Structural & Functional Investigations of Hepatitis B Virus Pol Activity in a Native-like Context

Hepatitis B Virus is the major cause of liver cancer worldwide. Despite the availability of an effective, safe and cheap vaccine roughly a million people are newly infected each year. They join a cohort of about 240 million patients who suffer from chronic HBV infection after failing to fully clear an initial exposure to the virus. The current outlook for these people is poor since the major clinical anti-viral drugs, directed at the reverse transcriptase active site of the viral polymerase, do not eradicate virus, and therefore imply lifelong treatment. Sadly, death from HBV induced liver cancer is lowered only by a maximum of 4-fold after decades of this treatment. Over 700,000 people die roughly every year as a result of infection which causes liver failure, cirrhosis and liver cancer. The WHO have issued a Global Challenge to make HBV infection a treatable disease by 2030. A major barrier to meeting this challenge is the difficulty in studying the viral polymerase in its native environment - a virally-induced protein container composed of virally-encoded core protein, in which the viral nucleic acid is converted from a single-stranded RNA form to a nicked double-stranded DNA version, the source of continued infection in the liver. This difficulty is principally due to the polymerase, which is only poorly soluble in most conditions. We have developed a unique system that can be studied safely in the bacterium E.coli, in which the polymerase is stabilised by binding to its native RNA target, the epsilon stem-loop, within a native-like viral protein shell. We will use this system to investigate how the enzyme achieves these potentially lethal nucleic acid transformations within this specialised protein container using spectroscopic techniques to assay the enzymatic reactions. These will be coupled with state-of-the-art structural studies in our world-class electron microscopy laboratory, and a specialised technique we have developed for studying the structures of nucleic acids within viruses. These will allow us to explore the mechanisms of action of leading novel anti-viral drug candidates, and make this system widely available for rapid therapeutic exploitation.

We have discovered a way to produce nucleocapsid-like particles (NCPs) of Hepatitis B Virus (HBV) core protein (Cp) in which the viral polymerase (Pol) and transcripts encompassing most of the viral genome self-assemble in E.coli. The enzymatic reactions required to create the nicked dsDNA version of the viral genome, i.e. the essential replicative steps of HBV infection, seem to occur within these artificial NCPs. This is a first in the field and opens exciting opportunities to study the processes of reverse transcription, RNase H template degradation, and DNA polymerisation in a near native particle. Such NCPs could potentially be the basis of future drug discovery screens, as well as providing mechanistic insights into the basic pathogenic enzymology. We will use this set-up here to interrogate the Pol activities in the NCPs, monitoring the impacts of enzyme inhibitors (drugs), and gRNA on these reactions, studying immobilised NCPs by smTIRF microscopy. Using atomic resolution cryo-EM reconstructions of NCPs at differing stages of these replication reactions, we will identify the structural consequences of Pol action. We will also use X-ray RNA/DNA footprinting of the functional NCPs to understand how Pol action interfaces with features known to be important in NCP assembly. This will provide unprecedented insights into the reverse-transcription and other Pol functions within a near native environment, and thus pave the way for the future development of Pol targeting anti-HBV drugs.