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 antiviral, 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 has not been investigated. In this study, we undertake 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 silencing of host methylases and demethylase enzymes involved in epitranscriptomic homeostasis, we establish 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 studied for translation of HBV core protein and reverse transcription function. Results were striking, in that, translation was negatively regulated but reverse transcription was positively regulated by m6A modification. Based on these exciting data, we propose to characterize m6A modification of HBV RNAs and identify the functional relevance of this modification in the HBV infection. Several aspects of their role in translation of HBsAg, HBeAg and HBx proteins will be studied. Effect of m6A modification in the Core associated pregenomic RNAs will be fully characterized. This study has opened a new avenue of HBV research in which to fully characterize how epitranscriptomic regulation may contribute to the unique viral life cycle and the progression to chronicity associated with HBV.

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Project Summary/Narrative Hepatitis B virus (HBV) infection causes chronic hepatitis, a major health burden worldwide. A clear understanding of host and viral factors is necessary to eliminate chronic hepatitis B. Here, we propose to investigate epitranscriptomic regulation of hepatitis B virus gene expression with particular modification of N6- methylyadenosine (m6A). This study will establish the functional role of this modification in viral life cycle and liver disease pathogenesis.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
High Priority, Short Term Project Award (R56)
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Virology - B Study Section (VIRB)
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Koshy, Rajen
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University of California, San Diego
Internal Medicine/Medicine
Schools of Medicine
La Jolla
United States
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