N6-methyladenosine modification of the 5' epsilon structure of the HBV pregenome RNA regulates its encapsidation by the viral core protein

Abstract

Hepatitis B virus (HBV) contains a partially double-stranded DNA genome. During infection, its replication is mediated by reverse transcription (RT) of an RNA intermediate termed pregenomic RNA (pgRNA) within core particles in the cytoplasm. An epsilon structural element located in the 5' end of the pgRNA primes the RT activity. We have previously identified the N6-methyladenosine (m⁶A)-modified DRACH motif at 1905 to 1909 nucleotides in the epsilon structure that affects myriad functions of the viral life cycle. In this study, we investigated the functional role of m⁶A modification of the 5' ε (epsilon) structural element of the HBV pgRNA in the nucleocapsid assembly. Using the m⁶A site mutant in the HBV 5' epsilon, we present evidence that m⁶A methylation of 5' epsilon is necessary for its encapsidation. The m⁶A modification of 5' epsilon increased the efficiency of viral RNA packaging, whereas the m⁶A of 3' epsilon is dispensable for encapsidation. Similarly, depletion of methyltransferases (METTL3/14) decreased pgRNA and viral DNA levels within the core particles. Furthermore, the m⁶A modification at 5' epsilon of HBV pgRNA promoted the interaction with core proteins, whereas the 5' epsilon m⁶A site-mutated pgRNA failed to interact. HBV polymerase interaction with 5' epsilon was independent of m⁶A modification of 5' epsilon. This study highlights yet another pivotal role of m⁶A modification in dictating the key events of the HBV life cycle and provides avenues for investigating RNA-protein interactions in various biological processes, including viral RNA genome encapsidation in the context of m⁶A modification.

Keywords: HBV pgRNA encapsidation; N6-methyladenosine; RNA–protein interaction; hepatitis B virus.

Fig. 1.

The cellular m⁶A methyltransferases affect the HBV pgRNA encapsidation. (A) The secondary structure of the epsilon elements is shown with the functional requirements for RNA packaging. They are represented as follows: Blue lines indicate structural requirements for encapsidation, blue nucleotides indicate specific sequence requirements for encapsidation, red nucleotides indicate DRACH motif, and *A is the m⁶A site. (B) HepAD38 cells stably expressing HBV were grown in the absence or presence of tetracycline for 72 h, and the cells were then transfected with the METTL3/14-specific siRNAs. After 48 h, cellular lysates, core-associated pgRNA, and total RNA were extracted from these cells for Western blotting and Northern blotting, respectively. (C) Huh7 cells were transfected with pHBV Y63D plasmid (RT-defective mutant) for 24 h, and then the siRNAs of METTL3/14 were transfected into Huh7 cells expressing pHBV Y63D plasmid. After 48 h, cellular lysates, core-associated pgRNA, and total RNA were extracted from these cells. The indicated proteins were analyzed by Western blotting. The encapsidated and cellular HBV RNA were analyzed by Northern blotting. (D–G) The siRNAs of METTL3/14 were transfected into stably expressing HBV HepAD38 cells grown in the absence or presence of tetracycline for 72 h. After 48 h, cells and supernatant were harvested. The core-associated pgRNA was analyzed by RT-qPCR (D). Hirt’s extract was prepared and subjected to Southern blot assays (E). The core-associated DNA and extracellular DNA levels were analyzed by qPCR (F and G). (H–K) HepG2-NTCP cells were infected with 2.5 × 10³ genome equivalents per cell of HBV particles. After 10 d, cellular lysates, total RNA, and core-associated pgRNA and DNA were extracted from these cells. The indicated proteins were analyzed by Western blotting (H). The cellular HBV RNA and core-associated pgRNA were analyzed by RT-qPCR (I and J). The HBV core-associated DNA was assayed by qPCR (K). In E, F to G, and I to K, the error bars represent the SDs of three independent experiments. The P values are calculated via an unpaired Student’s t test. **P < 0.01. siMETTL3/14, siRNAs of METTL3/14; I.B, immunoblotting; N.B, Northern blotting; N.D, not detected.

Fig. 2.

The m⁶A modification of the 5′ epsilon elements promotes HBV pgRNA encapsidation. (A) Schematics indicate the 5′ and 3′ m⁶A sites in the epsilon structures of the HBV pgRNA. Green circles indicate the m⁶A site, and red circles indicate A1907C mutation in the HBV pgRNA. pHBV 1.3-mer 5′-3′ MT contains the A1907C mutation at the 5′ and 3′ ends, pHBV 1.3-mer 5′ MT within the epsilon structure contains the A1907C mutation at the 5′ end, and the pHBV 1.3-mer contains the 3′ MT at the 3′ end. (B–F) The indicated pHBV 1.3 plasmids were transfected into Huh7 cells. After 72 h, cellular lysates, total RNA, core-associated pgRNA, HBV DNA, and core-associated DNA were extracted. The indicated proteins or HBV RNAs were each analyzed by Western or Northern blotting, respectively (B). Encapsidated pgRNA was detected by Northern blotting (C). The core-associated pgRNA was analyzed by RT-qPCR (D). Hirt’s DNA extracts were prepared, and the protein-free (PF) rcDNA and cccDNA were analyzed by Southern blotting (E). The core-associated DNA was analyzed by qPCR (F). (G–J) PHHs were infected with 2.5 × 10³ genome equivalents per cell of each HBV WT, 5′-3′ MT, 5′ MT, or 3′ MT infectious particles. After 10 d, PHHs were harvested to assess the expression of viral protein and RNA, core-associated pgRNA, and DNA, respectively. In D, F, G, H, and J, the error bars represent the SDs of three independent experiments. The P values are calculated via an unpaired Student’s t test. ***P < 0.001. I.B, immunoblotting; N.B, Northern blotting; N.D, not detected; S.B, Southern blotting.

