Human hepatitis B virus (HBV causes chronic hepatitis. Chronic hepatitis B is a major risk factor in the progression to the onset of hepatocellular carcinoma (HCC). In spite of an effective vaccine and currently used antivirals, there are estimated 350 million infected carriers worldwide. Antivirals do not eliminate HBV DNA in the nucleus. A better understanding of the role of host and viral factors contributing to chronic hepatitis is needed. Epitranscriptomic regulation of HBV gene expression provides another layer of regulatory schemes of gene expression. In this study, we propose to characterize the posttranscriptional modification of viral RNAs at the N6 position of the adenosine base termed m6A. Our extensive preliminary studies described in the grant application show that HBV RNAs are methylated as m6A. Using the strategy of silencing of host methylases and demethylase enzymes involved in epitranscriptomic homeostasis, we present evidence that HBV RNAs are m6A modified. There was clearly an effect on translation of viral transcripts in the absence of enzyme that catalyze m6A modification. Our results with MeRIP immunoprecipitation followed by Tru-seq analysis identified a potential range of sequences enriched in a peak of m6A residues. We mutated about 13 `A' residues in this region that spans from nucleotides 1700 to 2000 within the HBV genome. These were examined for translation of HBV proteins and reverse transcription function. Results were striking, in that, the translation was negatively regulated but reverse transcription was positively regulated by m6A modification. Based on these exciting data, we propose to continue characterization of m6A modification of HBV RNAs and identify the functional relevance of this modification in the HBV infection. This study opens a new avenue of HBV research to investigate epitranscriptomic regulation of the unique HBV life cycle and possible contribution to the progression to chronicity associated with infection.
Project Summary/Narrative Hepatitis B virus (HBV) infection causes chronic hepatitis, a major health burden worldwide. Here, we propose to investigate epitranscriptomic regulation of hepatitis B virus gene expression with particular modification of N6-methylyadenosine (m6A) of viral RNAs. This study will establish the functional role of m6A modification in viral life cycle and liver disease pathogenesis.