Epigenetic Regulation of Hepatitis B Virus in vivo

Chronic hepatitis B virus (HBV) infection is a global public health concern responsible for approximately 1 million deaths annually and rising. Current mainstays of HBV treatment, nucleoside analogs and interferon, are associated with drug resistance and negative side effects. Most importantly, these treatments are not curative and only slow the progression of chronic HBV, which frequently leads to significant liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Chronic HBV infection is maintained by viral covalently closed circular (ccc) DNA, a highly stable replication product and transcriptional template which exists as an episome in the nucleus. As such, the epigenetic silencing of nuclear HBV DNA represents one potential mechanism that may address curing chronic HBV infection. As the limited coding capacity of the virus leaves few viral drug targets, targeting host cellular factors that contribute to successful HBV replication is imperative. In the HBV transgenic mouse model of chronic infection, the forkhead box protein A/hepatocyte nuclear factor 3 (Foxa/HNF3) family of pioneer transcription factors and Ten-eleven translocation (Tet) methylcytosine dioxygenases are required to support postnatal viral demethylation and subsequent HBV transcription and replication. Liver-specific Foxa-deficient mice with hepatic expression of only Foxa3 do not support HBV replication but display biliary epithelial hyperplasia with bridging fibrosis. However, liver-specific Foxa-deficient mice with hepatic expression of only Foxa1 or Foxa2 also successfully restrict viral transcription and replication but display only minimal alterations in liver physiology. These observations suggest that the level of Foxa activity, rather than the combination of specific Foxa genes, is a key determinant of HBV biosynthesis. Liver-specific Tet2 and 3-deficient HBV transgenic mice exhibit a similar phenotype: The levels of viral transcription and replication intermediates are dramatically reduced. Hepatitis B core antigen is only observed in a very limited number of pericentral hepatocytes in a pattern that is similar to glutamate-ammonia ligase (Glul), a β-catenin target gene. HBV transcript abundance in adult Tet-deficient mice resembles that observed in wild-type neonatal mice. Additionally, HBV transgene DNA displays increased 5-methylcytosine (5mC) frequency at CpG sequences consistent with neonatal Tet deficiency being responsible for decreased developmental viral DNA demethylation mediated by 5mC oxidation to 5-hydroxymethylcytosine. Together, these data support a model wherein Foxa factors bind to HBV DNA and recruit Tet enzymes to mediate the demethylation of the viral genome and support subsequent viral biosynthesis. Consequently, Foxa and/or Tet inhibitors may potentially be developed into therapeutic agents capable of inducing and/or maintaining HBV covalently closed circular DNA methylation, resulting in transcriptional silencing and the resolution of chronic viral infection.