A genome-wide siRNA screen identifies previously unknown proviral and antiviral host factors in HBV infection

Xiaoming Cheng; Yuchen Xia; Chen Li; Min Zhang; Tadashi Inuzuka; Sirisha Chakka; Quan Chen; Yan Teng; Jianwei Mao; Xiaoyun Li; Wei Dong; Karim Mouzannar; Ken Chih-Chien Cheng; T Jake Liang

doi:10.1016/j.jhep.2025.11.017

A genome-wide siRNA screen identifies previously unknown proviral and antiviral host factors in HBV infection

J Hepatol. 2025 Nov 26:S0168-8278(25)02656-X. doi: 10.1016/j.jhep.2025.11.017. Online ahead of print.

Authors

Xiaoming Cheng¹, Yuchen Xia², Chen Li³, Min Zhang⁴, Tadashi Inuzuka⁴, Sirisha Chakka⁵, Quan Chen³, Yan Teng⁶, Jianwei Mao³, Xiaoyun Li³, Wei Dong⁷, Karim Mouzannar⁴, Ken Chih-Chien Cheng⁵, T Jake Liang⁸

Affiliations

¹ Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD 20814, USA; Department of Pathology, TaiKang Medical School, Zhongnan Hospital, Wuhan University, Wuhan, China. Electronic address: Xiaoming.cheng@whu.edu.cn.
² Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD 20814, USA; State Key Laboratory of Virology and Biosafety, Hubei Province Key Laboratory of Allergy and Immunology, Institute of Medical Virology, TaiKang Center for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan, China.
³ Department of Pathology, TaiKang Medical School, Zhongnan Hospital, Wuhan University, Wuhan, China; State Key Laboratory of Virology and Biosafety, Hubei Province Key Laboratory of Allergy and Immunology, Institute of Medical Virology, TaiKang Center for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan, China.
⁴ Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD 20814, USA.
⁵ National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA.
⁶ State Key Laboratory of Virology and Biosafety, Hubei Province Key Laboratory of Allergy and Immunology, Institute of Medical Virology, TaiKang Center for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan, China.
⁷ Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; Hubei Key Laboratory of Hepato-Pancreato-Biliary Diseases, Wuhan, Hubei, China.
⁸ Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD 20814, USA. Electronic address: jake.liang@nih.gov.

PMID: 41314348
PMCID: PMC12922363 (available on 2026-11-26)
DOI: 10.1016/j.jhep.2025.11.017

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.

Keywords: CHD4; Epigenetic regulation; Genome-wide siRNA screen; HBV infection; NCOA5; NRAS.

Abstract

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