A Relaxed Discrimination of 2′-O-Methyl-GTP Relative to GTP between de Novo and Elongative RNA Synthesis by the Hepatitis C RNA-dependent RNA Polymerase NS5B

Dutartre, Hélène; Boretto, Joëlle; Guillemot, Jean Claude; Canard, Bruno

doi:10.1074/jbc.m410191200

Cited by 34 publications

(45 citation statements)

References 42 publications

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“…NS5B is able to initiate RNA synthesis de novo; i.e., in the absence of a primer (15). The initiation reaction is fragile, and the complex composed of NS5B and RNA template dissociates frequently (16). The transition to the highly processive elongation mode is evident after incorporation of two to four nucleotides.…”

Section: Resultsmentioning

confidence: 99%

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: Resultsmentioning

confidence: 99%

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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“…Reactions with concentrations of PP i that exceeded 500 ⌴ showed decreases in primer rescue, presumably by depleting concentrations of free divalent metal ions required for catalysis. Small RNA products were also resistant to PP i -mediated excision, which probably reflects the frequent dissociation of the NS5B-RNA complex during early stages of RNA synthesis (10,15,35). Stability of the elongation complex.…”

Section: Resultsmentioning

confidence: 99%

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase

Deval¹,

Powdrill²,

D’Abramo³

et al. 2007

Antimicrob Agents Chemother

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Nonobligate chain terminators, such as 2-C-methylated nucleotides, block RNA synthesis by the RNAdependent RNA polymerase (RdRp) of hepatitis C virus (HCV). Previous studies with related viral polymerases have shown that classical chain terminators lacking the 3-hydroxyl group can be excised in the presence of pyrophosphate (PP i ), which is detrimental to the inhibitory activity of these compounds. Here we demonstrate that the HCV RdRp enzyme is capable of removing both obligate and clinically relevant nonobligate chain terminators. Pyrimidines are more efficiently excised than are purines. The presence of the next complementary templated nucleotide literally blocks the excision of obligate chain terminators through the formation of a dead-end complex (DEC). However, 2-C-methylated CMP is still cleaved efficiently under these conditions. These findings show that a 2-methylated primer terminus impedes nucleotide binding. The S282T mutation, associated with resistance to 2-C-methylated nucleotides, does not affect the excision patterns. Thus, the decreased susceptibility to 2-C-methylated nucleotides appears to be based solely on improved discrimination between the inhibitor and its natural counterpart. In conclusion, our data suggest that the phosphorolytic excision of nonobligate, pyrimidine-based chain terminators can diminish their potency. The templated nucleotide does not appear to provide protection from excision through DEC formation.Hepatitis C virus (HCV) infection is a serious public health concern that affects 170 million people worldwide (33, 42). Among those infected, approximately 20 to 30% develop severe liver disease, such as chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma (2). The combined use of the nucleoside analogue ribavirin and pegylated alpha interferon is the current treatment standard; however, success in treatment depends largely on the viral genotype, and this drug combination has also been associated with severe side effects (30,43). Thus, the development of novel, more potent, specific drugs is urgent.HCV belongs to the Flaviviridae family. The HCV RNA genome consists of approximately 10 kb, encoding a polyprotein which is processed into several smaller polypeptides, including the capsid protein (C), the envelope proteins (E1 and E2), and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Initial cis cleavage through NS2-NS3 releases the NS3 protein, which in turn continues to process the precursor. Promising compounds with the ability to inhibit the viral protease (NS3) and the polymerase (NS5B) have been identified.NS5B is a 65-kDa RNA-dependent RNA polymerase capable of initiating RNA synthesis de novo in the absence of a primer (16,17,25,27,45). Three classes of inhibitors of HCV NS5B have been developed, namely, nucleoside analogue inhibitors, nonnucleoside analogue inhibitors, and pyrophosphate (PP i ) analogues. Nonnucleoside inhibitors and PP i analogues are still under preclinical evaluation, while a 2Ј-modified nucleoside analogue has advanc...

