A Comprehensive Structure-Function Comparison of Hepatitis C Virus Strain JFH1 and J6 Polymerases Reveals a Key Residue Stimulating Replication in Cell Culture across Genotypes

Schmitt, Melanie; Scrima, Nathalie; Radujkovic, Danijela; Caillet‐Saguy, Célia; Simister, Philip C.; Friebe, Peter; Wicht, Oliver; Klein, Rahel; Bartenschlager, Ralf; Lohmann, Volker; Bressanelli, Stéphane

doi:10.1128/jvi.02177-10

Cited by 56 publications

(58 citation statements)

References 52 publications

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“…Despite previous data showing the central role of 5BSL3.2 in the life cycle of the virus by long-range RNA-RNA interaction with other genomic sequences such as SL2 (Friebe et al 2005;Murayama et al 2010;Schmitt et al 2011) and the region centered on nucleotide 9110 (Diviney et al 2008), surprisingly, there were no data obtained by kinetic methods demonstrating direct interaction between these RNA motifs. NMR spectroscopy has been used to characterize SL2 and 5BSL3.2 (Friebe et al 2005), but the kissing complex potentially formed between these two imperfect hairpins could not be observed.…”

Section: Discussioncontrasting

confidence: 40%

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

Palau

Masante

Ventura

et al. 2013

RNA

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Surface plasmon resonance was used to investigate two previously described interactions analyzed by reverse genetics and complementation mutation experiments, involving 5BSL3.2, a stem-loop located in the NS5B coding region of HCV. 5BSL3.2 was immobilized on a sensor chip by streptavidin-biotin coupling, and its interaction either with the SL2 stem-loop of the 3 ′ end or with an upstream sequence centered on nucleotide 9110 (referred to as Seq9110) was monitored in real-time. In contrast with previous results obtained by NMR assays with the same short RNA sequences that we used or SHAPE analysis with longer RNAs, we demonstrate that recognition between 5BSL3.2 and SL2 can occur in solution through a kissing-loop interaction. We show that recognition between Seq9110 and the internal loop of 5BSL3.2 does not prevent binding of SL2 on the apical loop of 5BSL3.2 and does not influence the rate constants of the SL2-5BSL3.2 complex. Therefore, the two binding sites of 5BSL3.2, the apical and internal loops, are structurally independent and both interactions can coexist. We finally show that the stem-loop SL2 is a highly dynamic RNA motif that fluctuates between at least two conformations: One is able to hybridize with 5BSL3.2 through loop-loop interaction, and the other one is capable of self-associating in the absence of protein, reinforcing the hypothesis of SL2 being a dimerization sequence. This result suggests also that the conformational dynamics of SL2 could play a crucial role for controlling the destiny of the genomic RNA.

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Section: Discussioncontrasting

confidence: 40%

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

Palau

Masante

Ventura

et al. 2013

RNA

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“…The high infection rate has been attributed to, at least in part, the NS5B polymerase (19). It is possible that the JFH-1 NS5B can tolerate amino acid substitutions even in highly conserved residues due to extra hydrophobic interactions between the thumb and finger domains, as shown by crystallographic studies (23,25). On the other hand, other nonstructural proteins and the 3Ј untranslated region could also play a role in regulating the fitness of the replicons.…”

Section: Discussionmentioning

confidence: 99%

Hepatitis C Virus Nucleotide Inhibitors PSI-352938 and PSI-353661 Exhibit a Novel Mechanism of Resistance Requiring Multiple Mutations within Replicon RNA

Lam¹,

Espiritu²,

Bansal³

et al. 2011

J Virol

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PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of ␤-D-2-deoxy-2-␣-fluoro-2-␤-C-methylguanosine-5-monophosphate. Both compounds are metabolized to the same active 5-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both wild-type and S282T NS5B polymerases. In order to identify mutations that confer resistance to these compounds, in vitro selection studies were performed using HCV replicon cells. While no resistant genotype 1a or 1b replicons could be selected, cells containing genotype 2a JFH-1 replicons cultured in the presence of PSI-352938 or PSI-353661 developed resistance to both compounds. Sequencing of the NS5B region identified a number of amino acid changes, including S15G, R222Q, C223Y/H, L320I, and V321I. Phenotypic evaluation of these mutations indicated that single amino acid changes were not sufficient to significantly reduce the activity of PSI-352938 and PSI-353661. Instead, a combination of three amino acid changes, S15G/C223H/V321I, was required to confer a high level of resistance. No cross-resistance exists between the 2-F-2-C-methylguanosine prodrugs and other classes of HCV inhibitors, including 2-modified nucleoside/-tide analogs such as PSI-6130, PSI-7977, INX-08189, and IDX-184. Finally, we determined that in genotype 1b replicons, the C223Y/H mutation failed to support replication, and although the A15G/C223H/V321I triple mutation did confer resistance to PSI-352938 and PSI-353661, this mutant replicated at only about 10% efficiency compared to the wild type.According to the World Health Organization, hepatitis C virus (HCV) currently infects more than 170 million people worldwide. The majority of these patients develop chronic liver disease, with a 20% rate of liver cirrhosis and fibrosis, and up to 5% could progress to hepatocellular carcinoma. There is no vaccine available, and the current standard of care (SOC), which combines pegylated alpha interferon (peg-IFN-␣) and ribavirin, has limited efficacy and may be associated with a number of side effects (3,5). Efforts to develop direct-acting antiviral agents (DAA) have focused on compounds that target viral proteins critical for HCV replication. Recently, two DAA targeting the NS3/4A protease, boceprevir (Victrelis) and telaprevir (Incivek), in combination with the SOC have been approved as treatment for hepatitis C. Other antiviral compounds currently in clinical development include NS5A inhibitors, nucleoside/nucleotide analogs, and nonnucleoside inhibitors that target the NS5B polymerase. While DAA strategies have shown promising results in reducing viral load, viral breakthrough related to the emergence of resistance has been observed and has since become a majo...

