Figure 5.

m⁶A-Abrogating Mutations in E1 Increase Infectious HCV Particle Production

(A) Schematic of the HCV genome with the mutation scheme for altering A or C residues (red arrows) to make the E1^mut virus. Consensus m⁶A motifs (green) and inactivating mutations (red) are shown. Dashes represent nucleotides not shown. Genomic indices match the HCV JFH-1 genome (AB047639).

(B) FFA of supernatants harvested from Huh7 cells after electroporation of WT or E1^mut HCV RNA (48 hr) and analyzed as the percentage of viral titer relative to WT.

(C) Gaussia luciferase assay to measure levels of the WT, E1^mut, or Pol⁻ HCV luciferase reporter (JFH1-QL/GLuc2A) transfected in Huh7.5 CD81 KO cells.

(D) Immunoblot analysis of extracts of WT, E1^mut, or Pol⁻ JFH1-QL/GLuc2A transfected in Huh7.5 CD81 KO cells.

(E) Enrichment of WT or E1^mut reporter RNA or SON mRNA by immunoprecipitation of FLAG-YTHDF2 or vector from extracts of Huh7 cells. Captured RNA was quantified by qRT-PCR and graphed as the percentage of input. Right: immunoblot analysis of anti-FLAG immunoprecipitated extracts and input.

(F) Enrichment of WT or E1^mut HCV RNA by immunoprecipitation of HCV Core from extracts of Huh7 cells electroporated with the indicated viral genomes (48 hr). Lower: immunoblot analysis of anti-Core immunoprecipitated extracts and input.

Data are representative of two (D and E) or three (B, C, and F) experiments and presented as the mean ± SD (n = 3). ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001 by unpaired Student’s t test. See also Figure S5.