Surfaceome CRISPR screen identifies OLFML3 as a rhinovirus-inducible IFN antagonist

Figures

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  Fig. 1. Genome-wide and surfaceome-wide screens for identification of host factors of RV. a Venn diagram showing the composition of surfaceome library. b Flow chart showing the procedures of genome-wide and surfaceome CRISPR screens. c Analyses of enriched sgRNA, or gene knockout, in genome-wide and surfaceome screens. d Analyses of the compositions of positive hits, as defined by a FDR of less than 0.01, in genome-wide and surfaceome screens. e Bubble plot showing the results of CRISPR screen. Top 10 candidate hits are shown. Significance of enrichment was calculated by MAGeCK

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  Fig. 2. Validation of the top 10 hits from surfaceome and genome-wide screens. a Cell viability assay for determination of the protective effects of identified gene knockout on RV-B14-induced cell death. The assay is performed at 24 h post infection of RV-B14 at an MOI of 2. b RT-qPCR quantification of viral loads in medium supernatant. The supernatant is harvested at 24 h post infection of RV-B14 at an MOI of 2. c Immunofluorescence (IF) staining of RV-B14 envelope protein for evaluation of infection rates at individual cell lines. IF staining is performed at 16 h post infection of RV-B14 at an MOI of 2. Each biological replicate contains the quantification results from 2000 cells. d, e RT-qPCR quantification of viral loads in medium supernatant (d) and the lysate (e) of mock and knockout single clones. The supernatant and cell lysate are harvested at 24 h post infection of RV-B14 at an MOI of 2. The significant difference between knockout cells and non-targeting sgRNA mock groups are determined using two-tailed unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

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  Fig. 3. Validation of the effects of ICAM-1, RAB5C, and OLFML3 on RV infection. a, b Time-dependent viral replication of RV-B14 in mock and knockout cells, as determined by viral loads in medium supernatant (a) and cell lysate (b). Cells are infected with RV-B14 at an MOI of 2. Viral RNA in cell lysates (b) is normalized to RPLP0 expression. Significant difference between test groups and non-targeting sgRNA group is determined using two-way ANOVA with Dunnett’s multiple comparisons test. c, d Rescued susceptibility of knockout cells to the infection of RV-B14 (c) and RV-A16 (d) by overexpression of RAB5C and OLFML3 respectively. e, f Rescued replication of RV-B14 (e) and RV-A16 (f) in knockout cells by overexpression of RAB5C and OLFML3 respectively, as determined by viral loads in medium supernatant. g, h Rescued replication of RV-B14 (g) and RV-A16 (h) in knockout cells by overexpression of RAB5C and OLFML3 respectively, as determined by viral loads in cell lysates. Viral RNA in cell lysates is normalized to RPLP0 expression. i Representative images of CPEs induced by clinical RV strain. Scale bar, 100 μm. j Cell viability of mock and knockout cells upon challenge of clinically…

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  Fig. 4. Dissection of the functions of RAB5C and OLFML3 in RV infection. a, b Viral loads in medium supernatant (a) and cell lysates (b) at 24 h after transfection of RV-A16 genome RNA. c, d Viral RNA in cell lysates of mock and knockout cells at 1, 3, and 6 h after infection with RV-B14 (c) and RV-A16 (d) at an MOI of 20 in the presence of 2 mM GuHCl. For a, b, significant difference between mock and test groups is determined using two-tailed Student’s t test. For c, d, significant difference between mock and RAB5C groups is determined using two-tailed Student’s t test. e Volcano plot showing differentially expressed genes (DEGs). RV infection-induced gene upregulation and downregulation are first calculated and the differentially upregulated or downregulated genes in mock and knockout cells are defined as DEGs. Cells are harvested and analyzed at 24 h after infection of RV-B14 at an MOI of 2. f GO analyses of biological processes of DEGs identified in E. g, h Heat map showing ISG expression in mock and OLFML3−/− cells at 24 h post infection of RV-B14 (g) and RV-A16 (h) at an MOI of 2. These results are derived from RT-qPCR quantification. Gene expression is normalized to RPLP0

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  Fig. 5. OLFML3 is a RV-inducible suppressor of type I IFN signaling during RV infection. a Structural organization of OLFML proteins. b RT-qPCR quantification of OLFML3 expression levels in H1-Hela cells in the absence or presence of RV-B14 or RV-A16. Samples are collected at 24 h post infection with a RV MOI of 2. c RT-qPCR quantification of IFIT2, OAS2, and ISG15 expression in WT and OLFML3−/− cells at 0, 12, and 24 h after RV-B14 infection at an MOI of 2. d RT-qPCR quantification of IFNB, STAT1, and STAT2 expression in WT and OLFML3−/− cells at 0, 12, and 24 h after RV-B14 infection at an MOI of 2. For c and d, gene expression is normalized to RPLP0 and significant difference was determined using two-way ANOVA with Sidak’s multiple comparisons test. Gene abbreviations are as follows: IFNB, interferon β; ISG15, IFN-stimulating genes 15; IFIT2, interferon induced protein with tetratricopeptide repeats 2; OAS2, 2′-5′-oligoadenylate synthetase 1; SOCS3, suppressor of cytokine signaling 3

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  Fig. 6. OLFML3 antagonizes type I IFN signaling in an SOCS3-dependent mechanism. a mRNA expression of SOCS3 in response to RV-B14 infection. Samples are collected at 0, 12, and 24 h after infection at an MOI of 2 and the fold change of SOCS3 expression before and after RV infection is shown. b RT-qPCR quantification of SOCS3 expression in mock or OLFML3−/− cells in the absence and presence of SOCS3 siRNA. Samples are collected at 24 h post RV-B14 infection at an MOI of 2. c RT-qPCR quantification of viral RNA in cell lysate in mock or OLFML3−/− cells in the absence and presence of SOCS3 siRNA. Samples are collected at 24 h post RV-B14 infection at an MOI of 2. Significant difference between non-target and each other group is determined unless indicated otherwise. d RT-qPCR quantification of STAT1, STAT2, IFNB, OAS2, ISG15, and IFIT2 expression in the absence and presence of SOCS3 siRNA. Samples are collected at 24 h post RV-B14 infection at an MOI of 2. Significant difference between OLFML−/− and each other group is determined unless indicated otherwise. For a–d, gene expression is normalized to RPLP0 and significant difference is determined using Student’s t test. NS, no significance. e…