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“…Various small molecule HCV RdRp inhibitors such as nucleoside analogues (16,17) and non-nucleoside inhibitors (NNI) (15, 18 -22) were synthesized and reported to be efficient NS5B inhibitors. After conversion to nucleoside triphosphate by cell host machinery, nucleoside analogue competes with natural NTP at the catalytic site of RdRp and terminates the elongation on incorporation.…”

Section: Hepatitis C Virus (Hcv)mentioning

confidence: 99%

Functional Characterization of Fingers Subdomain-specific Monoclonal Antibodies Inhibiting the Hepatitis C Virus RNA-dependent RNA Polymerase

Nikonov

Juronen

Ustav

2008

Journal of Biological Chemistry

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The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), encoded by nonstructural protein 5B (NS5B), is absolutely essential for the viral replication. Here we describe the development, characterization, and functional properties of the panel of monoclonal antibodies (mAbs) and specifically describe the mechanism of action of two mAbs inhibiting the NS5B RdRp activity. These mAbs recognize and bind to distinct linear epitopes in the fingers subdomain of NS5B. The mAb 8B2 binds the N-terminal epitope of the NS5B and inhibits both primer-dependent and de novo RNA synthesis. mAb 8B2 selectively inhibits elongation of RNA chains and enhances the RNA template binding by NS5B. In contrast, mAb 7G8 binds the epitope that contains motif G conserved in viral RdRps and inhibits only primer-dependent RNA synthesis by specifically targeting the initiation of RNA synthesis, while not interfering with the binding of template RNA by NS5B. To reveal the importance of the residues of mAb 7G8 epitope for the initiation of RNA synthesis, we performed site-directed mutagenesis and extensively characterized the functionality of the HCV RdRp motif G. Comparison of the mutation effects in both in vitro primer-dependent RdRp assay and cellular transient replication assay suggested that mAb 7G8 epitope amino acid residues are involved in the interaction of template-primer or template with HCV RdRp. The data presented here allowed us to describe the functionality of the epitopes of mAbs 8B2 and 7G8 in the HCV RdRp activity and suggest that the epitopes recognized by these mAbs may be useful targets for antiviral drugs. Hepatitis C virus (HCV)2 is a small positive strand RNA virus of the Flaviviridae family that is associated specifically with non-A and non-B hepatitis post-transfusion blood infections in humans (1). HCV, a noncytopathic hepatotropic virus, is a major causative agent of acute and chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (2). Recently, the World Health Organization estimated the prevalence of HCV antibodies approximating 2%, indicating that 123 million persons worldwide are affected by this virus (3). In infected cells, HCV genomic single-stranded ϳ9600-nucleotide RNA messenger directs the synthesis of the ϳ3000-amino acid polyprotein precursor (4), which is coand post-translationally cleaved by cellular and viral proteases producing mature structural and nonstructural proteins (5-7). The same genomic ssRNA serves as a template for the synthesis of the full-length minus strand, which is used for the overproduction of the virus-specific genomic ssRNA. The RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is a single subunit catalytic component of the viral replication machinery responsible for both of these steps.The catalytic domain of HCV RdRp has the "right-hand" configuration closely resembling those of HIV-1 reverse transcriptase (RT) (8) and the RdRps of poliovirus (9), reovirus (10), and phage 6 (11). Similarly to these polymerases, HCV NS5B is divide...

show abstract

A Relaxed Discrimination of 2′-O-Methyl-GTP Relative to GTP between de Novo and Elongative RNA Synthesis by the Hepatitis C RNA-dependent RNA Polymerase NS5B

Cited by 34 publications

References 42 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

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase

Functional Characterization of Fingers Subdomain-specific Monoclonal Antibodies Inhibiting the Hepatitis C Virus RNA-dependent RNA Polymerase

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