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

doi:10.1016/j.antiviral.2016.09.009

Antiviral Research

Volume 135, November 2016, Pages 24-30

https://doi.org/10.1016/j.antiviral.2016.09.009 Get rights and content

Highlights

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Genetic diversity within the HBV RNaseH domain causes variable enzymatic activity of the encoded RNaseH enzymes.
•
RNaseH enzymes from genotype C isolates are typically more enzymatically active than isolates from genotypes B and D.
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RNaseHs from HBV genotypes B, C, and D are equivalently sensitive to inhibitors from three chemotypes.
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Consensus RNaseHs are active and their sensitivity to RNaseH inhibitors is similar to that of natural isolates.

Abstract

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.

Introduction

Hepatitis type B is caused by hepatitis B virus (HBV), a member of the Hepadnaviridae family. HBV chronically infects up to 350 million people worldwide and kills over 600,000 patients annually, and it is the leading infectious cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma worldwide (Ganem and Prince, 2004, Lavanchy, 2004, Shepard et al., 2006, Sorrell et al., 2009). HBV is an enveloped virus that contains an icosahedral nucleocapsid core particle surrounding the viral DNA genome. The nucleocapsid also contains the viral polymerase protein (P). P is a reverse transcriptase that copies the RNA form of the viral genome into partially double-stranded DNA. The principal therapy for hepatitis B employs nucleos(t)ide analog drugs that inhibit DNA polymerization by P. This treatment suppresses HBV levels in serum to near or below the clinical limit of detection (Cox and Tillmann, 2011, Kwon and Lok, 2011), but viral replication is not completely eliminated (Coffin et al., 2011, Zoulim, 2004) and viremia rebounds in the vast majority of patients when the drugs are withdrawn. However, HBV infections are cleared in a few percent of HBV patients after years of nucleos(t)ide analog therapy (Marcellin et al., 2008, van Bommel et al., 2010, Woo et al., 2010, Wursthorn et al., 2010), implying that the infection could be cleared in more patients by suppressing HBV further. Greater suppression of HBV will require new drugs that will probably be used in combination with the nucleos(t)ide analogs treatments that already exist (Block et al., 2013, Tavis et al., 2013b).

HBV reverse transcription is catalyzed by two enzymatic activities that are both located on the viral P protein. The DNA polymerase activity synthesizes new DNA, and the ribonuclease H (RNaseH) destroys the RNA template after it has been copied into DNA (Seeger et al., 2013). Blocking either activity prevents synthesis of mature viral genomes, including both the cccDNA nuclear form of the genome that is the template for all viral RNAs and the partially double-stranded form found in infectious virions. Inhibiting the RNaseH causes viral genomic replication to stall, leading to an incomplete minus-polarity DNA strand and failure to synthesize the plus-polarity DNA strand (Chen and Marion, 1996, Chen et al., 1994, Gerelsaikhan et al., 1996). Drugs have not been developed against the HBV RNaseH despite it being a logical target, in large part due to technical difficulties in developing screening assays. We recently developed a low-throughput screening pipeline to identify inhibitors of the HBV RNaseH (Tavis et al., 2013a, Tavis and Lomonosova, 2015). This pipeline has been used to identify over 60 compounds that block viral DNA replication by suppressing the RNaseH.

RNaseH enzymes cleave RNA within a DNA:RNA heteroduplex (Hostomsky et al., 1993). RNaseH enzymes belong to the nucleotidyl transferase superfamily, whose members share a similar protein fold and enzymatic mechanisms (Nowotny, 2009, Yang and Steitz, 1995). This large family of proteins includes the retroviral RNaseHs and integrases, including the HIV enzymes (Dyda et al., 1994). The RNaseH active site contains four conserved carboxylates (the “DEDD” motif) that coordinate two divalent cations, usually Mg⁺⁺ (Nowotny et al., 2005). The RNase cleavage mechanism requires both cations to promote a hydroxyl-mediated nucleophilic scission reaction (Klumpp et al., 2003, Nowotny and Yang, 2006, Yang and Steitz, 1995), and the HBV RNaseH inhibitors appear to function by binding to the Mg⁺⁺ ions and impeding their function during catalysis (Tavis and Lomonosova, 2015).

HBV has 8 genotypes (A-H), plus provisional identification of genotypes I and J, that differ by >8% at the sequence level (Kramvis et al., 2005, Kurbanov et al., 2010b, Osiowy et al., 2006). Most genotypes are divided into subtypes that differ by >4.5%. Distribution of the genotypes varies around the world, with genotypes B and C being dominant in southeast Asia and genotype A and D being more common in the USA and Europe. Genotypes C and D are somewhat more virulent than other genotypes (Li et al., 2015, Lu et al., 2013, Osiowy et al., 2006). Despite the large genetic variation between and among the genotypes, there is little evidence that they differ significantly in their sensitivity to nucleos(t)ide analog drugs (Osiowy et al., 2015).

