Fig. 5.

Nuclear localization of HBx is required for inducing m⁶A modification of viral transcripts. (A) Subcellular localization of HBx, NES-fused HBx, or NLS-fused HBx protein transiently expressed in Huh7 cells for 48 h was analyzed by immunoblotting. (B–E) Huh7 cells were transfected with HBV 1.3 x-null together with pSI-x, pSI-NES-x, or pSI-NLS-x for 48 h prior to quantification of MeRIP qRT-PCR (B), precore/pgRNA (C), core-associated DNA (D), or immunoblotting analysis for the indicated proteins (E). (F) Analysis of m⁶A methyltransferase levels in the cytoplasmic or nuclear fraction of HBV 1.3 x-null–expressed Huh7 cells, which were cotransfected with pSI-x, pSI-NES-x, or pSI-NLS-x. The indicated proteins were analyzed from cytoplasmic and nuclear lysates by immunoblotting. (G–I) HepG2-NTCP cells were infected with HBV WT or x-null particles. After 7 d, cells were transfected with pSI-x, pSI-NLS-x, or pSI-NES-x plasmid for 3 d. Cells were harvested to assess m⁶A methylation levels of HBV RNAs and PTEN mRNA (G) or for immunoblotting analysis for the indicated proteins (H) or HBeAg levels (I). In all panels, data are mean ± SD (**P < 0.01 by unpaired one-tailed Student’s t test).