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“…(i) Expression of NS5B proteins. NS5B with a C-terminal deletion of 21 residues (NS5B⌬C21) from HCV strains Con1, JFH1, J4, and J6 was expressed as already reported (30).…”

Section: Methodsmentioning

confidence: 99%

“…Introducing the central JFH1 V405I polymorphism into closely related J6_NS5B recently allowed us to boost J6_NS5B's de novo RNA synthesis in vitro and J6_NS5B-based chimeras' replication in cells. An infectious J6_NS5B-based chimeric virus was thereby generated with just two nucleotide substitutions (30). We hypothesized that by stabilizing a very closed NS5B conformation, the V405I mutation favors the very first step in de novo synthesis (formation of the first phosphodiester bond), with limiting impairment of the subsequent steps (elongation of this dinucleotide primer).…”

Section: T He Hepatitis C Virus (Hcv) Is An Enveloped Positive-strandmentioning

confidence: 99%

Two Crucial Early Steps in RNA Synthesis by the Hepatitis C Virus Polymerase Involve a Dual Role of Residue 405

Scrima

Caillet‐Saguy

Ventura

et al. 2012

J Virol

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The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5B initiates RNA synthesis by a de novo mechanism and then processively copies the whole RNA template. Dissections of de novo RNA synthesis by genotype 1 NS5B proteins previously established that there are two successive crucial steps in de novo initiation. The first is dinucleotide formation, which requires a closed conformation, and the second is the transition to elongation, which requires an opening of NS5B. We also recently published a combined structural and functional analysis of genotype 2 HCV-NS5B proteins (of strains JFH1 and J6) that established residue 405 as a key element in de novo RNA synthesis ( T he hepatitis C virus (HCV) is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae (28). The genome of HCV is a 9,600-nucleotide (nt)-long RNA molecule encompassing a single open reading frame (ORF) that is translated primarily into one polyprotein and flanked by nontranslated regions (NTRs). The NTRs are the most conserved parts of the viral genome and play important roles in viral translation and replication. The polyprotein precursor is cleaved by cellular and viral proteases into at least 10 different mature proteins. The nonstructural proteins NS3 to NS5B are associated within a membrane replication complex in which NS5B is the RNA-dependent RNA polymerase (RdRp). NS5B copies the RNA genome into a complementary negative strand and subsequently uses this negative strand as the template for the synthesis of new HCV genomes (for a review, see reference 21).In vitro, recombinant NS5B is a highly processive RdRp that copies RNA templates up to the size of the HCV genome (4). C-terminal deletions of the 21-residue transmembrane helix (NS5B⌬C21) are fully active and much easier to produce and purify than the full-length enzyme (37), so that these deletions are used almost exclusively for structural and functional characterization of NS5B. These studies have shown that NS5B can initiate RNA synthesis by a de novo mechanism (19) allowing full copy of the template in a single polymerization round (14,29). De novo initiation from the 3= end of the viral positive-and negative-strand RNA is likely to be the physiological mode of initiation of RNA synthesis in infected cells (8). Recombinant NS5B is very poorly active overall despite its high processivity and a turnover number comparable to that of other polymerases (7). Dissections of the early steps of RNA synthesis in vitro (12,14,29) have shown that this is due to two inefficient steps in de novo RNA synthesis by NS5B; the first step is de novo initiation proper, i.e., formation of the first dinucleotide complementary to the last two 3= bases of the template, and the second step is use of this dinucleotide as a primer for further RNA synthesis.Structural analysis of NS5B led us to propose that these two steps are closely linked to two conformational st...

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A Comprehensive Structure-Function Comparison of Hepatitis C Virus Strain JFH1 and J6 Polymerases Reveals a Key Residue Stimulating Replication in Cell Culture across Genotypes

Cited by 56 publications

References 52 publications

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

Hepatitis C Virus Nucleotide Inhibitors PSI-352938 and PSI-353661 Exhibit a Novel Mechanism of Resistance Requiring Multiple Mutations within Replicon RNA

Two Crucial Early Steps in RNA Synthesis by the Hepatitis C Virus Polymerase Involve a Dual Role of Residue 405

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