Characterization of the Elongation Complex of Dengue Virus RNA Polymerase

Zhang, Jin; Deval, Jérôme; Johnson, Kenneth A.; Swinney, David C.

doi:10.1074/jbc.m110.162685

Cited by 49 publications

(39 citation statements)

References 49 publications

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“…The values are comparable with kinetic parameters determined for other related viral RdRps (17,18). For mismatch incorporations, we observe both decreases in k pol and increases in K d values for each of the 12 possible combinations.…”

Section: Resultssupporting

confidence: 73%

Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance

Powdrill

Tchesnokov²,

Kozak³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

135

104

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The development of resistance to direct-acting antivirals (DAAs) targeting the hepatitis C virus (HCV) can compromise therapy. However, mechanisms that determine prevalence and frequency of resistance-conferring mutations remain elusive. Here, we studied the fidelity of the HCV RNA-dependent RNA polymerase NS5B in an attempt to link the efficiency of mismatch formation with genotypic changes observed in vivo. Enzyme kinetic measurements revealed unexpectedly high error rates (approximately 10 −3 per site) for G∶U∕U∶G mismatches. The strong preference for G∶U∕U∶G mismatches over all other mistakes correlates with a mutational bias in favor of transitions over transversions. Deep sequencing of HCV RNA samples isolated from 20 treatment-naïve patients revealed an approximately 75-fold difference in frequencies of the two classes of mutations. A stochastic model based on these results suggests that the bias toward transitions can also affect the selection of resistance-conferring mutations. Collectively, the data provide strong evidence to suggest that the nature of the nucleotide change can contribute to the genetic barrier in the development of resistance to DAAs.A pproximately 170 million people worldwide are infected with hepatitis C virus (HCV) (1). Chronically infected individuals are at risk of developing severe liver disease, including cirrhosis and hepatocellular carcinoma (2). Although the infection can be cured with a combination of interferon-α and ribavirin, severe side effects and complicated treatment schedules can compromise therapy (3). Moreover, the response is particularly poor in patients infected with HCV genotype 1, which is the most prevalent genotype in North America and Europe (4).Several direct-acting antivirals (DAAs) targeting important viral and host factors are currently undergoing clinical evaluation. Drugs acting against the viral NS5B RNA-dependent RNA polymerase (RdRp) or NS3 protease are in advanced clinical trials, with the protease inhibitors (PIs) telaprevir and boceprevir recently gaining FDA approval. Combining interferon-α and ribavirin with either PI can produce increases in sustained virological response when compared with the combination of interferon-α and ribavirin without PI (5, 6). However, the emergence of resistance-conferring mutations can lead to treatment failure (7). Like other RNA viruses, HCV shows quasispecies-like characteristics (8). Accordingly, resistant variants are selected from a genetically highly diverse population. Resistance to PIs is rapidly selected in cell-based replicon systems and in vivo (9). Similar observations have been made with the various classes of nonnucleoside analogue inhibitors (NNIs) of NS5B (9). Moreover, mutations that decrease susceptibility to highly potent inhibitors of the RNA binding protein NS5A also emerge rapidly in the replicon system (10). In contrast, the barrier to the development of resistance to nucleoside analogue inhibitors (NIs) appears to be significantly higher. The 2′-C-methylated NIs were shown to select in v...

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Section: Resultssupporting

confidence: 73%

Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance

Powdrill

Tchesnokov²,

Kozak³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

135

104

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“…Thus, the catalytic competence of the polymerase would not be a preexisting state of the enzyme, but the nucleic acid binding would induce it. A very slow binding step in the association of the isolated DENV polymerase domain-template-primer complex seems to support this model and was proposed to result from the temperature-dependent, structural rearrangement of the closing loops (13).…”

Section: Discussionmentioning

confidence: 66%

“…Thus, the interactions with the ssRNA are sufficient to initiate and continue the nucleic acid synthesis. Both the isolated polymerase domain and the full-length enzyme can catalyze the RNA synthesis in vitro (9,13). Nevertheless, the isolated polymerase domain shows decreased enzymatic activity (9).…”

mentioning

confidence: 99%

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Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes

