Hepatitis B virus X protein recruits methyltransferases to affect cotranscriptional N6-methyladenosine modification of viral/host RNAs

Abstract

Chronic hepatitis B virus (HBV) infections are one of the leading causes of cirrhosis and hepatocellular carcinoma. N6-methyladenosine (m⁶A) modification of cellular and viral RNAs is the most prevalent internal modification that occurs cotranscriptionally. Previously, we reported the dual functional role of m⁶A modification of HBV transcripts in the viral life cycle. Here, we show that viral HBV X (HBx) protein is responsible for the m⁶A modifications of viral transcripts. HBV genomes defective in HBx failed to induce m⁶A modifications of HBV RNAs during infection/transfection, while ectopic expression of HBx restores m⁶A modifications of the viral RNAs but not the mutant HBx carrying the nuclear export signal. Using chromatin immunoprecipitation assays, we provide evidence that HBx and m⁶A methyltransferase complexes are localized on the HBV minichromosome to achieve cotranscriptional m⁶A modification of viral RNAs. HBx interacts with METTL3 and 14 to carry out methylation activity and also modestly stimulates their nuclear import. This role of HBx in mediating m⁶A modification also extends to host phosphatase and tensin homolog (PTEN) mRNA. This study provides insight into how a viral protein recruits RNA methylation machinery to m⁶A-modify RNAs.

Keywords: METTL3/14; N6-methyladenosine modification; cotranscriptional modification; hepatitis B virus; hepatitis B virus X protein.

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).

Fig. 2.

ChIP assay to determine the HBx protein-mediated recruitment of m⁶A methyltransferases onto HBV cccDNA and PTEN locus on the chromosome. (A and B) PHHs were infected with HBV or HBV x-null particles. Cross-linked chromatins prepared 10 d after infection were immunoprecipitated with anti-HBx or anti-METTL3 or anti-METTL14 antibodies and analyzed by qRT-PCR (A) or semiquantitative PCR (B). (C and D) Chromatins extracted from HBV- or HBV Δx-infected HepG2-NTCP cells were immunoprecipitated with anti-HBx or anti-METTL3 or anti-METTL14 antibodies. Immunoprecipitated chromatins were analyzed by qRT-PCR (C) or semiquantitative PCR (D). In all panels, data are mean ± SD (**P < 0.01; ***P < 0.001; unpaired one-tailed Student’s t test).

Fig. 3.

HBx protein-mediated m⁶A modification affects the stability of viral RNAs and PTEN RNA. (A–C) Huh7 cells, which were transfected with pHBV 1.3 or pHBV 1.3 x-null, were treated with control or METTL3/14 siRNAs. After 72 h, total RNA and lysates were extracted to assess HBV precore/pgRNA (A), PTEN mRNA (B), or the indicated protein expressions (C). (D–G) Analysis of HBV precore/pgRNA (D and E) and PTEN mRNA (F and G) stabilities in Huh7 cells transfected with pHBV 1.3 or pHBV 1.3 x-null. After 24 h, the transfected cells were treated with control or METTL3/14 siRNAs by incubation for 2 d, and cells were incubated with actinomycin D. The cells were harvested at 0, 6, 12, or 24 h following actinomycin D treatment, and relative levels of remaining HBV pgRNA (D and E) and PTEN mRNA (F and G) were analyzed. In all panels, data are mean ± SD (*P < 0.05 by unpaired one-tailed Student’s t test). n.s., not significant.

Fig. 4.

HBx protein interacts with METTL3 and 14 and stimulates their nuclear import. (A) Huh7 cells, which were transfected with pHBV 1.3 x-null, were treated with control or pcDNA 3.1 FLAG-HBx. Cytoplasm and nuclear lysates were immunoprecipitated with anti-FLAG, followed by immunoblotting for the indicated proteins. (B–E) PHHs were infected with HBV WT or x-null particles for 10 d (B and C). HepG2-NTCP cells were infected with the same amount of virus particles of HBV WT or x-null for 24 h and then further incubated for 9 d (D and F). The HBV-infected PHHs and HepG2-NTCP cells were harvested to assess immunoblotting from whole lysates (B and D) or isolated nuclear and cytoplasmic biochemical fractions (C and E). (F and G) Huh7 cells were transfected with pHBV 1.3 or HBV 1.3 x-null together with pcDNA3.1 FLAG-HBx or pcDNA3.1 (control). The indicated proteins were analyzed by immunoblotting (F). Immunoblot analysis of isolated nuclear and cytoplasmic biochemical fraction from indicated cells (G). In C, E, and G, the METTL3 and 14 expression levels relative to FTO from three independent experiments were quantified using ImageJ.

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).

Fig. 6.

HBx amino acids 51 to 100 are essential for interaction with METTL3/14 and regulating m⁶A modification of HBV RNAs. (A–C) Huh7 cells, which expressed pHBV 1.3 x-null, were transfected with control, pcDNA 3.1 FLAG-HBx, or HBx truncated mutant plasmids. Cells were harvested to assess immunoblotting analysis for the indicated proteins (A), HBV precore/pgRNA levels (B), or m⁶A methylation levels of HBV RNAs (C). (D and E) Huh7 cells, which were transfected with pHBV 1.3 x-null, were treated with control; truncated HBx 50 to 154, 1 to 100, or 100 to 154; or HBx plasmid. The indicated proteins were analyzed by immunoblotting (D). Lysates were immunoprecipitated with anti-FLAG, followed by immunoblotting for the indicated proteins (E). In B and C, data are mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001; unpaired one-tailed Student’s t test).