Identification of Antinorovirus Genes in Human Cells Using Genome-Wide CRISPR Activation Screening

Abstract

Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in human cells. Our screens identified with high confidence 49 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprisingly, the majority of the genes identified are neither associated with innate or adaptive immunity nor associated with any antiviral activity. Confirmatory studies of eight of the genes 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.IMPORTANCE Norovirus is one of the leading causes of food-borne illness worldwide. 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 screens to induce gene overexpression at the native locus through recruitment of transcriptional activators to individual gene promoters. We found 49 genes that could block murine norovirus replication in 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 are a resource for those studying noroviruses, and we provide a robust approach to identify novel antiviral genes.

Keywords: CRISPR; noroviruses; virology.

FIG 1

CRISPRa screen for antinorovirus genes. (A) Schematic of the CRISPRa system used in this study. Dead Cas9 (dCas9) fused to VP64 interacts with sgRNAs at the transcriptional start site of a gene. sgRNA have PP7 hairpins that recruit a chimeric protein containing heat shock factor (HSF), p65 transcription factor, and phage coat protein (PCP) fused together. In combination, this leads to the transcriptional activation of the targeted gene. (B) HeLa-CD300lf cells expressing dead Cas9-VP64 fusion were transduced with the indicated sgRNA plasmids and assayed for CD4 expression 1 week after antibiotic selection. At baseline CD4 levels are low, but they dramatically increase upon expression of the CRISPRa machinery. (C) Cartoon overview of the cell survival CRISPRa screen performed with HeLa-CD300lf cells. sgRNAs from cells surviving MNoV challenge were analyzed and compared to relative abundance of a mock-infected sample.

FIG 2

Identification of anti-MNoV genes in human cells. (A) Heat map showing the enrichment of genes in the two indicated conditions. Genes are color coded based upon their STARS score. (B) Comparison of STARS scores from HeLa-CD300lf cells challenged with MNoVCW3 (x axis) and MNoV^CR6 (y axis). Genes that did not meet the criteria to receive a STARS score are assigned a value of 0.

FIG 3

Validation of anti-MNoV genes in human cells. (A and B) HeLa-CD300lf cells expressing dCas9-VP64 and the indicated sgRNAs were challenged with MNoVCW3 (A) or MNoVCR6 (B) at an MOI of 50. Cellular viability was assessed 72 h postinfection (hpi). Values are normalized for each cell line to uninfected control wells to determine the percent viability. The data are shown as means ± the standard errors of the mean (SEM) from three independent experiments, and data were analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (C) Graph depicting the correlation between the relative viabilities of cell lines after 72 h of MNoV^CW3 (x axis) and MNoV^CR6 (y axis) infection. Each dot represents the average relative viability over three independent experiments that the indicated sgRNA-expressing cell line exhibited. (D and E) HeLa-CD300lf cells expressing dCas9-VP64 and the indicated sgRNAs were infected with MNoVCW3 (D) or MNoVCR6 (E) at an MOI of 0.05. Viral production was assessed at 24 hpi by plaque assay. The data are shown as means ± the SEM from three independent experiments, and data were analyzed by one-way ANOVA with Tukey’s multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (F) Graph depicting the correlation between the viral production 24 h after MNoVCW3 (x axis) and MNoV^CR6 (y axis) infection. Each dot represents the average PFU/ml value across over three independent experiments that the indicated sgRNA-expressing cell line exhibited.

FIG 4

A specific TRIM7 isoform inhibits MNoV replication in HeLa cells. (A, top) Cartoon diagram of TRIM7 isoform 1 and isoform 4. RING, B-box, coiled-coil (CC), and SPRY domains are displayed. Isoform 4 has an alternative, shorter coiled-coil domain and does not encode a SPRY domain. (A, bottom) Representative Western blot for the indicated protein expression in HeLa-CD300lf cells expressing either an empty vector, TRIM7 isoform 1, or TRIM7 isoform 4. (B and C) HeLa-CD300lf cells expressing the indicated constructs infected at an MOI of 50 with MNoVCW3 (B) or MNoVCR6 (C). Cellular viability was assessed at 72 hpi. Values are normalized for each cell line to uninfected control wells to determine the percent viability. The data are shown as means ± the SEM from three independent experiments, and data were analyzed by one-way ANOVA with Tukey’s multiple-comparison test. **, P < 0.01; ***, P < 0.001; ns, not significant. (D and E) HeLa-CD300lf cells expressing the indicated constructs infected at an MOI of 0.05 with MNoVCW3 (D) or MNoVCR6 (E). Viral production was assessed at 24 hpi by plaque assay. The data are shown as means ± the SEM from three independent experiments, and data were analyzed by one-way ANOVA with Tukey’s multiple-comparison test. ****, P < 0.0001; ns, not significant.

FIG 5

TRIM7 E3-ligase activity but not MSK1 phosphorylation is required to inhibit MNoV replication. (A, top) Cartoon diagram of TRIM7 isoform 1 with the indicated point mutations highlighted. C29 and C32 are required to catalyze ubiquitination, while S107 is phosphorylated by MSK1. (A, bottom) Representative Western blots of the indicated protein expressions in HeLa-CD300lf cells. (B and C) HeLa-CD300lf cells expressing indicated constructs infected at an MOI of 0.05 with MNoVCW3 (D) or MNoVCR6 (E). Viral production was assessed at 24 hpi as measured by plaque assay. The data are shown as means ± the SEM from three independent experiments, and data were analyzed by one-way ANOVA with Tukey’s multiple-comparison test. ****, P < 0.0001; ns, not significant.

FIG 6

TRIM7 blocks MNoV replication in mouse BV2 cells postentry. (A) Representative Western blot of human TRIM7 expression in BV2 cells transduced with the indicated constructs. (B and C) BV2 cells expressing the indicated constructs infected at an MOI of 5 with MNoVCW3 (B) or MNoVCR6 (C). Cellular viability was assessed at 24 hpi. Values are normalized for each cell line to the uninfected control wells to determine the percent viability. The data are shown as means ± the SEM from three independent experiments, and data were analyzed by one-way ANOVA with Tukey’s multiple-comparison test. ***, P < 0.001; ****, P < 0.0001; ns, not significant. (D and E) BV2 cells expressing indicated constructs infected at an MOI of 0.05 with MNoVCW3 (D) or MNoVCR6 (E). Viral production was assessed at 12 hpi by plaque assay. The data are shown as means ± the SEM from three independent experiments, and data were analyzed by one-way ANOVA with Tukey’s multiple-comparison test. ****, P < 0.0001; ns, not significant. (F) BV2 expressing the indicated constructs were transfected with viral RNA from MNoVCW3 and harvested at 12 h posttransfection. Viral production was measured by plaque assay. The data are shown as means ± the SEM from three independent experiments, and data were analyzed by one-way ANOVA with Tukey’s multiple-comparison test. ****, P < 0.0001; ns, not significant.