ABS 125: Genome-wide CRISPR screen reveals host genes that regulate Hepatitis B Virus life cycle

Alan Nesterenko ¹ Yaron Bram ¹ , Rachel Tiersky ¹ , Robert E Schwartz ¹ ²

¹ Department of Medicine, Weill Cornell Medicine, New York, NY, United States
² Department of Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, New York, NY, USA

Van Wickle (2025) Volume 1, ABS 125

Introduction: Hepatitis B virus (HBV) is difficult to eradicate due to its ability to persist in host cells and reactivate under certain conditions. Current treatments suppress viral replication but rarely result in a complete cure. A major obstacle to developing curative therapies is the limited understanding of how HBV interacts with host genetic factors to promote replication and evade immune responses.
To address this, we developed a cellular model that replicates the full HBV life cycle and allows for the identification of chronically infected cells and precise quantification of viral gene expression. Using genome-wide CRISPR-Cas9 screens, we identified host genes that influence HBV replication and antigen production. We then used knockout models to investigate the impact of these genes on viral components such as HBsAg, HBeAg, and pregenomic RNA (pgRNA).

Through this approach, we identified both positive and negative regulators of HBV gene expression and analyzed the cellular pathways that mediate these effects. Our findings highlight novel host factors involved in the regulation of viral replication, especially those affecting Hepatitis B surface antigen (HBsAg) expression.

Moving forward, we plan to examine how these regulatory pathways function in HBV-infected primary human hepatocytes. This will help determine whether the identified host factors can serve as potential therapeutic targets and provide further insight into host-virus interactions in chronic HBV infection.

Methods: construct into the host genome. Cre recombinase was then introduced to trigger HBV reactivation and covalently closed circular DNA (cccDNA) formation. Upon activation, HBV protein expression was confirmed by detecting viral markers including HBsAg and HBeAg. We then performed a genome-wide CRISPR-Cas9 knockout screen to identify host genes that regulate HBV expression. Top candidate genes were selected for hit validation using individual knockouts, followed by ELISA and qPCR to measure viral protein and RNA levels. Finally, pathway analysis was conducted with a focus on genes in the UFMylation pathway and innate immune signaling. We specifically investigated how HBx suppresses MAVS, a key antiviral adaptor, through a UFM1-dependent mechanism to reveal host-virus interactions driving HBV persistence.

Results: Through genome-wide CRISPR screening, we identified genes in the UFMylation pathway, including UBA5, UFC1, and UFL1, as key regulators of HBV surface antigen production. UFMylation is a post-translational modification process that attaches the small protein UFM1 to target proteins, regulating their stability and function, particularly during cellular stress and ER homeostasis. Notably, we found that HBx suppresses MAVS, a central adaptor protein in the innate antiviral response, through a UFM1-dependent mechanism, revealing a potential strategy used by HBV to evade immune detection and maintain chronic infection.

Discussion: We plan to further investigate the UFMylation pathway and MAVS signaling to better understand their roles in supporting chronic HBV infection and viral immune evasion. By studying these regulatory mechanisms in more depth, including in primary hepatocytes, we aim to clarify how host pathways are manipulated by HBV. Ultimately, our goal is to use this knowledge to identify and develop targeted antiviral therapies that disrupt these interactions and offer more effective treatment options for chronic HBV infection.