Identifying Host Transcription Factors Governing Hepatitis B Virus Covalently Closed Circular DNA Expression

Abstract

Hepatitis B Virus (HBV) is a human hepatotropic pathogen that causes acute and chronic hepatitis. It is one of the most prevalent chronic viral infections worldwide, leading to liver fibrosis, cirrhosis, decompensated liver disease, and hepatocellular carcinoma (HCC), collectively accounting for approximately 786,000 deaths annually. Although HBV can be prevented through vaccination, current antiviral therapies remain non-curative due to the persistence of a long-lasting episomal DNA, covalently closed circular DNA (cccDNA). cccDNA is formed in the nucleus of infected hepatocytes from lesion-bearing relaxed circular DNA delivered by the HBV virion and serves as the transcriptional template for most viral RNAs essential to the viral cycle. Because HBV gene expression depends entirely on host transcriptional machinery, we aimed to comprehensively characterize host factors that regulate cccDNA transcription, as these could subsequently be exploited for antiviral therapies. Thus, we generated recombinant reporter cccDNA molecules to study HBV promoter activity (Core, S, and X) in HBV infection-permissive (HepG2) and non-permissive (293T) embryonic cells. We found that core promoter expression was robust in hepatic cells but barely detectable in 293T cells. Based on these observations, we hypothesized that hepatocyte-specific transcription factors play a critical role in regulating HBV core promoter expression. We therefore investigated hepatocyte-enriched transcription factors previously reported to bind the core promoter. Using a focused CRISPRi screen, we identified HNF4α as a potential key regulator of HBV infection. However, overexpression of three HNF4α isoforms in 293T cells did not restore core promoter activity, suggesting that these isoforms alone are insufficient, and that additional isoforms and/or cofactors are required to induce cccDNA transcription. We further identified three HNF4α binding sites within the HBV cccDNA sequence and generated different single, double, and triple mutants. Transfection of these mutant cccDNAs revealed that disruption of a single binding site within the X promoter is sufficient to reduce HBV antigen release. Altogether, these findings provide new insights into the regulation of HBV gene transcription and the tools generated establish a versatile platform for large-scale screens to identify and systematically characterize host factors governing this process. Ultimately, these advances may inform the development of new antiviral strategies aimed at treating, and potentially curing, HBV infection—a major global health burden.