Host-cell interactions in HBV infection and pathogenesis: the emerging role of m6A modification

Abstract

Hepatitis B virus (HBV) is a DNA virus with a complex life cycle that includes a reverse transcription step. HBV is poorly sensed by the immune system and frequently establishes persistent infection that can cause chronic infection, the leading cause of liver cancer and cirrhosis worldwide. Recent mounting evidence has indicated the growing importance of RNA methylation (m6A modification) in viral replication, immune escape, and carcinogenesis. The value of m6A RNA modification for the prediction and clinical management of chronic HBV infection remains to be assessed. However, a number of studies indicate the important role of m6A-marked transcripts and factors of m6A machinery in managing HBV-related pathologies. In this review, we discuss the fundamental and potential clinical impact of m6A modifications on HBV infection and pathogenesis, as well as highlight the important molecular techniques and tools that can be used for studying RNA m6A methylome.

Keywords: ALKBH; Epitranscriptomics; HCC; METTL; hypoxia; interferon; liver cancer.

Figure 1.

HBV life cycle. HBV enters hepatocytes through the initial attachment to heparan sulfate proteoglycans and then by highly-specific interaction with sodium taurocholate cotransporting polypeptide (NTCP) receptor followed by virion uncoating and transport to the nucleus. In the nucleus, rcDNA is converted into cccDNA by a multi-step process with the assistance of host factors. cccDNA is a template for transcription of all viral RNAs (preC RNA, pgRNA, preS RNA, X RNA and additional spliced isoforms). All viral RNAs contain 3′-end ϵ stem loop, while pgRNA and preC mRNA also harbour 5′-end ϵ stem loop. HBV RNAs are exported in the cytoplasm for protein translation. pgRNA is then selectively packaged into newly formed nucleocapsids, where it is reverse transcribed to produce rcDNA. Nucleocapsids can be enveloped to form a mature virion and released from cells or transported to the nucleus to replenish cccDNA pool. If reverse transcription is interrupted, virions containing pgRNA are released from cells. Double-stranded linear HBV DNA (dslDNA), produced due to aberrant reverse transcription, serves as the major source of viral integration. HBV DNA integrations represent a replicative dead-end for the virus, but serves as a template for viral RNA and protein production [1].

Figure 1.

HBV life cycle. HBV enters hepatocytes through the initial attachment to heparan sulfate proteoglycans and then by highly-specific interaction with sodium taurocholate cotransporting polypeptide (NTCP) receptor followed by virion uncoating and transport to the nucleus. In the nucleus, rcDNA is converted into cccDNA by a multi-step process with the assistance of host factors. cccDNA is a template for transcription of all viral RNAs (preC RNA, pgRNA, preS RNA, X RNA and additional spliced isoforms). All viral RNAs contain 3′-end ϵ stem loop, while pgRNA and preC mRNA also harbour 5′-end ϵ stem loop. HBV RNAs are exported in the cytoplasm for protein translation. pgRNA is then selectively packaged into newly formed nucleocapsids, where it is reverse transcribed to produce rcDNA. Nucleocapsids can be enveloped to form a mature virion and released from cells or transported to the nucleus to replenish cccDNA pool. If reverse transcription is interrupted, virions containing pgRNA are released from cells. Double-stranded linear HBV DNA (dslDNA), produced due to aberrant reverse transcription, serves as the major source of viral integration. HBV DNA integrations represent a replicative dead-end for the virus, but serves as a template for viral RNA and protein production [1].

Figure 2.

m6A modification of HBV RNA and its functional effects. (A) HBV RNA is methylated only at a single position, in the typical DRACH motif (D = A, G, or U; R = A or G; and H = A, C, or U), at A1907 at the 3′ end. As pgRNA contains two copies of the DRACH motif due to terminal redundancy, it is m6A methylated both at 3′ and 5′ ends. (B) HBV RNA/pgRNA is m6A modified by m6A writers (METTL3/METTL14) where METTL3 uses S-adenosyl methionine as a methyl donor, and METTL14 functions as an RNA-binding platform. M6A-modified HBV RNA/pgRNA is then recognized by specific reader proteins, such as YTHDC1-2, YTHDF1-3, IGF2BP1-3, eIF3, FMRP, FXR1, FXR2, SND1, hnRNPA2B1, hnRNPC, hnRNPG, etc. The effects of m6A modification typically depend on the recognition by different m6A readers. Interaction with YTHDF2 and YTHDF3 reduces stability and accelerates the decay of m6A-modified HBV RNA and reduces HBV protein production. YTHDC1 and FMRP readers promote export of HBV RNA from the nucleus into the cytoplasm and affect HBV pgRNA encapsidation process. M6A modification at the 5′ end of HBV pgRNA promotes reverse transcription. (C) HBx induces nuclear import of METTL3/METTL14 and recruits the m6A writers to the HBV cccDNA, inducing co-transcriptional m6A modification of HBV RNA/pgRNA.

Figure 2.

m6A modification of HBV RNA and its functional effects. (A) HBV RNA is methylated only at a single position, in the typical DRACH motif (D = A, G, or U; R = A or G; and H = A, C, or U), at A1907 at the 3′ end. As pgRNA contains two copies of the DRACH motif due to terminal redundancy, it is m6A methylated both at 3′ and 5′ ends. (B) HBV RNA/pgRNA is m6A modified by m6A writers (METTL3/METTL14) where METTL3 uses S-adenosyl methionine as a methyl donor, and METTL14 functions as an RNA-binding platform. M6A-modified HBV RNA/pgRNA is then recognized by specific reader proteins, such as YTHDC1-2, YTHDF1-3, IGF2BP1-3, eIF3, FMRP, FXR1, FXR2, SND1, hnRNPA2B1, hnRNPC, hnRNPG, etc. The effects of m6A modification typically depend on the recognition by different m6A readers. Interaction with YTHDF2 and YTHDF3 reduces stability and accelerates the decay of m6A-modified HBV RNA and reduces HBV protein production. YTHDC1 and FMRP readers promote export of HBV RNA from the nucleus into the cytoplasm and affect HBV pgRNA encapsidation process. M6A modification at the 5′ end of HBV pgRNA promotes reverse transcription. (C) HBx induces nuclear import of METTL3/METTL14 and recruits the m6A writers to the HBV cccDNA, inducing co-transcriptional m6A modification of HBV RNA/pgRNA.

