Clare Gilmour, University Of Alberta

Clare Gilmour1, Kira Sviderskaia2, Lucy Luo3, Olivier Julien2-3, Vanessa Meier-Stephenson1-2,4 1Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, 2Department of Medicine, University of Alberta, Edmonton, AB, 3Department of Biochemistry, University of Alberta, Edmonton, AB 4Li Ka Shing institute of Virology, University of Alberta, Edmonton, AB

Discovering & Evolving

Background: Hepatitis B Virus (HBV) infects over 300 million people globally and contributes to more than 50% of hepatocellular carcinoma (HCC) cases. Despite its global impact, the mechanisms by which HBV drives HCC remain incompletely understood. Two mutations in the pre-core promoter (PCP) region, A1762T and G1764A, are strongly linked to increased HCC risk. Due to gene overlap, these mutations also result in K130M and V131I substitutions in the HBV X protein (HBx). It is unclear whether this increased risk arises from enhanced promoter activity, altered HBx function, or both, which is what this study aims to determine. Methods: Site-directed mutagenesis was used to introduce PCP mutations into a luciferase reporter plasmid under the control of the HBV PCP. Wild-type (WT) and mutant constructs were transfected into HepG2-NTCP cells, and luciferase activity was measured. To evaluate HBx's role, co-transfections were performed using WT or mutant HBx tagged at either the N- or C-terminus. Additionally, proteomic studies were initiated using anti-FLAG magnetic beads with N-terminally tagged HBx. Results: A two-way ANOVA revealed significant effects of both the PCP mutations alone and in combination with HBx. Notably, N-terminally tagged HBx increased promoter activity, while C-terminal tagging had no effect. Preliminary proteomic analysis of WT and mutant HBx is underway to investigate potential protein interactions. Conclusions: Although PCP mutations suppress promoter activity, N-terminally tagged HBx significantly increases it. These findings highlight HBx as a key driver of HBV-associated HCC and underscore the importance of its structural integrity in viral pathogenesis.