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. Author manuscript; available in PMC: 2019 Oct 15.
Published in final edited form as: Nat Microbiol. 2019 Apr 15;4(7):1096–1104. doi: 10.1038/s41564-019-0425-6

Figure 4. IRF1-regulated RARRES3 acyl transferase restricts HAV replication by downregulating mTOR.

Figure 4.

(a) Lentivirus transduction of catalytically-active RARRES3 restricts HAV infection in PH5CH8 cells expressing IRF1 sgRNA #2 (left panels) or Huh-7.5 cells (right panels). RARRES3, but not its catalytically-inactive RARRES3/C113S, inhibited HAV infection in both cell lines. *p<0.05, **p<0.01 vs. vector control (two-way ANOVA with Dunnett’s multiple comparisons test). (b) Huh-7.5 cells stably expressing indicated lentiviral vectors were challenged with HAV carrying NanoLuc (NLuc) with 30 μM 2’CMA (DAA) or vehicle (DMSO). NLuc activities at indicated time points post-infection are shown. **p<0.01 (two-way ANOVA with Dunnett’s multiple comparisons test). (c) Transfection of subgenomic HAV-Luc RNA or its replication-incompetent mutant (Δ3D) in Huh-7.5 cells expressing wild-type RARRES3 versus RARRES3/C113S. **p<0.01 vs. vector control (two-way ANOVA with Dunnett’s multiple comparisons test). (d) Infection of HAV/NLuc in Huh-7.5 cells expressing RARRES3 sgRNA. Immunoblots are shown on top. **p<0.01 vs. vector control (two-way ANOVA with Dunnett’s multiple comparisons test). (e) Steady-state levels of mTOR-related factors in Huh-7.5 cells stably expressing RARRES3 and RARRES3/C113S. (f) Immunoblots of P70S6K siRNA-transfected Huh-7.5/RARRES3 cells. (g) Phosphorylation of p70S6K and mTOR in Huh-7.5 cells expressing IRF1 sgRNA. (h) Impacts of mTOR inhibitors on HAV/NLuc vs. HCV/GLuc vs. DENV/NLuc replication and the cell viability. Inhibitory effects of all these inhibitors on HAV/NLuc vs. other reporter viruses differed significantly. **p<0.01 (two-way ANOVA with Dunnett’s multiple comparisons test). (i) Inhibition of transfected subgenomic HAV-Luc RNA replication in Huh-7.5 cells by the mTOR inhibitors. DAA, 30 μM 2’CMA. **p<0.01 vs. DMSO control (one-way ANOVA with Dunnett’s multiple comparisons test). Data are means ± s.d. from 3 independent experiments (b, d, i) or from 3 technical replicates representative of 2 (a, e-h) or 3 independent experiments (c). The precise p-values are shown in Supplementary Table 9.