A genome-wide CRISPR screen identifies UFMylation and TRAMP-like complexes as host factors required for hepatitis A virus infection

Figures

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  Figure 1.. Genome-wide CRISPR KO screen identifies host cellular factors important for HAV infection (A) Schematic of the genome-wide CRISPR KO screen. A library of Huh7.5.1-Cas9 CRISPR KO cells was infected with cytopathic HAV HM175/18f and virus-resistant cells were harvested 12 days post-infection (dpi). gRNA abundance was measured in the original and selected cell population by next-generation sequencing. (B) Significance of enriched genes in CRISPR KO screen based on RIGER analysis. Hits were clustered and colored by function. The complete ranked gene list and RIGER scores can be found in Table S1. (C) Selected terms from gene ontology (GO) analysis of genes enriched in CRISPR KO screen (RIGER p < 0.001). The complete list of GO terms can be found in Table S2.

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  Figure 2.. KO of identified host factor genes reduces HAV replication (A) Quantification of HAV RNA in WT and clonal KO Huh7.5.1 cell lines by quantitative reverse transcriptase PCR (qRT-PCR) at 3 dpi. Of note, this time point likely supports multiple rounds of viral replication and spread. (B) Quantification of HAV RNA in WT, PAPD5 KO, PAPD7 KO, and PAPD5/PAPD7 double-KO (DKO) cells 3 dpi by qRT-PCR. (C) Viral growth curves using recombinant HAV expressing nanoluciferase (HAV-Nluc). WT, SLC35A1 KO, UBA5 KO, PAPD5/7 DKO, and ZCCHC14 KO cells were infected and lysed at indicated time points, and luciferase levels were measured. Dotted line indicates luminescence levels in WT cells at 72 hpi. Data are displayed as mean ± SD (n = 3 biological replicates). (D–F) Quantification of HAV RNA in WT, UBA5 KO (D), PAPD5 KO (E), or ZCCHC14 KO (F) cells and KO cells complemented with respective cDNAs 3 dpi by qRT-PCR. (G and H) Quantification of HAV, human rhinovirus A2 (RV-A2), enterovirus 71 (EV-71), and encephalomyocarditis virus (EMCV) RNA in WT versus UFM1 KO cells (G) or WT versus DKO cells (H) by qRT-PCR. Cells infected with HAV or RV-A2 were harvested after 48 hpi, with EV-71 after 24 hpi and…

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  Figure 3.. HAV depends on functional UFM1 conjugation of the ribosomal protein RPL26 for optimal translation (A) Schematic of cellular UFMylation pathway. #, rank in gene enrichment analysis of CRISPR screen. (B) UFM1 immunoblot of WT, UBA5 KO, UFSP2 KO, UFM1 KO, and UFM1 KO cells complemented with WT, Gly83Ala, or ΔC3 UFM1 as well as UBA5 KO cells complemented with WT or catalytically inactive (cat) UBA5. Top bands represent UFM1 conjugation products, and the bottom band represents free UFM1. P84 was used as loading control. (C) Quantification of HAV RNA in WT, UFSP2 KO, UBA5 KO, UFM1 KO, and UFM1 KO cells complemented with WT, Gly83Ala, or ΔC3 UFM1. Viral RNA was normalized to 18S RNA, and results are displayed relative to infection in WT cells. (D) Time course of luciferase activity expressed by electroporated HAV-FLuc subgenomic replicon RNA in WT, UBA5 KO, and UFSP2 KO cells. (E) Time course of luciferase activity expressed by electroporated HAV-FLuc subgenomic replication-incompetent replicon RNA in WT, UBA5 KO, and UFSP2 KO cells. (F) Immunoblots of uninfected and HAV-infected WT, UBA5 KO, UFM1 KO, and UFSP2 KO cells. Membranes were stained with anti-UFM1, anti-HAVVP1, or anti-RPL26…

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  Figure 4.. Catalytic activity of TRAMP-like complexes is important for HAV translation, independent of regulating viral poly(A) tail length or RNA stability (A) Time course of luciferase activity expressed by electroporated HAV-FLuc subgenomic replicon RNA in WT, DKO and ZCCHC14 KO cells. Datasets represent mean ± SD (n = 2 biological replicates) for each time point. (B) Time course of luciferase activity expressed by electroporated HAV-FLuc replication-incompetent replicon RNA in WT, DKO cells, and ZCCHC14 KO. Datasets represent mean ± SD (n = 2 biological replicates) for each time point. (C) Northern blot of HAV RNA from WT or PAPD5/7 DKO cells, which were electroporated with replication-deficient HAV replicon and harvested at indicated time points post-electroporation. Membrane was stained using an in vitro-transcribed DIG-labeled anti-sense RNA probe targeting nucleotides 7118–7465 of the HAV genome. 18S ribosomal RNA was visualized using SYBR Safe dye on agarose gel prior to transfer onto membrane. (D) Poly(A) tail length assay. Total RNA from infected WT or KO cells was G/I tailed at 3′ ends, reverse transcribed and PCR amplified by either genome-specific (GS) forward and GS reverse…

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  Figure 5.. Small-molecule inhibition of PAPD5 and PAPD7 reduces HAV infection in human hepatocyte cells and human liver organoids (A) Chemical structure of RG7834. (B) Dose-response curve of HAV RNA levels in Huh7.5.1 cells treated with different RG7834 concentrations as measured by qRT-PCR 3 days post-infection/treatment. Non-linear curve was fitted with least-squares regression using GraphPad Prism 8, and EC50 was determined. (C) Quantification of HAV RNA in control and RG7834 (100 nM)-treated Huh7.5.1 cells 3 dpi by qRT-PCR. (D) Quantification of HBV total RNA in control and RG7834 (100 nM)-treated HepG2-NTCP cells 15 dpi by qRT-PCR. Drug was replenished every 3–4 days. (E) Quantification of HCV RNA in control and RG7834 (100 nM)-treated Huh7.5.1 cells 3 dpi by qRT-PCR. (F) Quantification of HEV RNA in control and RG7834 (100 nM)-treated Huh7.5.1 cells by qRT-PCR 6 days post-transfection of viral RNA. (G) Quantification of HAV RNA in control and RG7834 (100 nM)-treated human liver organoids 7 dpi by qRT-PCR. Organoids were either stem-like or differentiated to adult hepatocytes prior to infection. For all qRT-PCR experiments, viral RNA was normalized to 18S RNA, and results are displayed…