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

Abstract

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 Mg2+ 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.

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

“…The inhibitors were employed in ODN-directed RNaseH assays at their respective IC 50 values against the MBP-HRHgtB, C, and D variants: 30 µM for compounds #1 and #46, and 15 µM for compound #12. Note that these IC 50 values are higher than we originally reported because although the MBP-tagged version of the HBV RNaseH is a more robust enzyme and is much easier to purify to homogeneity than our original version of the recombinant HBV RNaseH (Villa et al, 2016), it is 3- to 5-fold less sensitive to inhibition by these compounds. Four variants were selected for analysis from each genotype (variants with the highest and lowest activity plus two intermediates).…”

“…Sensitivity of the consensus RNaseH enzymes to compound #12 at its IC 50 value (15 µM) was determined with ODN-directed RNaseH assays, with activity being compared to the activity of a genotype C reference RNaseH. Only the P2 product band was quantified because the P1 band is subject to preferential degradation by the HBV RNaseH’s 3’-5’ exonuclease activity (Villa et al, 2016) and hence is present in sub-stoicheometric amounts. The Y axis is arbitrary absorption units measured by densitometry of the autoradiograms.…”

Inhibition of hepatitis B virus replication by N -hydroxyisoquinolinediones and related polyoxygenated heterocycles

“…The lack of sensitivity in the oligonucleotide directed RNA cleavage assay led us to improve the purification of the recombinant RNaseH (Villa et al, 2016) and develop an alternative RNaseH assay that employs a molecular beacon (Chen et al, 2008). In this assay, a hairpin DNA oligonucleotide labeled with fluorescein on one end and a quencher on the other is held in a linear conformation by annealing to a complementary RNA; cleavage of the RNA causes the DNA to fold into a hairpin, suppressing fluorescence (Fig.…”

“…To improve sensitivity of the RNaseH assay, we improved the recombinant RNaseH purification (Villa et al, 2016) and developed an alternative assay that employs a molecular beacon (Chen et al, 2008). Using the molecular beacon assay, we identified four compounds as RNaseH inhibitors because they reduced the rate of substrate degradation with a dose-dependent pattern.…”

“…HuRNaseH1 and HBV RNaseH were expressed in Escherichia coli and purified by nickel-affinity chromatography as described (Villa et al, 2016). …”