Szymanski

Jezewska

Bujalowski

et al. 2011

Journal of Biological Chemistry

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Fundamental aspects of interactions of the Dengue virus type 3 full-length polymerase with the single-stranded and doublestranded RNA and DNA have been quantitatively addressed. The polymerase exists as a monomer with an elongated shape in solution. In the absence of magnesium, the total site size of the polymerase-ssRNA complex is 26 ؎ 2 nucleotides. In the presence of Mg 2؉ , the site size increases to 29 ؎ 2 nucleotides, indicating that magnesium affects the enzyme global conformation. The enzyme shows a preference for the homopyrimidine ssRNAs. Positive cooperativity in the binding to homopurine ssRNAs indicates that the type of nucleic acid base dramatically affects the enzyme orientation in the complex. Both the intrinsic affinity and the cooperative interactions are accompanied by a net ion release. The polymerase binds the dsDNA with an affinity comparable with the ssRNAs affinity, indicating that the binding site has an open conformation in solution. The lack of detectable dsRNA or dsRNA-DNA hybrid affinities indicates that the entry to the binding site is specific for the sugar-phosphate backbone and/or conformation of the duplex. The dengue virus (DENV)2 is a member of the Flaviviridae family and the etiological agent of dengue fever, a disease affecting worldwide ϳ100 million people each year (1-5). The Flaviviridae family includes such human pathogens as tick-borne encephalitis virus, hepatitis C virus, West Nile virus, yellow fever virus, and Japanese encephalitis virus (1-5). The dengue virus exists in four distinct types, DENV1, -2, -3, and -4, which show ϳ60% genomic sequence identity (1). The dengue fever often develops into the dengue hemorrhagic fever, an acute form of the disease, possibly as a result of sequential infection with different types of the virus. It is characterized by a significant mortality, particularly for individuals with a weakened or undeveloped immune system. The small, 10.7-kbp positive strand RNA genome of the dengue virus encodes only 10 proteins: three structural proteins (capsid, premembrane, and envelope proteins) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins) (3, 6 -8). The entire viral genome is translated as a single polyprotein, subsequently cleaved by viral and cellular proteases into functional entities.The NS5 protein is the viral replicative RNA-dependent RNA polymerase (1, 3, 6 -8). The primary structure of the fulllength polymerase encompasses 900 amino acids (i.e. it is the largest of the nonstructural proteins of the dengue virus) (6 -10). The enzyme is built of two functional domains (6 -10). The N-terminal domain includes the first 263 amino acids. It functions as a methyltransferase (MTase), whose activity is necessary for viral RNA recognition by the host cell translational apparatus. Correspondingly, amino acids ϳ273-906 form the polymerase domain, containing the active site of the RNA synthesis (Fig. 1a) (6 -8). The crystal structures of the isolated N-terminal and polymerase domains have been determined up t...

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“…Ribavirin has been used to select high-fidelity variants of EV71 (21,43), footand-mouth disease virus (FMDV) (44,45), and chikungunya virus (22), suggesting that these viruses occupy the lower end of the fidelity spectrum. One might expect that the need to retain crossspecies infectivity in zoonotic arboviruses necessitates higher-fidelity polymerases, as implied by yellow fever virus fidelity data (46), although data from dengue virus polymerase suggest that it has a fidelity comparable to those of PV and FMDV RdRPs based on single nucleotide incorporation assays (47). Mutations in motif A of West Nile virus polymerase that were designed to stabilize the active-site structure diminish virus growth (48), suggesting that this virus has some tolerance for slowing the polymerase, but biochemical data to assess the effects on the polymerase rate and fidelity are not available.…”

Section: Fig 8 Comparison Of Coxsackievirus and Poliovirus Polymerasementioning

confidence: 99%

Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases

Campagnola

McDonald

Beaucourt

et al. 2015

J Virol

106

144

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Viral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination, and these effects are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNAdependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity. IMPORTANCEPositive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects on in vitro nucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growth in vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness. P ositive-strand RNA viruses replicate by using virally encoded RNA-dependent RNA polymerases (RdRPs) that provide a direct RNA-to-RNA replication function not found in host cells. The structures of many RdRPs, primarily from picornaviruses, caliciviruses, and flaviviruses, have been solved to reveal a core structure composed of the classic right-hand arrangement of finger, palm, and t...

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Characterization of the Elongation Complex of Dengue Virus RNA Polymerase

Cited by 49 publications

References 49 publications

Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance

Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance

Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes

Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases

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