Identification of anti-norovirus genes in mouse and human cells using genome-wide CRISPR activation screening

Noroviruses (NoVs) are a leading cause of gastroenteritis world-wide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation (CRISPRa) genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in either mouse or human cells. Our screens identified with high confidence 57 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprising, the majority of the genes identified are not associated with innate or adaptive immunity nor with any antiviral activity. Confirmatory studies of eight of the genes in validate the initial screening data. Mechanistic studies on TRIM7 demonstrated a conserved role of the molecule in mouse and human cells in restricting MNoV in a step of infection after viral entry. Furthermore, we demonstrate that two isoforms of TRIM7 have differential antiviral activity. Taken together these data provide a resource for understanding norovirus biology and demonstrate a robust methodology for identifying new antiviral molecules across cell types and species. Author Summary Norovirus is one of the leading causes of foodborne illness world-wide. Despite its prevalence, our understanding of norovirus biology is limited due to the difficulty in growing human norovirus in vitro and a lack of an animal model. Murine norovirus (MNoV) is a model norovirus system because MNoV replicates robustly in cell culture and in mice. To identify host genes that can restrict norovirus replication when overexpressed we performed genome-wide CRISPR activation (CRISPRa) screens to induce gene overexpression at the native locus through recruitment of transcriptional activators to individual gene promoters. We found 57 genes could block murine norovirus replication in either mouse or human cells. Several of these genes are associated with classical immune signaling pathways, while many of the molecules we identified have not been previously associated with antiviral activity. Our data is a resource for those studying norovirus and we provide a robust approach to identify novel antiviral genes.


Abstract:
16 Noroviruses (NoVs) are a leading cause of gastroenteritis world-wide, yet host 17 factors that restrict NoV replication are not well understood. Here, we use a CRISPR 18 activation (CRISPRa) genome-wide screening to identify host genes that can inhibit  cause of viral gastroenteritis worldwide and is estimated to have an economic burden of 50 $1 billion annually [2]. Despite the high infectivity of HNoV between individuals, it has 51 proven difficult to culture in vitro with significant advances made only in the past few 52 years [3,4]. Because of the challenges in growing HNoV in cell culture and lack of an 53 animal model, murine norovirus (MNoV) has emerged as a system for understanding 54 norovirus biology. MNoV grows robustly in cell culture, is amenable to reverse genetics, 55 and is a natural pathogen of laboratory mice. MNoV  only on sgRNA homology for specificity, it overcomes many of these obstacles. We 79 therefore utilized CRISPRa to identify genes whose overexpression block MNoV 80 replication. 81 We use recently developed CRISPRa genome-wide libraries to screen for host 82 genes that antagonize MNoV replication in both mouse and human cells. Our screens 83 and subsequent validation identify numerous anti-MNoV genes including canonical 84 interferon stimulated genes and several genes with novel antiviral activity. We 85 demonstrate that the antiviral activity of one such gene, TRIM7, is highly specific to only 86 one of the two major TRIM7 isoforms, highlighting the utility and power of the CRISPRa 87 screening approach. Further mechanistic studies demonstrate that TRIM7 inhibits   94 We set out to identify genes in mouse and human cells that have anti-norovirus 95 activity when overexpressed. By screening cell lines from two different species, we 96 sought to capture a wider diversity of antiviral genes and potentially identify those 97 molecules with evolutionary conserved antiviral function. We first generated cell lines HeLa-CD300lf cell lines (BV2-dCas9-VP64 and HeLa-CD300lf-dCas9-VP64).

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Before proceeding with a genome-wide screen, we developed a simple assay to 107 test transcriptional activation activity in BV2-dCas9-VP64 and HeLa-CD300lf-dCas9-108 VP64 cells relying on the low level of expression of CD4 in these cells. sgRNAs  We next introduced a pooled, genome-wide CRISPRa library into BV2-dCas9-113 VP64 (Caprano library) and HeLa-CD300lf-dCas9-VP64 (Calabrese library) cells.  To explore the possibility that individual hits function in a similar pathway, we 159 took the 49 genes that scored for either viral strains and analyzed them using GSEA 160 [25,26]. Comparison of the hallmark genes with our dataset indicated a significant 161 enrichment in genes related to interferon alpha responses, including GBP2, which has 162 been recently shown to block MNoV replication complex formation [27]. When 163 analyzing the function of the genes by GO terms we also found an enrichment for 164 interferon and cytokine responses along with genes involved in embryogenesis and 165 RNA polymerase gene expression (Table 2). These data demonstrate the ability to 166 discover expected candidate antiviral genes (e.g. those regulated by inflammatory 167 cytokines) and candidate genes involved in other pathways (e.g. embryogenesis).

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Analysis and comparison of screen in BV2 and HeLa cells 170 We compared the results from the BV2 and HeLa-CD300lf CRISPRa screens.

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The top hit in all virus and cell type combinations was TRIM7 (Figure 2A and Tables S1-  (Tables S1-S4). When looking at the 175 GSEA both screens had an interferon signature (Table 1 and 2). The lack of 176 concordance between other hits could be due to cell type, species, library, or selective 177 pressure differences between screens. Additionally, we find that false-negatives are 178 common (see below) and our data likely underrepresents the true overlap of function 179 across the cellular systems. None the less, these data highlight the importance of   for testing strain specificity for potentially antiviral molecules.

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As the screen was designed to enrich for genes that protected cells from virus  Finally, we determined which step of the viral life cycle was targeted by TRIM7.

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Since ubiquitination can lead to proteaseome-mediated degradation of proteins, we first 229 tested whether the MNoV receptor, CD300lf, was reduced upon TRIM7 overexpression.

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In HeLa-CD300lf we found no effect of TRIM7 on CD300lf protein levels ( Figure 4A). 231 We next tested the hypothesis that TRIM7 targets a critical step of MNoV entry.

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Transfection of MNoV CW3 RNA was unable to rescue MNoV production in human TRIM7 233 isoform 1 expressing BV2 cells indicating that TRIM7 can target a post-entry step of the 234 MNoV life cycle to inhibit viral replication ( Figure 4J).    for each gene of rank-ordered sgRNA hits that was above 10% of total sequenced 372 sgRNAs. A STAR score was assigned to genes that had a sgRNA score above this 373 threshold in either of the two independent pools.

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For GSEA analysis, screen hits were queried at the BROAD GSEA portal The data that support the findings of this study are available from the corresponding 414 authors upon request.