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N6-methyladenosine (m6A) is the most prevalent modification of mammalian cellular RNAs. m6A methylation is linked to epigenetic regulation of several aspects of gene expression, including RNA stability, splicing, nuclear export, RNA folding, and translational activity. m6A modification is reversibly catalyzed by methyltransferases (m6A writers) and demethylases (m6A erasers), and the dynamics of m6A-modified RNA are regulated by m6A-binding proteins (m6A readers). Recently, several studies have shown that m6A methylation sites have been identified in hepatitis B virus (HBV) transcripts and the hepatitis C virus (HCV) RNA genome. Here, we review the role of m6A modification in HBV/HCV replication and its contribution to liver disease pathogenesis. A better understanding of the functions of m6A methylation in the life cycles of HBV and HCV is required to establish the role of these modifications in liver diseases associated with these viral infections.

Background and aimsEpitranscriptomic modification of RNA has emerged as the most prevalent form of regulation of gene expression that affects development, differentiation, metabolism, viral infections, and most notably cancer. We have previously shown that hepatitis B virus (HBV) transcripts are modified by N6 methyladenosine (m6 A) addition. HBV also affects m6 A modification of several host RNAs, including phosphatase and tensin homolog (PTEN), a well-known tumor suppressor. PTEN plays a critical role in antiviral innate immunity and the development of hepatocellular carcinoma (HCC). Reports have shown that PTEN controlled interferon regulatory factor 3 (IRF-3) nuclear localization by negative phosphorylation of IRF-3 at Ser97, and PTEN reduced carcinogenesis by inhibiting the phosphatidylinositol-3-kinase (PI3K)/AKT pathway.Approach and resultsHere, we show that HBV significantly increases the m6 A modification of PTEN RNA, which contributes to its instability with a corresponding decrease in PTEN protein levels. This is reversed in cells in which the expression of m6 A methyltransferases is silenced. PTEN expression directly increases activated IRF-3 nuclear import and subsequent interferon synthesis. In the absence of PTEN, IRF-3 dephosphorylation at the Ser97 site is decreased and interferon synthesis is crippled. In chronic HBV patient biopsy samples, m6 A-modified PTEN mRNA levels were uniformly up-regulated with a concomitant decrease of PTEN mRNA levels. HBV gene expression also activated the PI3K/AKT pathway by regulating PTEN mRNA stability in HCC cell lines.ConclusionsThe m6 A epitranscriptomic regulation of PTEN by HBV affects innate immunity by inhibiting IRF-3 nuclear import and the development of HCC by activating the PI3K/AKT pathway. Our studies collectively provide new insights into the mechanisms of HBV-directed immune evasion and HBV-associated hepatocarcinogenesis through m6 A modification of the host PTEN mRNAs.

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.

Interferon (IFN) stimulates a whole repertoire of cellular genes, collectively referred to as ISGs (Interferon-stimulated genes). ISG20, a 3´-5´ exonuclease enzyme, has been previously shown to bind and degrade hepatitis B Virus (HBV) transcripts. Here, we show that the N6-methyladenosine (m6A)-modified HBV transcripts are selectively recognized and processed for degradation by ISG20. Moreover, this effect of ISG20 is critically regulated by m6A reader protein, YTHDF2 (YTH-domain family 2). Previously, we identified a unique m6A site within HBV transcripts and confirmed that methylation at nucleotide A1907 regulates HBV lifecycle. In this report, we now show that the methylation at A1907 is a critical regulator of IFN-α mediated decay of HBV RNA. We observed that the HBV RNAs become less sensitive to ISG20 mediated degradation when methyltransferase enzymes or m6A reader protein YTHDF2 are silenced in HBV expressing cells. By using an enzymatically inactive form ISG20D94G, we further demonstrated that ISG20 forms a complex with m6A modified HBV RNA and YTHDF2 protein. Due to terminal redundancy, HBV genomic nucleotide A1907 position is acquired twice by pregenomic RNA (pgRNA) during transcription and therefore the sites of methylation are encoded within 5´ and 3´ epsilon stem loops. We generated HBV mutants that lack m6A site at either one (5´ or 3´) or both the termini (5´& 3´). Using these mutants, we demonstrated that m6A modified HBV RNAs are subjected to ISG20-mediated decay and propose sequence of events, in which ISG20 binds with YTHDF2 and recognizes m6A-modified HBV transcripts to carry out the ribonuclease activity. This is the first study, which identifies a hitherto unknown role of m6A modification of RNA in IFN-α induced viral RNA degradation and proposes a new role of YTHDF2 protein as a cofactor required for IFN-α mediated viral RNA degradation.

N6-methyladenosine (m6A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m6A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m6A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m6A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m6A site in the HBV RNA and found that a conserved m6A consensus motif situated within the epsilon stem loop structure, is the site for m6A modification. The epsilon stem loop is located in the 3' terminus of all HBV mRNAs and at both the 5' and 3' termini of the pgRNA. Mutational analysis of the identified m6A site in the 5' epsilon stem loop of pgRNA revealed that m6A at this site is required for efficient reverse transcription of pgRNA, while m6A methylation of the 3' epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m6A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m6A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.

BACKGROUND: Chronic stroke can impair cardiopulmonary function, mobility, and daily activities. This study assessed the impact of robot-assisted gait training (RAGT) on such impairments. OBJECTIVE: To investigate the effects of robot-assisted gait training on cardiopulmonary function, walking ability, lower extremity function and strength, activities of daily living (ADLs), and blood test results among individuals with chronic stroke. METHODS: A multicenter, prospective, single-blinded, randomized controlled trial with 22 chronic stroke participants compared RAGT against a control exercise regimen. RAGT involved three days weekly sessions of high-intensity interval training for 8 weeks (24 sessions) with a Morning Walk® device. The control group also performed home exercises. (24 sessions) Measures included VO2max, Functional Ambulatory Category, 2-minute walk test, 10-meter walk test, Motricity Index-Lower, Korean version of the Fugl-Meyer Assessment Scale, Modified Barthel Index, Berg Balance Scale, muscle strength, InBody body composition, and blood tests (cholesterol, lipid, glucose). RESULTS: RAGT significantly improved VO2max, gait, balance, and lower limb strength compared with controls, with significant improvements in 2-minute walk test, 10-meter walk test, Motricity Index-Lower, and Fugl-Meyer Assessment outcomes. No changes were seen in muscle mass or blood markers. CONCLUSION: RAGT enhances cardiopulmonary function and ambulatory capacity in chronic stroke patients, underscoring its potential in stroke rehabilitation. [ABSTRACT FROM AUTHOR]