CPB1
73
5
0.07
ADD2
291
336
1.15
C1R
109
38
0.35

NUP188
176
12
0.07
ST6GALNAC3
289
332
1.15
DHRS4L1
609
212
0.35

MAPK12
88
6
0.07
KIF4A
216
246
1.14
KIAA1731
230
80
0.35

ALDH16A1
44
3
0.07
hsa-mir-4729
53
60
1.13
PSMC6
72
25
0.35

SPSB3
177
12
0.07
CCM2L
107
121
1.13
PRM1
242
84
0.35

ABL1
266
18
0.07
LATS2
23
26
1.13
PRKAA1
75
26
0.35

ASB16
133
9
0.07
IPCEF1
346
390
1.13
TEX101
252
87
0.35

CBX2
284
19
0.07
CCDC132
342
385
1.13
BOK
271
93
0.34

ODF3B
180
12
0.07
OR4K2
289
325
1.12
LMO1
191
65
0.34

B3GNT5
135
9
0.07
SERPINI1
76
85
1.12
SLC6A16
391
133
0.34

CCKAR
120
8
0.07
NPY4R
186
208
1.12
WNT10A
300
102
0.34

PLCB2
75
5
0.07
GNRH2
110
123
1.12
CECR6
381
129
0.34

LOC286238
166
11
0.07
ZNF431
414
459
1.11
GGCT
101
34
0.34

SPATA6
61
4
0.07
SCYL3
74
82
1.11
CD28
119
40
0.34

SLFN14
168
11
0.07
CAV3
205
227
1.11
TBX3
499
167
0.33

FAM175B
550
36
0.07
AGL
357
394
1.10
GPR83
273
91
0.33

ZNF706
306
20
0.07
hsa-mir-224
132
144
1.09
INTU
165
55
0.33

WASF2
153
10
0.07
SPTLC3
335
365
1.09
MPZ
99
33
0.33

MAGEA8
123
8
0.07
PHLDA3
123
134
1.09
GRB7
239
79
0.33

FABP6
400
26
0.07
CCDC11
758
824
1.09
C5orf51
106
35
0.33

NCAPH2
31
2
0.06
IL1F10
35
38
1.09
TAS2R43
155
51
0.33

hsa-mir-3684
280
18
0.06
RAD51B
95
103
1.08
TMEM165
349
114
0.33

MFSD1
140
9
0.06
UNG
107
116
1.08
SERPINB4
98
32
0.33

0R4E2
327
21
0.06
ARF4
337
365
1.08
EDAR
92
30
0.33

HCN3
234
15
0.06
NR2F2
53
57
1.08
TGM5
210
68
0.32

SETMAR
236
15
0.06
IFIT5
29
31
1.07
SLC16A6
351
113
0.32

FXYD5
571
36
0.06
SEC61B
239
255
1.07
FOXB2
499
159
0.32

ENAM
143
9
0.06
STK17B
255
272
1.07
RAC1
400
127
0.32

ACTL6B
32
2
0.06
OR2M3
554
586
1.06
CHRNA6
334
106
0.32

hsa-mir-4328
403
25
0.06
ZNF540
323
341
1.06
EBLN2
462
146
0.32

PLA2G5
178
11
0.06
CCDC27
274
289
1.05
DCXR
285
90
0.32

ZKSCAN4
260
16
0.06
NKX3-1
148
156
1.05
SLC30A9
57
18
0.32

BPIFB6
65
4
0.06
hsa-mir-376b
97
102
1.05
APOLD1
73
23
0.32

HCK
278
17
0.06
MAP3K13
125
130
1.04
ERI1
127
40
0.31

SPINT2
131
8
0.06
POU2F3
80
83
1.04
RSBN1
143
45
0.31

CHST11
279
17
0.06
LCE6A
192
196
1.02
UNC13C
387
121
0.31

hsa-mir-4263
149
9
0.06
hsa-mir-526a-1
48
49
1.02
FKBP10
253
79
0.31

OTOP1
414
25
0.06
ZMYND8
280
285
1.02
AMOT
264
82
0.31

ZFYVE21
829
50
0.06
RAB3IL1
260
264
1.02
OR2AT4
248
77
0.31

FNDC1
166
10
0.06
hsa-mir-30a
294
297
1.01
ABRA
406
126
0.31

UBE2G2
100
6
0.06
CTNNA3
297
300
1.01
VTA1
87
27
0.31

NRXN2
50
3
0.06
TAS2R13
148
149
1.01
C13orf45
243
75
0.31

RAD51AP1
367
22
0.06
MAP1LC3C
97
97
1.00
MED6
26
8
0.31

NQO2
419
25
0.06
GDF6
47
47
1.00
CASR
186
57
0.31

TABLE 2

List of gene hits and scores.

