Fig. 1.

HBx protein regulates m⁶A modification of HBV RNAs and host PTEN mRNA during HBV infection of PHHs and HepG2 NTCP cells and HBV 1.3 transfection. (A–C) PHHs and HepG2-NTCP cells were infected with 2.5 × 10³ genome equivalents per cell of HBV WT or x-null particles. After 10 d, total RNA and protein were extracted from HBV-infected PHHs. m⁶A-modified HBV transcripts and PTEN mRNA levels were quantified by MeRIP qRT-PCR (A). The indicated protein expression levels were analyzed by immunoblotting (B). HBeAg levels in media were analyzed using culture media by ELISA (C). (D–F) HepG2-NTCP cells were infected with HBV WT or x-null particles. After 7 d, pSI-x plasmids were transfected into HBV x-null–infected HepG2-NTCP cells. After 3 d, cells were harvested to assess m⁶A modification levels of HBV transcripts and PTEN mRNA (D) or to perform immunoblotting analysis for the indicated proteins (E) or HBeAg levels (F). (G–K) Huh7 cells were transfected with pHBV 1.3 or pHBV 1.3 x-null or cotransfected with pHBV 1.3 x-null and pcDNA3.1 FLAG-HBx. After 72 h, cells were harvested to assess m⁶A-methylated HBV transcripts and PTEN mRNA (G), HBV precore/pgRNA (H), PTEN mRNA (I), HBV core-associated DNA (J), or the indicated protein expressions (K). In all panels, data are mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001; unpaired one-tailed Student’s t test).