Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region

Stuyver, Lieven; Locarnini, Stephen; Lok, Anna S.; Richman, Douglas D.; Carman, William F.; Dienstag, Jules L.; Schinazi, Raymond F.

doi:10.1053/jhep.2001.22166

Cited by 351 publications

(120 citation statements)

References 69 publications

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“…Furthermore, mutations in drug-resistant hPOL overlap the open reading frames of HBV surface antigens. This raises the specter that escape mutants might arise against approved vaccines and existing therapeutics that could make them ineffective (84,85).…”

Section: Discussionmentioning

confidence: 99%

Large-Scale Production and Structural and Biophysical Characterizations of the Human Hepatitis B Virus Polymerase

Vörös

Urbanek

Rautureau

et al. 2014

J Virol

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Hepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies-broad-spectrum antivirals-are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in Escherichia coli, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly ␣-helical structures with small amounts of ␤-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity in vitro, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the in vitro priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV. IMPORTANCEThe viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of muchneeded new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in Escherichia coli and also methods for purifying them in soluble forms that have activity in vitro. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV. Hepatitis B virus (HBV) is a highly infectious, species-specific pathogen that causes serious liver diseases, including cancer, and causes 600,000 deaths annually (1). While excellent prophylactic HBV vaccines exist, these are ineffective against extant chronic HBV infections which affect 350 million people worldwide. Approved therapies for chronic HBV infections include immuno-modulators (e.g., interferon therapies) and nucleoside analogues that inhibit the reverse transcriptase domain of hPOL, the multifunctional viral polymerase of human HBV. These reverse transcriptase inhibitors, currently our best weapons against HBV, are not complete cures and have unwanted side effects (2, 3). While reverse transcriptase inhibitors are initially very effective at...

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Section: Discussionmentioning

confidence: 99%

Large-Scale Production and Structural and Biophysical Characterizations of the Human Hepatitis B Virus Polymerase

Vörös

Urbanek

Rautureau

et al. 2014

J Virol

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“…Due to the functional similarity, the structure of the HIV RT is used for modelling the spatial structure of the HBV polymerase [6,7]. Comparison of HBV polymerase and HIV-1 RT on an amino acid sequence basis revealed only around 14% homologous sites [6], but the YMDD motif is conserved (HBV pol sequence position 203-206 according to the nomenclature of Stuyver et al [8]). The enzymatic pocket is formed by certain structurally exposed amino acids (described above), which enable a possible identical conformation of the pockets of HIV-1 and HBV polymerases (fig.…”

Section: Structure Of the Hiv Reverse Transcriptase And Hepatitis B Vmentioning

confidence: 99%

Effect of Antiretroviral HIV Therapy on Hepatitis B Virus Replication and Pathogenicity

Gürtler¹

2014

Intervirology

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Coinfections with hepatitis B virus (HBV) and HIV are very frequent. Although HBV is a DNA virus, it replicates via reverse transcription like HIV. Structural similarities between the enzymatic pocket of the HBV DNA polymerase and HIV-1 reverse transcriptase are the basis that certain drugs inhibit both enzymes and thus the replication of both viruses. HBV components increase the pathogenic action of HIV and vice versa directly by certain proteins like HBsAg in the case of HBV and HIV-encoded Tat and Vpr and by disturbing the cytokine balance in affected cells. Antiretroviral therapy is highly beneficial for HIV/HBV-coinfected patients, but carries the risk of drug-induced resistance development and hepatotoxicity. Even with restoration of the immune capacity, signs of hepatic inflammation may develop even after 10 years of treatment.

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“…Mutations that result in replacement of methionine in the tyrosine-methionine-aspartate (YMDD) catalytic site motif with either valine or isoleucine confer resistance to LMV [4,5]. These changes are designated rtM204V/I, using the genotype-independent nomenclature system proposed previously [6]. rtM204I substitution has been detected in isolation, but the rtM204V change in the HBV DNA in those patients failing LMV therapy is found in association with other changes, which are essentially compensatory; these include rtL180M and rtV173L [7,8,9], and rtL80V/I [10].…”

Section: Introduction To Nucleos(t)ide Analogue Resistancementioning

confidence: 99%

Mechanisms of Hepatitis B Virus Resistance Development

Warner

Locarnini²

2014

Intervirology

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Hepatitis B virus (HBV) resistance to nucleos(t)ide analogue (NA) therapy is essentially structure specific, with each NA falling within three main structural groups. Resistance to each of these is characterized by specific mutations in the reverse transcriptase domains of the HBV polymerase, and may be associated with compensatory mutations which can increase replication. HBV polymerase is considered to have a traditional ‘right-handed' structural conformation, and each of the resistance mutations is predicted to cause a specific structural change of the polymerase, thereby preventing incorporation of NA into replicating DNA. The selection of resistance occurs at different rates for each NA, and is affected by the high mutational rate of HBV and the ability of the drug to suppress viral replication. Some mutations or combinations of mutations may be associated with multidrug resistance, limiting treatment options. In contrast to most other viruses, resistance in HBV is confounded by the overlapping surface gene, the major NA-resistant mutations also altering the surface proteins in most cases, potentially altering virus secretion and neutralization, which may pose a public health threat in the future.

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Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region

Cited by 351 publications

References 69 publications

Large-Scale Production and Structural and Biophysical Characterizations of the Human Hepatitis B Virus Polymerase

Large-Scale Production and Structural and Biophysical Characterizations of the Human Hepatitis B Virus Polymerase

Effect of Antiretroviral HIV Therapy on Hepatitis B Virus Replication and Pathogenicity

Mechanisms of Hepatitis B Virus Resistance Development

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