Screen 1—Technical repeat 1
Screen 1—Technical repeat 2
Screen 2

gene
rank
effect_size
gene
rank
effect_size
gene
rank
effect_size

SEC63
1
6.65686
SEC63
1
6.63909
STT3A
1
4.57001

SPCS3
2
8.28261
STT3A
2
4.58143
SEL1L
2
7.67986

SEC61B
3
3.83774
SEC61B
3
4.19879
EMC4
3
5.78404

RAP1GAP
4
9.75488
OSTC
4
4.57055
SEC63
4
6.28183

SLC35G5
5
8.25877
SERP1
5
3.91901
EMC3
5
3.45899

OR6T1
6
7.74729
SPCS3
6
10.6345
MARK3
6
9.25255

SEC61A1
7
7.69873
ZYX
7
8.21384
CYP11B2
7
7.27094

RRAS
8
7.5599
PARVG
8
7.79001
RPL23A
8
7.24956

RAB2B
9
7.45755
RAPSN
9
7.13626
KLRK1
9
7.15577

SERP1
10
3.79056
SLIT2
10
7.00096
SERP1
10
6.79395

SMAD4
11
6.70044
CYB5A
11
6.59212
FAM212A
11
6.7087

CCSER2
12
6.72758
TPTE2
12
6.50752
CTSD
12
6.60584

OSTC
13
6.68833
FBXL20
13
6.3948
SLC26A9
13
6.58576

ATOH7
14
6.34179
CYP1A2
14
6.36727
ESPN
14
6.54546

LRRC37A3
15
6.2001
AP1S3
15
6.21934
PCP2
15
6.56191

OST4
16
5.84208
CPSF3L
16
6.01095
USB1
16
6.4

FEZ2
17
5.71344
ZKSCAN4
17
5.8358
FIG4
17
6.35348

TBPL1
18
5.57081
hsa-mir-4421
18
5.78248
TRERF1
18
6.31503

DAPL1
19
5.56721
HSPA13
19
5.64703
PCDH9
19
6.27122

CHCHD7
20
5.54647
ALDH16A1
20
5.52185
FBXL20
20
6.25805

MMGT1
21
3.29686
ASB16
21
5.48721
GORAB
21
6.1351

PRH2
22
5.38824
KCTD13
22
5.43248
GKN2
22
6.1157

CHMP1B
23
5.37789
PCDHGA6
23
5.45522
UNKL
23
6.07086

SSR3
24
5.32014
HIST1H2BI
24
5.35364
HSPA13
24
5.95852

hsa-mir-6775
25
5.26677
SCLT1
25
5.13587
NCOA7
25
5.75767

GGTLC1
26
5.08853
ATG4D
26
5.13277
C12orf50
26
5.63995

RBMX2
27
4.91974
APBB3
27
4.99539
DNAJC25
27
5.54295

MORN2
28
4.84886
ZSCAN23
28
4.88516
KCP
28
5.46617

C11orf65
29
4.84833
NCAPH2
29
4.90572
ASPSCR1
29
5.49734

TMEM232
30
4.57723
FAM111A
30
4.78874
CD63
30
5.43052

TAAR8
31
4.51472
DDIT4
31
4.78743
RGMB
31
5.30957

GZMM
32
4.49035
C1orf110
32
4.7761
SSR3
32
5.09917

PPP1R13B
33
4.42226
DNAJ B1
33
4.74875
ADAMTS15
33
5.09282

IL26
34
4.39677
DCTN5
34
4.728
PASK
34
4.97399

DNAJB14
35
4.28583
CYP17A1
35
4.67326
IFT20
35
4.88396

NDST1
36
4.24682
MED26
36
4.54364
PNP
36
4.80465

hsa-mir-802
37
4.21247
TBK1
37
4.5155
PHF10
37
4.76692

RER1
38
4.20327
OR5L2
38
4.49653
RAB39A
38
4.68146

CES4A
39
4.18567
AP4S1
39
4.48447
TRIM21
39
4.66975

COA5
40
4.10895
ATP6AP1L
40
4.41445
DDR2
40
4.5992

EMC6
41
2.45528
FNBP4
41
4.41489
GGT1
41
4.57118

MOGS
42
3.92186
hsa-mir-4442
42
4.48664
SYT10
42
4.56983

PANX2
43
3.92521
ABCC9
43
4.44742
ANKIB1
43
4.48158

RASA1
44
3.91723
THRB
44
4.39707
BCAS1
44
4.41301

EXO1
45
3.85964
MKL2
45
4.34966
ESF1
45
4.42205

TABLE 3

sgRNA sequence used for gene validation.

SEQ

Gene
Spacer Sequence
ID NO:

