Identifying factors regulating production and expression of HBV cccDNA and the minichromosome using in vitro and in vivo models of HBV replication

Abstract

Chronic Hepatitis B (CHB) affects more 250 million people globally, causing significant morbidity and mortality from associated sequelae. The etiological agent, hepatitis B virus (HBV) contributes to the majority of cirrhosis and hepatocellular carcinoma (HCC) cases, resulting in nearly a million deaths annually. Carriage of the virus (HBsAg positivity) is associated with elevated risk of developing these liver complications, so while available nucleos(t)ide analogue (NUC/NA) therapy can effectively control viral replication, viral clearance to the point of ‘functional cure’ is required to significantly lower the risk. Moreover, development of a cure remains paramount as treatment is typically life-long, and cessation of therapy can lead to reactivation of the virus via the nuclear reservoir of a viral genome template known as the covalently closed circular DNA (cccDNA) minichromosome. Unaffected by conventional therapies, cccDNA is the required template for the synthesis of all viral transcripts, including greater-than-genome transcript pregenomic RNA (pgRNA), which in turn, is the template for reverse transcription to form the relaxed circular DNA (rcDNA) genome of progeny virions. Despite immense efforts, much remains to be elucidated about the virus and its replication cycle, largely due to the fact that the HBV research field is restricted by a severe lack of suitable/appropriate models for investigating all stages of the viral cycle, with the cccDNA minichromosome being particularly difficult to interrogate.

This PhD project addressed this knowledge gap by characterising a monomeric/genome-length based transfection system both in vitro and in vivo, and investigating its utility as a novel model enabling investigation of cccDNA-like molecules. The model was utilised to further explore the impact of different/specific viral-host protein interactions on viral replication. Specifically, this was centred on the viral HBx and cellular DDB1 proteins, as the interaction between these two was recently demonstrated to facilitate viral transcription following viral infection. Finally, a broad proteomics approach involving microarray screening was applied to identify novel host factors/mechanisms associated with HBV replication. HBV interacts with many cellular proteins and factors, redirecting the associated processes to benefit viral replication and propagation. While some of these cellular components have been described previously, likely many more are yet to be discovered, and this project aimed to identify additional potential therapeutic targets that could ultimately contribute to HBV cure.