Ultradeep Pyrosequencing and Molecular Modeling Identify Key Structural Features of Hepatitis B Virus RNase H, a Putative Target for Antiviral Intervention

Abstract

Last-generation nucleoside/nucleotide analogues are potent against hepatitis B virus (HBV) and have a high barrier to resistance. However, delayed responses have been observed in patients previously exposed to other drugs of the same class, long-term resistance is possible, and cure of infection cannot be achieved with these therapies, emphasizing the need for alternative therapeutic approaches. The HBV RNase H represents an interesting target because its enzyme activity is essential to the HBV life cycle. The goal of our study was to characterize the structure of the HBV RNase H by computing a 3-dimensional molecular model derived from E. coli RNase H and analyzing 2,326 sequences of all HBV genotypes available in public databases and 958,000 sequences generated by means of ultradeep pyrosequencing of sequences from a homogenous population of 73 treatment-naive patients infected with HBV genotype D. Our data revealed that (i) the putative 4th catalytic residue displays unexpected variability that could be explained by the overlap of the HBx gene and has no apparent impact on HBV replicative capacity and that (ii) the C-helix-containing basic protrusion, which is required to guide the RNA/DNA heteroduplex into the catalytic site, is highly conserved and bears unique structural properties that can be used to target HBV-specific RNase H inhibitors without cross-species activity. The model shows substantial differences from other known RNases H and paves the way for functional and structural studies as a prerequisite to the development of new inhibitors of the HBV cell cycle specifically targeting RNase H activity.

Recent Advance of the Hepatitis B Virus Inhibitors: A Medicinal Chemistry Overview

“…Southern blot analysis showed that R703A, D777A and R781A mutants significantly reduced amounts of viral DNAs, which revealed that three charged residues of the HBV polymerase RNaseH domain contribute to the catalysis of RNaseH in removing the RNA template, but not in the RNA encapsidation [133,134]. HBV reverse transcription requires the viral RNase H to destroy the viral RNA after it has been translated into minus strand DNA.…”

The hepatitis B virus ribonuclease H as a drug target

“…HBV is a genetically diverse virus with eight or nine genotypes (A – I) that differ from each other by ≥8% at the nucleotide level (Schaefer, 2007). The RNaseH sequences differ between genotypes by ~6% at the amino acid level (Hayer et al, 2014). Currently, active HBV RNaseH can be produced from HBV genotypes B, C, D, and H.…”

Predicted structure of the hepatitis B virus polymerase reveals an ancient conserved protein fold

“…The RT models are accurate enough in the active site to permit interpretation of the mechanisms of resistance to nucleoside analog drugs, but little is known of their accuracy outside of the active site. Three homology models exist for the RNase H domain 28–30 . None of them contain the full domain 31 and only one 28 correctly predicts the last D‐E‐D‐D active site residue 8,32 …”

“…Three homology models exist for the RNase H domain. [28][29][30] None of them contain the full domain 31 and only one 28 correctly predicts the last D-E-D-D active site residue. 8,32 AlphaFold is an ab initio protein structure prediction program.…”