AUP1
AACCTGCGAAGGACGCTGTC
1

AUP1
AGAGATTCTGTGCTTCCACG
2

AUP1
CTGCTGCTCTACGCGCCAGT
3

B4GALT2
TGCAGTCGGGCGGTGTGTAT
4

B4GALT2
CCCTGTCCTGACTCGCCACC
5

B4GALT2
GACCGCAACCTATACCGCTG
6

BRD3
CGACGTGACGTTTGCAGTGA
7

BRD3
ATTATTACCCCCTGCTCCAA
8

BRD3
CATCACTGCAAACGTCACGT
9

C11orf65
ATTCATGTGCGATTCAGATT
10

C11orf65
TTAGCACAGAGATCTTCAAT
11

C11orf65
TATCATCGTATAGAAAACAA
12

C1QL1
CCGCGTCGTAGTTGTTGCCT
13

C1QL1
CTTGATGAAGACCTCGTCGC
14

C1QL1
CACGCGCGGCACCGTGGTGT
15

CLCC1
TGGCGATTCGAAGATTCCTT
16

CLCC1
GGATCCATATAATGTGTTAA
17

CLCC1
TCTTTGTCTGCTCTGCATCG
18

CTSV
CGTGACGCCAGTGAAGAATC
19

CTSV
CATGTCACCAAAAGCATTCA
20

CTSV
ACAATGGCCATGAATGCTTT
21

CTSV
CATGAATCTTTCGCTCGTCC
22

CTSV
ACACAGAAGATTATATGGCG
23

EMC2
CACAGAGTCAAGCGATTAAC
24

EMC2
GATTGCCATTCGAAAAGCCC
25

EMC2
TAATGAATATGCTTCTAAGC
26

EMC3
GTCCTCCCTATGATTCTTAT
27

EMC3
TCCGAAGCCCAAATACATTG
28

EMC3
CATCCACCAATAAGAATCAT
29

EMC4
TGCTTGTCCAAGTAACCGAC
30

EMC4
AACCAATCCGATGCATGTGT
31

EMC4
AGCTGTTGCCATGACGGCCC
32

EMC6
ACGGCCGCCTCGCTGATGAA
33

EMC6
GACCTCGGTGTCAGCGCTGT
34

EMC6
AGACGTGCACCATGCCGTAG
35

FAM151B
GTATCATGCAGCTAACCACA
36

FAM151B
AGAACACAGCCAGCCAATTA
37

FAM151B
GCGCTTACCTGGGCCTCCAG
38

FAM210A
GTCCACGCCGCCACCCGTCA
39

FAM210A
GTGAAGTATCTGCGCAGTCA
40

FAM210A
AACCCAATGAGTTCTAGAAA
41

FBXL20
ATTATACCTCAATATCCCTC
42

FBXL20
TTACCGTAACAGGAGTTCTT
43

FBXL20
TTCCCAAAGAACTCCTGTTA
44

GPR161
CACAGTCGTCATCGTGGAGG
45

GPR161
CACCTGCCATGAGCGCAGTG
46

GPR161
AGAGACTCCACGTCCCGCTC
47

HSPA12B
ACGTGGAGACCGCTCTGCGC
48

HSPA12B
CACTGGGGACCGCTCCGGGC
49

HSPA12B
GGGCAATGCCGCAGCTTTCC
50

HSPA13
CAAACCCACTGTTCACGCGA
51

HSPA13
CCAAGTCTATCACCAAGACG
52

HSPA13
GGCTGACGTCTTCCACGTCT
53

HSPA13