Figure 3.

Interplay between innate immune responses and m6A-modified HBV RNA. (A) HBV RNA devoid of m6A marks is readily recognized by cytoplasmic RNA sensor RIG-I followed by activation of the downstream RIG-I signalling, dephosphorylation of IRF-3 and activation of anti-viral IFN responses which contribute to HBV RNA degradation. In contrast, m6A-modified HBV RNA is coupled with YTHDF2 which shields viral RNA from recognition by RIG-I. While RIG-I was found to recognize 5′-ϵ region of HBV pgRNA inducing type III IFN response and preventing interaction of pgRNA with viral Polymerase [33], some evidence suggests that RIG-I is unable to recognize HBV RNA [34,35]. Additionally, HBV can efficiently suppress or bypass RIG-I recognition [36,37]. (B) Treatment of HBV-infected cells with IFNα activates expression of interferon-stimulated genes, including an RNA exonuclease molecule ISG20. In turn, ISG20 can interact only with m6A-modified and YTHDF2-bound HBV RNA, resulting in rapid RNA degradation. (C) In uninfected cells, PTEN mRNA can activate IRF-3 and induce IFN signalling, thus contributing to antiviral immune responses. Upon HBV infection, HBV HBx protein mediates recruitment of METTL3/METTL14 complex to PTEN mRNA and its m6A methylation. Methylated PTEN mRNA is then bound by YTHDF2 reader protein, reducing its stability and promoting PTEN mRNA decay.

Figure 3.

Interplay between innate immune responses and m6A-modified HBV RNA. (A) HBV RNA devoid of m6A marks is readily recognized by cytoplasmic RNA sensor RIG-I followed by activation of the downstream RIG-I signalling, dephosphorylation of IRF-3 and activation of anti-viral IFN responses which contribute to HBV RNA degradation. In contrast, m6A-modified HBV RNA is coupled with YTHDF2 which shields viral RNA from recognition by RIG-I. While RIG-I was found to recognize 5′-ϵ region of HBV pgRNA inducing type III IFN response and preventing interaction of pgRNA with viral Polymerase [33], some evidence suggests that RIG-I is unable to recognize HBV RNA [34,35]. Additionally, HBV can efficiently suppress or bypass RIG-I recognition [36,37]. (B) Treatment of HBV-infected cells with IFNα activates expression of interferon-stimulated genes, including an RNA exonuclease molecule ISG20. In turn, ISG20 can interact only with m6A-modified and YTHDF2-bound HBV RNA, resulting in rapid RNA degradation. (C) In uninfected cells, PTEN mRNA can activate IRF-3 and induce IFN signalling, thus contributing to antiviral immune responses. Upon HBV infection, HBV HBx protein mediates recruitment of METTL3/METTL14 complex to PTEN mRNA and its m6A methylation. Methylated PTEN mRNA is then bound by YTHDF2 reader protein, reducing its stability and promoting PTEN mRNA decay.

Figure 4.

m6A-mediated mechanisms of HBV-induced liver carcinogenesis. (A) HBx upregulates METTL3 expression, resulting in m6A methylation of circ-ARL3 and its interaction with YTHDC1 reader protein. M6A modification enhances circ-ARL3 biogenesis and sponging of a strong tumour-suppressive miR-1305. Sponging inactivates miR-1305 and contributes to HCC development. (B) PTEN is a potent tumour suppressor. HBx-mediated m6A modification of PTEN mRNA results in PTEN mRNA degradation and loss of its function. (C) HBx stabilizes WD repeat-containing protein 5 (WDR5) protein, a subunit of histone H3 lysine 4 methyltransferase complex, promotes H3K4me3 histone modification of ALKBH5 promoter and upregulation ALKBH5 mRNA expression. ALKBH5 catalyses demethylation of HBx mRNA, increasing its stability and production of pro-oncogenic HBx protein. As such, HBx and ALKBH5 form a positive feedback loop and can be regarded a self-accelerating mechanism of HCC development.

Figure 4.

m6A-mediated mechanisms of HBV-induced liver carcinogenesis. (A) HBx upregulates METTL3 expression, resulting in m6A methylation of circ-ARL3 and its interaction with YTHDC1 reader protein. M6A modification enhances circ-ARL3 biogenesis and sponging of a strong tumour-suppressive miR-1305. Sponging inactivates miR-1305 and contributes to HCC development. (B) PTEN is a potent tumour suppressor. HBx-mediated m6A modification of PTEN mRNA results in PTEN mRNA degradation and loss of its function. (C) HBx stabilizes WD repeat-containing protein 5 (WDR5) protein, a subunit of histone H3 lysine 4 methyltransferase complex, promotes H3K4me3 histone modification of ALKBH5 promoter and upregulation ALKBH5 mRNA expression. ALKBH5 catalyses demethylation of HBx mRNA, increasing its stability and production of pro-oncogenic HBx protein. As such, HBx and ALKBH5 form a positive feedback loop and can be regarded a self-accelerating mechanism of HCC development.