Fig. 4.

Mutations of 5′ and 3′ DRACH motifs and their effect on HBV protein expression and DNA synthesis. (A) Schematics indicate the location of the A1907C mutations of 5′ and 3′ m⁶A sites in HBV RNAs. Open circles represent WT (green) and A1907C mutations (red) in HBV plasmid DNA. Green solid circles indicate m⁶A modification while red solid circles represent lack of m⁶A (due to A1907C mutations) in encoded RNAs. HBV-M1 having the A1907C mutation at both termini, HBV-M2 only at the 5′ end, and HBV-M3 only at the 3′ end. (B–G) Immunoblot analysis of HBV proteins following transfection of indicated HBV constructs in HepG2 cells with relative quantifications below the respective blots [(B) M1, (D) M2, (F) M3] and core-associated DNA isolated from core particles and quantified by qPCR using equal amounts of purified DNA in the input. Relative levels of core-associated DNA from the various constructs [(C) M1, (E) M2, (G) M3] graphed as percent relative to HBV-WT. (H) RT-qPCR analysis of relative levels of remaining HBV transcript relative to GAPDH isolated at indicated times following 24 h of actinomycin D treatment from HBV 1.3mer (WT or M3)-expressing HepG2 cells. (I) Model for how m⁶A at the 5′ or 3′ epsilon stem loops of HBV transcripts differentially regulates their stability and reverse transcription. Data here are presented from three independent experiments and the bars represent mean ± SD. ***P ≤ 0.001 by unpaired Student’s t test, and n.s., not significant.