AGTCGAGAGCCAACATATTC
54

HSPA13
TACTGACAATGATGTATATG
55

MAN1B1
AGCAACTGTCGAGATTGCAG
56

MAN1B1
ATCCGCAGAGGACAGTCATC
57

MAN1B1
CCTTCCGGGCGGGGATCCAC
58

MCCD1
GCTCCAACAACTCTTCAGCT
59

MCCD1
CTGCTCTTCCATGCTTGCTT
60

MCCD1
AAACTTAGGCGCCTCCTCCA
61

MLL5
TACAGCAGAGACGTCATACT
62

MLL5
ATGCTCATGACGTTCGCCTC
63

MLL5
GAGGACGAGCACCATAATTA
64

MLXIP
TGCCAAGTACCTCCGGCCGG
65

MLXIP
GGACCTCTCCAGCCTGGTCC
66

MLXIP
TGGCCCAATCCCCGGGAAAT
67

MMGT1
GCATCATGGCGCCGTCGCTG
68

MMGT1
CAGGCACTTACGCTGCGCAG
69

MMGT1
CAAGGACATTTGATACGTTA
70

OMD
CCATTTAACATACATTCGTG
71

OMD
GCCAATATGAAACTTATCAG
72

OMD
CCTGTTTGGTAATCATCATC
73

OR52E2
TATCCACAACTTCACACTTA
74

OR52E2
TAGCGCCATCCTCACCAACA
75

OR52E2
TTGAGTCGGGCTTCATGAGT
76

OST4
CGCCATCTTCGCCAACATGC
77

OST4
GAGCGACACGCCCAGCATGT
78

OST4
CGTCAACAATCCCAAGAAGC
79

OSTC
TCAGTCATAGAACCGACACT
80

OSTC
CGAATCCAATGAACAGAAGA
81

OSTC
AGATTCCTTCTTCTGTTCAT
82

PARVG
CGTGAACCGGAGTCTGCAGC
83

PARVG
CTCCCTCCCAACCAACGTCC
84

PARVG
GTGGCAGGCCAAGTGGAGCG
85

PLA2G10
AGTCCGGCTCACATAGGAAC
86

PLA2G10
CTGCTGCTGCTGCTTCTACC
87

PLA2G10
CCAGGATATTACGTGTGCAC
88

PVRL3
GTGCTTCGTGCGCCGAACTC
89

PVRL3
AACGGTCCCCGGAGCAAACC
90

PVRL3
TCTGAAGCCAATAAACCATC
91

RAP1GAP
GAAGTTGGACGCGATCATGT
92

RAP1GAP
CGGATGCGACCCTCCCAGAC
93

RAP1GAP
TGCTGAATATGCCTGCTACA
94

RAP1GAP
TCCCGGACCCCGCTGTGTTC
95

RAP1GAP
GCTTTGTCAGCAAAAATTCC
96

RASA1
TACCATCCGTCGTAAAACAA
97

RASA1
AATGGTTGACAACATTCATC
98

RASA1
CGATAGCAGAAGAACGCCTC
99

RASA1
GCTTATAATTTACTAATGAC
100

RASA1
ACAAGTACTCAATGACACAG
101

RBM25
GCCATAATGCTCATTGGTAC
102

RBM25
TTATTACATGACCTGCAAAT
103

SEC61B
TAGTGGCCCTGTTCCAGTAT
104

SEC61B
GTAGAATCGCCACATCCCCC
105

SEC61B
TCCTTACCTCTGCCGGACAG
106

SEC63
GGTGTATGTGGTATCGTTTA
