Hepatitis B virus genetic diversity has minimal impact on sensitivity of the viral ribonuclease H to inhibitors.

Hepatitis B virus (HBV) causes hepatitis, cirrhosis, liver failure, and liver cancer, but the current therapies that employ either nucelos(t)ide analogs or (pegylated)interferon α do not clear the infection in the large majority of patients. Inhibitors of the HBV ribonuclease H (RNaseH) that are being developed with the goal of producing anti-HBV drugs are promising candidates for use in combination with the nucleos(t)ide analogs to improve therapeutic efficacy. HBV is genetically very diverse, with at least 8 genotypes that differ by ≥8% at the sequence level. This diversity is reflected in the viral RNaseH enzyme, raising the possibility that divergent HBV genotypes or isolates may have varying sensitivity to RNaseH inhibitors. To evaluate this possibility, we expressed and purified 18 patient-derived RNaseHs from genotypes B, C, and D. Basal RNaseH activity and sensitivity to three novel RNaseH inhibitors from three different chemotypes were assessed. We also evaluated four consensus HBV RNaseHs to determine if such sequences would be suitable for use in antiviral drug screening. The patient-derived enzymes varied by over 10-fold in their basal RNaseH activities, but they were equivalently sensitive to each of the three inhibitors. Similarly, all four consensus HBV RNaseH enzymes were active and were equally sensitive to an RNaseH inhibitor. These data indicate that a wide range of RNaseH sequences would be suitable for use in antiviral drug screening, and that genotype- or isolate-specific genetic variations are unlikely to present a barrier during antiviral drug development against the HBV RNaseH.

Purification and enzymatic characterization of the hepatitis B virus ribonuclease H, a new target for antiviral inhibitors.

Hepatitis B virus (HBV) reverse transcription requires coordinated function of the reverse transcriptase and ribonuclease H (RNaseH) activities of the viral polymerase protein. The reverse transcriptase has been biochemically characterized, but technical difficulties have prevented both assessment of the RNaseH and development of high throughput inhibitor screens against the RNaseH. Expressing the HBV RNaseH domain with both maltose binding protein and hexahistidine tags led to stable, high-level accumulation of the RNaseH in bacteria. Nickel-affinity purification in the presence of Mg(2+) and ATP removed co-purifying bacterial chaperones and yielded nearly pure monomeric recombinant enzyme. The endonucleolytic RNaseH activity required an DNA:RNA duplex ≥14 nt, could not tolerate a stem-loop in either the RNA or DNA strands, and could tolerate a nick in the DNA strand but not a gap. The RNaseH had no obvious sequence specificity or positional dependence within the RNA, and it cut the RNA at multiple positions even within the minimal 14 nt duplex. The RNaseH also possesses a processive 3'-5' exoribonuclease activity that is slower than the endonucleolytic reaction. These results are consistent with the HBV reverse transcription mechanism that features an initial endoribonucleolytic cut, 3'-5' degradation of RNA, and a sequence-independent terminal RNA cleavage. These data provide support for ongoing anti-RNaseH drug discovery efforts.