Creating a Fluorescently-Tagged HBV POL Construct to Enable Live Visualization of POL in Natural Infection Contexts

Abstract

Chronic infection with hepatitis B virus (HBV) is an ongoing global health issue affecting an estimated 296 million people. Left untreated, HBV can lead to the development of decompensated cirrhosis and hepatocellular carcinoma (HCC). Immunizations administered at birth protect against chronic disease but are not universally accessible. Current treatments can slow disease progression and reduce HCC risk but require daily administration to sustain viral suppression. An important yet currently understudied part of the HBV replication cycle is the viral polymerase (POL), which mediates reverse transcription of pregenomic RNA (pgRNA) into relaxed circular DNA (rcDNA) within assembled nucleocapsids. POL remains particularly challenging to study in physiologically relevant contexts due to a lack of well-characterized monoclonal antibodies, low expression levels relative to other HBV viral proteins, and no current methods to visualize POL dynamics in real time. Existing methods for imaging POL require cell lysis or necessitate overexpression systems that are not representative of native viral contexts. In this thesis, we aimed to address these key limitations in the field by developing a recombinant covalently closed circular DNA (cccDNA) construct where HBV POL is fused to the fluorescent protein mStayGold (SG) and can be expressed through the native viral promoter along with other viral proteins. We initially generated and validated wildtype (WT) and knockout mutant cccDNA constructs (POL_KO and core_KO) to establish the recombinant cccDNA platform we would utilize. We then generated SG-encoding recombinant cccDNA constructs (POL-SG and POL_KO-SG), which also had their topological integrities and sequences verified. Transfection of HepG2 cells with POL-SG cccDNA produced detectable viral antigens intracellularly (HBcAg and HBsAg) and extracellularly (HBeAg and HBsAg), as well as mStayGold fluorescence, which provided initial evidence that the fusion protein was successfully being expressed. Unexpectedly, we also detected SG fluorescence in POL_KO-SG, which we hypothesize is the result of alternative splicing of the HBV pgRNA excising the introduced stop codon. Experiments are currently underway to confirm this hypothesis. If validated, POL-SG will present a novel tool that will allow for the spatiotemporal dynamics of HBV POL to be studied under conditions that better approximate natural HBV infection.