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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 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.

SUBMITTER: Villa JA

PROVIDER: S-EPMC4980287 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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

Villa Juan Antonio JA Pike Daniel P DP Patel Kunjan B KB Lomonosova Elena E Lu Gaofeng G Abdulqader Roz R Tavis John E JE

Antiviral research 20160617

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-leve ...[more]

PMID: 27321664

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Project description:Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 µM, the best compounds had low micromolar IC(50) values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 µM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development.

Dataset Information

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

Publications

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

Similar Datasets

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