107

SEC63
GTGATGAGGTTATGTTCATG
108

SEC63
TTGGTATTCTCGGTCTGTTT
109

SEL1L
AGCATATCGGTATCTCCAAA
110

SEL1L
GCAGAAATGATGTATCAAAC
111

SEL1L
CTTGGCTTTCTGTATGCCTC
112

SERP1
TCTTGGCGACGTTGCCGCGC
113

SERP1
CGAAGATGGTCGCCAAGCAA
114

SERP1
TCCTACAGACGCCTTCTCTT
115

SHC1
CCTCCAGTCAATGCGTGCCC
116

SHC1
TTACCAATGTAGCTCCCAAG
117

SHC1
GGCAACATAGGCGACATACT
118

SHC1
AGTCCAGGGCACGCATTGAC
119

SHC1
CCCTTCATACCTGGACAGGG
120

SPCS1
TGGCACTGCGCGTCAGTAGC
121

SPCS1
ACGTGGCTGAACAGTTCGGG
122

SPCS1
GAGAGGATGCCGGCGATAGA
123

SPCS3
CTCTGAACCAAGTTGTCCTA
124

SPCS3
TGTCCTGATCCTGTCACAAG
125

SPCS3
ACCTAGAGAAAGAAGTGATC
126

SPCS3
TCAAAACAATCTTGTCCCAT
127

SPCS3
AAAAATGTAGAAGATTTCAC
128

SSR3
CTATAACAACACTCTGTTCC
129

SSR3
GACCCTAGTAAGCACATATT
130

SSR3
TCTATTTGGAGCCAGTAGAC
131

SSR3
CAGGGTTATACTGGCGAATA
132

SSR3
AAGCAACAATGACCACGACC
133

STT3A
ACAGACATTCCGAATGTCGA
134

STT3A
AAGGTGGTACGTGACGATGG
135

STT3A
CTCGGTCATCAAACCAGTTA
136

SYVN1
GTATGCCATCCTGATGACGA
137

SYVN1
CCGCCATCATCACTGCCGTG
138

SYVN1
GGCCAGGGCAATGTTCCGCA
139

TMEM100
TGTTTGACTCTCCCGTCTCT
140

TMEM100
GGTGATCACAACTTCACTCT
141

TMEM100
TGTCTTCATCGCCGGCATCG
142

ZIC2
ACACGCACCCCAGCTCGCTG
143

ZIC2
GCTTCGCCAACAGCAGCGAC
144

ZIC2
CTATGAGTCGTCCACGCCCC
145

ZNF488
CTTTCGCCTAACGTCCGACC
146

ZNF488
ACACTACAGACCTCGCTTGT
147

ZNF488
AAAGTCGACCCCAACAAGCG
148

sgRNA control
CGCTTCCGCGGCCCGTTCAA
149

sgRNA control
ATCGTTTCCGCTTAACGGCG
150

	Number	Date	Country
	62239067	Oct 2015	US
	62239455	Oct 2015	US

METHODS OF INHIBITING VIRAL REPLICATION COMPRISING THE SIGNAL PEPTIDASE COMPLEX

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

CROSS REFERENCE TO RELATED APPLICATIONS

Provisional Applications (2)