Fig. 2: HBV m⁵C-1291 promotes viral RNA export and translation and prevents RIG-I recognition of viral RNA.

A HBV transcripts are associated with ALYREF. Huh7 cells were transfected with Flag-ALYREF and then with HBV 1.1-mer at 24 h post-transfection. HBV transcripts were immunoprecipitated by anti-Flag antibody at 48 h post-transfection. The enriched HBV RNA was measured by qPCR (A top). Immunoprecipitated ALYREF examined by western blot. (A bottom). B ALYREF promotes HBV RNA export. Subcellular distribution of HBV transcripts in the cytoplasmic and nuclear fractions examined by qPCR after overexpression of ALYREF for 48 h (B top panel). ALYREF expression examined by western blot (B, bottom). C–E The m⁵C-1291 promotes 0.7 kb mRNA export. A point mutation of C1291T is shown, which was introduced to m⁵C-1291 in the cDNA reporter of HBV 0.7 kb mRNA (C bottom). Huh7 cells were co-transfected with Flag-ALYREF, and cDNA of 0.7 kb mRNA-wt or 0.7 kb mRNA-mt for 48 h, respectively. Subcellular distribution of 0.7 kb mRNA-wt or 0.7 kb mRNA-mt were examined as described in (B) (C top). HBV 0.7 kb mRNA wt or mt were then immunoprecipitated by anti-Flag antibody against Flag-ALYREF and measured by qPCR (D). E–G The m⁵C-1291 promotes 0.7 kb mRNA translation. Huh7 cells were transfected with cDNA of 0.7 kb mRNA-wt or 0.7 kb mRNA-mutant for 48 h. Cell lysates were hyper-centrifuged in a sucrose gradient after cycloheximide treatment, and RNA was extracted from the layer with maximum absorbance at OD260. Ribosome-associated HBV 0.7 kb mRNA was assessed by qPCR (F). GAPDH was used as a control (E). The 5′-UTR of 0.7 kb mRNA-wt or 0.7 kb mRNA-mt were fused to firefly luciferase (Fluc) reporter, respectively (G bottom). Huh7 cells were transfected with wild-type or mutant 5′-UTR Fluc reporter. The relative luciferase activities were calculated by dual luciferase assay at 24 h post-transfection (G top). Renilla luciferase (Rluc) served as an internal control. (H) HBV m⁵C mutants increase IFN-β mRNA expression. Huh7 cells were co-transfected with poly I:C and HBV 1.1-mer wild type or mutants. The expression of IFN-β was assessed by RT-qPCR at 18 h post-transfection. (I) Expression of five ISGs assessed by RT-qPCR at 18 h post-transfection. J The m⁵C-1291 blocks RIG-I recognition. RNA-oligo pulldown was performed with huh7 cell lysate using biotin-labeled oligos, which contained m⁵C-1291, m⁵C-1255, or non-m⁵C, respectively. The pulled-down proteins were immunoblotted by anti-RIG-I antibody. Pull down without oligos (Beads) served as the control. The bar represents the mean from three independent experiments. ***P < 0.001; **P < 0.01; *P < 0.05.