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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.

Project description:Background: Identification and characterization of developed antiviral drug resistance mutations are key to the success of antiviral therapies against hepatitis C virus (HCV), which remains a worldwide highly prevalent pathogenic disease. Although most studies focus on HCV genotypes 1, 2 or 3, the investigation of drug resistance in HCV genotype 4, predominant in North Africa, is especially significant in Egypt.Methods: We performed mutational and genotypic analysis of the untranslated region (UTR) and nonstructural protein 5B (NS5B) drug resistance-associated regions of HCV for patients in the surrounding villages of Mansoura city, who were not responding to different antiviral treatments (sofosbuvir (SOF), ribavirin, and interferon). Furthermore, molecular modelling approaches (homology modelling and docking studies) were used to investigate the significance of the identified NS5B mutations for SOF and ribavirin binding in the HCV genotype 4a NS5B active site.Results: Genotypic analysis confirmed all samples to have genotype 4 with sub-genotype 4a predominant. Partial sequencing of the UTR and NS5B resistance-associated regions identified D258E, T282S and A307G mutations in all isolates of NS5B. The UTR mutation site at position 243 was associated with interferon resistance, whereas the NS5B T282S mutation was considered as significant for SOF and ribavirin resistance. Docking studies in the HCV genotype 4a homology model predict SOF and ribavirin to accommodate a nucleotide-like binding mode, in which the T282 residue does interfere with the binding as it would in HCV genotypes 1 and 2. Mutation energy calculations predict T282S to moderately destabilize the binding of SOF and ribavirin by 0.57 and 0.47 kcal/mol, respectively.Conclusion: The performed study identified and characterized several antiviral drug resistance mutations of HCV genotype 4a and proposed a mechanism by which the T282S mutation may contribute to SOF and ribavirin resistance.

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.

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