Little is known regarding how HBV's genetic diversity affects its sensitivity to the newly discovered RNaseH inhibitors, although cross-genotypic inhibition is possible because we found that RNaseH inhibitors can block replication of both genotype A and D isolates in cell culture (Hu et al., 2013, Tavis et al., 2013a). Therefore, we evaluated RNaseH activity and sensitivity to RNaseH inhibitors for 18 patient-derived HBV RNaseHs from genotypes B, C, and D, plus four consensus RNaseH sequences derived from genotypes B, C, and D. These genotypes were selected because they are among the most common (Kramvis et al., 2005, Kurbanov et al., 2010a) and they account for most of HBV's pathology (Tanwar and Dusheiko, 2012). The RNaseH inhibitors were selected from three different chemical classes (Cai et al., 2014, Hu et al., 2013, Tavis et al., 2013a) to evaluate potential chemotype-specific effects of HBV variation on inhibitor efficacy.

Section snippets

Compound acquisition and synthesis

The compounds employed are shown in Fig. 3. Compound #46 (β-thujaplicinol) was acquired from the NCI Developmental Therapeutics Program, compound #1 was purchased from Toronto Research Chemicals, and compound #12 was synthesized at Saint Louis University following established protocols (Williams et al., 2008, Williams et al., 2010). All compounds were ≥95% pure. They were dissolved in dimethyl sulfoxide (DMSO) at 10 mM and stored at −80 °C in small aliquots in opaque tubes.

HBV RNaseH genotype specific and consensus sequences

One hundred complete

Activity of variant RNaseHs

One hundred patient-derived HBV sequences for each genotype B, C and D were obtained from Genbank (Supplemental Table 1) and the RNaseH sequences were deduced from the genomic sequences. The sequences were aligned at the protein level and phylogenetic trees were constructed. Six representative sequences distributed throughout the trees were selected for biochemical analysis for each of genotypes B, C, and D, yielding a total of eighteen variant patient-derived sequences (Table 1 and Fig. 1).

Discussion

HBV is a very genetically diverse virus with at least eight genotypes that differ by >8% at the sequence level (Kramvis et al., 2005, Kurbanov et al., 2010a, Tanwar and Dusheiko, 2012). These differences are large enough to cause variable efficacy of direct-acting antiviral compounds that would be analogous to what was encountered with the earlier generations of protease inhibitors for Hepatitis C virus, which were largely specific for only genotype 1 (CADTH, 2014). Here, we evaluated the

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article. JET is an inventor on USA patent applications 14/647,331, 61/174,385, 62/174,350, and 62/309,332 that cover therapeutic use of RNaseH inhibitors against HBV.

Author contributions

JAV and GL designed and conducted most of the experiments, assisted with data interpretation, and wrote the manuscript. MJD conducted the genetic analyses and chose the variant RNaseHs for analysis. XC assisted with protein purification and data analyses. MJM and RH synthesized compound #12. JET conceived of the project, directed its execution, helped interpret all data, and edited the manuscript. All authors approved the final version.

Acknowledgements

This work was supported by NIH grants R03 AI109460 and R01 AI104494 to JET. We thank Elena Lomonosova for many helpful discussions and Roz Abdulqader for technical assistance.

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¹: These authors contributed equally.

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

Highlights

Abstract

Introduction

Section snippets

Compound acquisition and synthesis

HBV RNaseH genotype specific and consensus sequences

Activity of variant RNaseHs

Discussion

Conflict of interest

Author contributions

Acknowledgements

Antivir. Res.

Antivir. Res.

Antivir. Res.

FEBS Lett.

Vaccine

Cell

Antivir. Res.

Antivir. Res.

Bioorg. Med. Chem. Lett.

Structure

Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification

Proteins Struct. Funct. Bioinforma.

CADTH Therapeutic Review. Direct-acting Antiviral Agents for Chronic Hepatitis C Genotype 1 [Internet]

Genetic and biochemical diversity in the HCV NS5B RNA polymerase in the context of interferon alpha plus ribavirin therapy

J. Viral Hepat.

Amino acids essential for RNAse H activity of hepadnaviruses are also required for efficient elongation of minus-strand DNA

J. Virol.

Selected mutations of the duck hepatitis B virus P gene RNase H domain affect both RNA packaging and priming of minus-strand DNA synthesis

J. Virol.

Molecular characterization of intrahepatic and extrahepatic hepatitis B virus (HBV) reservoirs in patients on suppressive antiviral therapy

J. Viral Hepat.

Emerging pipeline drugs for hepatitis B infection

Expert. Opin. Emerg. Drugs

Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases

Science

Hepatitis B virus infection–natural history and clinical consequences

N. Engl. J. Med.

Hepatitis B virus nucleocapsid envelopment does not occur without genomic DNA synthesis

J. Virol.

Successes and challenges on the road to cure hepatitis C

PLoS Pathog.

Ribonuclease H

Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors

Nucleic Acids Res.