Abstract
BACKGROUND & AIMS: Hepatitis B virus (HBV) extensively exploits host cellular machinery for productive infection. This study aimed to comprehensively identify and validate host factors critical for HBV infection using a functional genomics approach. METHODS: A whole-genome small-interfering RNA screen was performed in HepG2-NTCP cells, utilizing high-throughput AlphaLISA detection of intracellular HBV core and e antigens as the readout. Selected genes underwent rigorous multi-tiered validation: in vitro assessment of viral infection/replication via knockdown, overexpression, and knockout in hepatoma cell lines or primary human hepatocytes; analysis of gene expression in tumor/non-tumor tissues from 21 patients with HBV-related hepatocellular carcinoma; and in vivo evaluation using an AAV-HBV mouse model with target modulation. RESULTS: Validation confirmed nuclear receptor coactivator 5 (NCOA5) and chromodomain-helicase-DNA-binding protein 4 (CHD4) as essential proviral host factors; their knockdown significantly reduced HBV replication. Conversely, neuroblastoma RAS (NRAS) was identified as an antiviral factor. Mechanistically, NCOA5 likely operates via the estrogen receptor and hepatocyte nuclear factor 4 alpha (HNF4α) axis, while CHD4 modulates cccDNA histone modifications. NRAS knockdown enhanced HBV transcription by elevating HNF4α expression and inducing cell cycle arrest, which may explain the observed restriction of HBV replication in HBV-associated hepatocellular carcinoma tissues, where RAS hyperactivation frequently occurs. CONCLUSIONS: This study identifies NCOA5 and CHD4 as crucial proviral cofactors and NRAS as a potent antiviral factor regulating HBV replication. The findings highlight HBV's profound host dependence, uncover specific molecular mechanisms (involving HNF4α, epigenetic regulation of cccDNA, and cell cycle), and reveal validated host targets for potential therapeutic strategies against HBV infection. IMPACT AND IMPLICATIONS: Hepatitis B virus (HBV) needs human liver cell machinery to infect and multiply. We used large-scale genetic screening to find human proteins that either help or block HBV infection. We discovered two key proteins (NCOA5 and CHD4) that help HBV replicate, and one protein (NRAS) that blocks it. We confirmed these findings in human liver cells and mice. Understanding how these proteins control HBV (through regulating viral transcription, modifying viral DNA, or affecting cell growth) reveals potential new targets for developing better treatments against this serious liver infection.