-methyladenosine (mA) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found mA on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that mA 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 mA to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the mA site in the HBV RNA and found that a conserved mA consensus motif situated within the epsilon stem loop structure, is the site for mA 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 mA site in the 5' epsilon stem loop of pgRNA revealed that mA at this site is required for efficient reverse transcription of pgRNA, while mA methylation of the 3' epsilon stem loop results in destabilization of all HBV transcripts, suggesting that mA has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how mA regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.

A majority of the human genome is transcribed into noncoding RNAs, of which the functions of long noncoding RNAs (lncRNAs) are poorly understood. Many host proteins and RNAs have been characterized for their roles in HIV/AIDS pathogenesis, but there is only one lncRNA, NEAT1, which is shown to affect the HIV-1 life cycle. We profiled 90 disease-related lncRNAs and found NRON (noncoding repressor of Nuclear Factor of Activated T cells [NFAT]) to be one of several lncRNAs whose expression was significantly altered following HIV-1 infection. The regulation of NRON expression during the HIV-1 life cycle was complex; its levels were reduced by the early viral accessory protein Nef and increased by the late protein Vpu. Consequently, Nef and Vpu also modulated activity of the transcription factor NFAT. The knockdown of NRON enhanced HIV-1 replication through increased activity of NFAT and the viral LTR. Using siRNA-mediated NFAT knockdown, we show the effects of NRON on HIV-1 replication to be mediated by NFAT, and the viral Nef and Vpu proteins to modulate NFAT activity through their effects on NRON. These findings add the lncRNA, NRON to the vast repertoire of host factors utilized by HIV for infection and persistence.

N6 methyladenosine (m6A), the methylation of the adenosine base at the nitrogen-6 position, is the most common epitranscriptomic modification of mRNA that affects a wide variety of biological functions. We have previously reported that hepatitis B viral RNAs are m6A modified, displaying a dual functional role in the viral life cycle. Here, we show that cellular m6A machinery regulates host innate immunity against hepatitis B and C viral (HBV/HCV) infections by inducing m6A modification of viral transcripts. The depletion of the m6A writer enzymes (METTL3 and METTL14) leads to an increase in viral RNA recognition by retinoic acid-inducible gene I (RIG-I), thereby stimulating type-I interferon production. This is reversed in cells, in which m6A METTL3 and METTL14 are overexpressed. The m6A modification of viral RNAs renders RIG-I signaling less effective, and while single nucleotide mutation of m6A consensus motif of viral RNAs, enhances RIG-I sensing activity. Importantly, m6A reader proteins (YTHDF2 and YTHDF3) inhibit RIG-I transduced signaling activated by viral RNAs by occupying m6A modified RNAs and inhibiting RIG-I recognition. Collectively, our results provide new insights into the mechanism of immune evasion via m6A modification of viral 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 (m 6 A)-modified HBV transcripts are selectively recognized and processed for degradation by ISG20. Moreover, this effect of ISG20 is critically regulated by m 6 A reader protein, YTHDF2 (YTH-domain family 2). Previously, we identified a unique m 6 A 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 m 6 A reader protein YTHDF2 are silenced in HBV expressing cells. By using an enzymatically inactive form ISG20 D94G , we further demonstrated that ISG20 forms a complex with m 6 A 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 m 6 A site at either one (5´or 3´) or both the termini (5´& 3´). Using these mutants, we demonstrated that m 6 A modified HBV RNAs are subjected to ISG20-mediated decay and propose sequence of events, in which ISG20 binds with YTHDF2 and recognizes m 6 A-modified HBV transcripts to carry out the ribonuclease activity. This is the first study, which identifies a hitherto unknown role of m 6 A 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.

Background and Aims Epitranscriptomic 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 (m6A) addition. HBV also affects m6A 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 Results Here, we show that HBV significantly increases the m6A 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 m6A 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, m6A‐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. Conclusions The m6A 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 m6A modification of the host PTEN mRNAs.

N6-methyladenosine (m6A) is the most prevalent and internal modification of eukaryotic mRNA. Multiple m6A methylation sites have been identified in the viral RNA genome and transcripts of DNA viruses in recent years. m6A modification is involved in all the phases of RNA metabolism, including RNA stability, splicing, nuclear exporting, RNA folding, translational modulation, and RNA degradation. Three protein groups, methyltransferases (m6A-writers), demethylases (m6A-erasers), and m6A-binding proteins (m6A-readers) regulate this dynamic reversible process. Here, we have reviewed the role of m6A modification dictating viral replication, morphogenesis, life cycle, and its contribution to disease progression. A better understanding of the m6A methylation process during viral pathogenesis is required to reveal novel approaches to combat the virus-associated diseases.

YTHDC1 and fragile X mental retardation protein (FMRP) bind N6-methyladenosine (m6A) modified RNAs and facilitate their transport to the cytoplasm. Here, we investigated the role of these proteins in Hepatitis B virus (HBV) gene expression and life cycle. We have previously reported that HBV transcripts are m6A-methylated and this modification regulates the viral life cycle. HBV is particularly interesting as its DNA genome upon transcription gives rise to a pregenomic RNA (pgRNA), which serves as a template for reverse transcription to produce the relaxed circular DNA that transforms into a covalently closed circular DNA (cccDNA). While m6A modification negatively affects RNA stability and translation of viral transcripts, our current results revealed the possibility that it may positively affect pgRNA encapsidation in the cytoplasm. Thus, it plays a differential dual role in the viral life cycle. YTHDC1 as well as FMRP recognize m6A-methylated HBV transcripts and facilitate their transport to the cytoplasm. In cells depleted with YTHDC1 or FMRP, viral transcripts accumulate in the nucleus to affect the viral life cycle. Most importantly, the core-associated DNA and subsequent cccDNA syntheses are dramatically affected in FMRP or YTHDC1-silenced cells. This study highlights the functional relevance of YTHDC1 and FMRP in the HBV life cycle with the potential to arrest liver disease pathogenesis. IMPORTANCE YTHDC1 and FMRP have been recently implicated in the nuclear export of m6A modified mRNAs. Here, we show that FMRP and YTHDC1 proteins bind with m6A-modified HBV transcripts and facilitate their nuclear export. In the absence of FMRP and YTHDC1, HBV transcripts accumulate inside the nucleus to reduce reverse transcription inside HBV core particles and subsequently the cccDNA synthesis. Our study shows how m6A binding proteins can regulate the HBV life cycle by facilitating the nuclear export of m6A-modified HBV RNA.

Autophagy is a self-eating process, in which the damaged or excessed cell organelles and misfolded protein aggregates are removed from the cellular microenvironment. Autophagy is generally thought of as a pro-survival mechanism which is not only important for balancing energy supply at times of nutrient deprivation but also in the removal of various stress stimuli to ensure homeostasis. In addition to the target materials of “self” origin, autophagy can also eliminate intracellular pathogens and acts as a defense mechanism to curb infections. In addition, autophagy is linked to the host cell’s innate immune response. However, viruses have evolved various strategies to manipulate and overtake host cell machinery to establish productive replication and maintain infectious process. In fact, replication of many viruses has been found to be autophagy-dependent and suppression of autophagy can potentially affect the viral replication. Thus, autophagy can either serve as an anti-viral defense mechanism or a pro-viral process that supports viral replication. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are known to co-opt cellular autophagy process as a pro-viral tool. Both viruses also induce mitophagy, which contributes to the establishment of chronic hepatitis. This review focuses on the roles of autophagy and mitophagy in the chronic liver disease pathogenesis associated with HBV and HCV infections.