Large-Scale Production and Structural and Biophysical Characterizations of the Human Hepatitis B Virus Polymerase

Abstract

Hepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies-broad-spectrum antivirals-are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in Escherichia coli, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly ␣-helical structures with small amounts of ␤-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity in vitro, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the in vitro priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV. IMPORTANCEThe viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of muchneeded new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in Escherichia coli and also methods for purifying them in soluble forms that have activity in vitro. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV. Hepatitis B virus (HBV) is a highly infectious, species-specific pathogen that causes serious liver diseases, including cancer, and causes 600,000 deaths annually (1). While excellent prophylactic HBV vaccines exist, these are ineffective against extant chronic HBV infections which affect 350 million people worldwide. Approved therapies for chronic HBV infections include immuno-modulators (e.g., interferon therapies) and nucleoside analogues that inhibit the reverse transcriptase domain of hPOL, the multifunctional viral polymerase of human HBV. These reverse transcriptase inhibitors, currently our best weapons against HBV, are not complete cures and have unwanted side effects (2, 3). While reverse transcriptase inhibitors are initially very effective at...

Towards an HBV cure: state-of-the-art and unresolved questions—report of the ANRS workshop on HBV cure

“…While the prospects of novel polymerase inhibitors remain to be determined, capsid inhibitors, inhibitors of cccDNA formation and DNA cleavage enzymes have great potential as novel antiviral strategies that could directly impact cccDNA pools. The recent achievement to produce recombinant HBV polymerase at a large scale may be instrumental to design novel improved polymerase inhibitors 116. However, these novel inhibitors, whether they inhibit the priming, the RNA-dependent or DNA-dependent DNA synthesis, or the RNAseH activity of the polymerase, will have to show significant advantage over the existing nucleos(t)ide analogues with a high antiviral potency and a high barrier to resistance 117 118.…”

Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase

“…1). It was notable that the predicted helix 6 spans the traditional boundary between the TP and spacer domains, which means that a more realistic boundary may be after this helix, with the TP domain ending at position 192, as has also been suggested recently (36).…”

Conservation of antiviral activity and improved selectivity in PMEO-DAPym upon pyrimidine to triazine scaffold hopping

“…Given that the viral DNA polymerases represent the eventual targets for the antireplicative action of these compounds, we tried to account for the selectivity profile displayed by 27 and 8 by focusing on the 3D structures of these viral enzymes. The well characterized dimeric HIV-1 and HIV-2 RTs and monomeric MSV RT are known to share a common active site architecture (Nowak et al, 2013) which, on the basis of multiple sequence alignments and functional data (Menendez-Arias et al, 2014), can be confidently extended to that of the still unresolved HBV DNA polymerase (Voros et al, 2014). Therefore, the same binding mode that was experimentally determined for the diphosphate of 3 (3-pp) in the active site of HIV-1 RT in complex with a templateprimer double helix (Tuske et al, 2004) can be safely assumed for 27-pp, as previously reported for 8-pp (Herman et al, 2010), and not only for HIV-1 and HIV-2 RTs but also for MSV RT and HBV DNA polymerase ( Fig.…”