Fig. 3.

The m⁶A modification of the 5′ epsilon of the HBV pgRNA does not affect viral pol–5′ epsilon interaction. (A) pHBV 1.3 Bulge MT was generated by the deletion of nucleotides from 1863 to 1868 nt of pHBV 1.3-mer. They are represented as follows: Blue lines indicate the position of deleted nucleotides, and *A is the m⁶A site. (B–D) Huh7 cells were cotransfected with FLAG–pol and pHBV 1.3 WT or 5′-3′ MT, 5′ MT, or 3′ MT plasmids. After 48 h, cells were washed with PBS, and then cells were irradiated with UV for RNA–protein cross-linking. Total RNA and cell lysates were extracted from these cells. FLAG-tagged HBV Pol proteins were immunoprecipitated using anti-FLAG M2 magnetic beads from UV-irradiated cell lysates. Immunoprecipitated RNAs were extracted by TRIzol. The input HBV pgRNA was analyzed by RT-qPCR (B). Immunoprecipitated HBV pgRNA levels were normalized by input HBV pgRNA levels by RT-qPCR (C). Immunoprecipitated FLAG–Pol and the indicated input proteins were analyzed by Western blotting (D). In B and C, the error bars represent the SDs of three independent experiments. The P values are calculated via an unpaired Student’s t test. *P < 0.05. IP, immunoprecipitation; N.D, not detected; n.s., nonsignificant.

Fig. 4.

The m⁶A modification of the 5′ epsilon element promotes the interaction with core protein. (A–C) HepAD38 cells stably expressing HBV were grown in the absence or presence of tetracycline for 72 h, and thecells were then transfected with the METTL3/14-specific siRNAs. After 48 h, cells were irradiated with UV for RNA–protein cross-linking. Cells were harvested to extract total RNA and cellular lysates. Cellular lysates were immunoprecipitated using an anti-core antibody. The input HBV pgRNA was analyzed by RT-qPCR (A). Enriched HBV pgRNA levels were normalized by input HBV pgRNA levels by RT-qPCR (B). The indicated proteins were analyzed by Western blotting (C). (D–F) pHBV 1.3 Pol-null plasmids were transfected into Huh7 cells for 24 h, and the cells were then transfected with the METTL3/14-specific siRNAs. After 48 h, cells were irradiated with UV for RNA–protein cross-linking and harvested to extract total RNAs and cellular lysates, respectively. Cellular lysates were immunoprecipitated using an anti-core antibody. The input HBV pgRNA was analyzed by RT-qPCR (D). Enriched HBV pgRNA levels were normalized by input HBV pgRNA levels by RT-qPCR (E). The indicated proteins were analyzed by Western blotting (F). (G–I) The indicated plasmids were transfected into Huh7 cells. After 72 h, cells were UV irradiated, and total RNA and cellular lysates were extracted. The UV cross-linked RNA–protein complexes were immunoprecipitated using anti-core antibodies. The HBV pgRNA levels were assayed by qRT-PCR (G). Immunoprecipitated pgRNA levels were normalized by input pgRNA using RT-qPCR (H). The indicated proteins were analyzed by Western blotting (I). (J) The A1907C mutation in the epsilon structure is predicted to create a bubble. The compensatory U1851G mutation (blue) was generated in the 5′ epsilon (pHBV-5′-MT-CM). (K–M) Huh7 cells were transfected with the indicated plasmids. After 72 h, RNA–protein complexes from these cells were cross-linked by UV irradiation, and total RNA and cellular lysates were extracted, respectively. RNA–protein complexes were immunoprecipitated using anti-core antibodies. The input pgRNA levels were analyzed by RT-qPCR (H). Immunoprecipitated pgRNA levels were normalized by input pgRNA levels using RT-qPCR (I). The indicated proteins were analyzed by Western blotting (J). In A and B, D and E, and H and I, the error bars represent the SDs of three independent experiments. The P values are calculated via an unpaired Student’s t test. *P < 0.05, **P < 0.01. IP, immunoprecipitation.

Fig. 5.

The m⁶A methylated pgRNA is enriched in the core particles. (A and B) Huh7 cells were transfected with pHBV Y63D (RT-defective pol mutant) plasmid for 24 h, and the cells were further transfected with the siRNAs of METTL3/14 for 48 h. Total RNA and encapsidated pgRNA, and cellular lysates were isolated from these cells. Methylated RNAs from total RNA and encapsidated pgRNA fraction were immunoprecipitated from using anti-m⁶A antibodies. Immunoprecipitated (m⁶A-methylated) RNA levels were normalized by input RNA levels by RT-qPCR (A). CREBBP and HPRT1 were used for either positive or negative control. The indicated proteins were analyzed by Western blotting (B). (C and D) Huh7 cells were transfected with pHBV 1.3-mer or pHBV 1.3-mer 5′-3′ MT plasmid. After 72 h, cellular lysates, total RNA, and encapsidated pgRNA were extracted. Methylated RNAs from total RNA and encapsidated pgRNA fractions were immunoprecipitated using anti-m⁶A antibodies. Immunoprecipitated RNAs were analyzed by RT-qPCR (C). The indicated proteins were analyzed by Western blotting (D). In A and C, the error bars represent the SDs of three independent experiments. The P values are calculated via an unpaired Student’s t test. **P < 0.01. I.B, immunoblotting.