The Crystal Structure of a Cardiovirus RNA-Dependent RNA Polymerase Reveals an Unusual Conformation of the Polymerase Active Site

Vives-Adrián, Laia; Lujan, Celia; Oliva, Baldo; Linden, Lonneke van der; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; Kuppeveld, Frank J. M. van; Ferrer-Orta, Cristina; Verdaguer, N.

doi:10.1128/jvi.03502-13

Cited by 26 publications

(32 citation statements)

References 61 publications

(91 reference statements)

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“…The polymerase Gly1 and Glu2 residues become part of a flexible 5-residue linker between the domains and the N-terminus binding pocket collapses slightly as a result of their removal, primarily through backbone movements of residues 61–66 that form the outside of the binding pocket (Figure 3D). Second, there is an EMCV 3D pol structure [ 4NYZ ] where, for unknown reasons, the very N-terminus moved out of its binding pocket (Vives-Adrian et al, 2014). In this structure there is a major rearrangement of residues 237–242, the structural link between motif A in the active site and the buried N-terminus, and Phe239 flips from being buried down in the palm domain to being tucked up into a shallow pocket in the fingers where it makes a cation–π interaction with Lys56 (Figure 3C).…”

Section: Picornaviral Polymerase Structurementioning

confidence: 99%

Picornaviral polymerase structure, function, and fidelity modulation

Peersen

2017

Virus Research

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Like all positive strand RNA viruses, the picornaviruses replicate their genomes using a virally encoded RNA-dependent RNA polymerase enzyme known as 3Dpol. Over the past decade we have made tremendous advances in our understanding of 3Dpol structure and function, including the discovery of a novel mechanism for closing the active site that allows these viruses to easily fine tune replication fidelity and quasispecies distributions. This review summarizes current knowledge of picornaviral polymerase structure and how the enzyme interacts with RNA and other viral proteins to form stable and processive elongation complexes. The picornaviral RdRPs are among the smallest viral polymerases, but their fundamental molecular mechanism for catalysis appears to be generally applicable as a common feature of all positive strand RNA virus polymerases.

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Section: Picornaviral Polymerase Structurementioning

confidence: 99%

Picornaviral polymerase structure, function, and fidelity modulation

Peersen

2017

Virus Research

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“…); the Picornaviridae, including Coxsackie virus B3, poliovirus, FMDV, HEV71, human rhinovirus, and encephalomyocarditis virus(Chen et al 2013;Gruez et al 2008;Love et al 2004;Ferrer-Orta 2004;Appleby et al 2005;Thompson and Peersen 2004;Hansen et al 1997;Vives-Adrian et al 2014); the Birnaviridae, including infectious bursal disease virus(Pan et al 2007); and the Orthomyxoviridae, including influenza A virus(He et al 2008). All these polymerases have a "right-handed" architecture, which is comprised of finger, palm, and thumb domains.…”

mentioning

confidence: 99%

Fidelity Variants and RNA Quasispecies

Bordería

Rozen-Gagnon

Vignuzzi

2015

Current Topics in Microbiology and Immunology

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By now, it is well established that the error rate of the RNA-dependent RNA polymerase (RdRp) that replicates RNA virus genomes is a primary driver of the mutation frequencies observed in RNA virus populations-the basis for the RNA quasispecies. Over the last 10 years, a considerable amount of work has uncovered the molecular determinants of replication fidelity in this enzyme. The isolation of high- and low-fidelity variants for several RNA viruses, in an expanding number of viral families, provides evidence that nature has optimized the fidelity to facilitate genetic diversity and adaptation, while maintaining genetic integrity and infectivity. This chapter will provide an overview of what fidelity variants tell us about RNA virus biology and how they may be used in antiviral approaches.

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“…In complex "UTPa," the phosphates are in close contact with both metal ions, resembling the classical binding mode first described for T7 DNA polymerase and HIV-1 reverse transcriptase. "UTPb" more closely resembles the binding mode found for wild-type HCV J4 NS5B (46), where the ␥-phosphate interacts with metal B on the left and basic residues on the finger domain (e.g., R48, K51, and R158). matching the catalytically relevant RdRp regions (encompassing motifs A, B, C, and E), while "peripheral" regions, such as the finger, fingertip, and thumb domains, were omitted from these superpositions.…”

Section: Figmentioning

confidence: 88%

Hydrophobic and Charged Residues in the C-Terminal Arm of Hepatitis C Virus RNA-Dependent RNA Polymerase Regulate Initiation and Elongation

Cherry

Dennis

Baron

et al. 2015

J Virol

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The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a ␤-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of ␤-loop and C-terminal arm mutants, which were used for in vitro analysis of RdRp de novo initiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the ␤-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general. IMPORTANCEHCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr¡Phe or Asp¡Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase. V iral RNA-dependent RNA polymerases (RdRps) represent the catalytic core that facilitates genome replication of RNA viruses. In order to maintain genome integrity, all RdRps must have a mechanism for initiating replication effectively. In vivo, viral RdRps can initiate genome synthesis by using either a primerdependent or a de novo (primer-independent) mechanism (1, 2). In the primer-dependent mechanism, a short oligonucleotide or a protein is used to provide an initial platform on which the nascent strand is built. In de novo initiation, the RdRp links the second incoming nucleotide to the already present nucleotide known as the initiating nucleotide (NTPi). The hepatitis C virus (HCV) RdRp and other related viral RdRps utilize such a mechanism in vivo (1, 2), although primer-dependent synthesis can be measured in vitro. Recently, short di-and trinu...

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The Crystal Structure of a Cardiovirus RNA-Dependent RNA Polymerase Reveals an Unusual Conformation of the Polymerase Active Site

Cited by 26 publications

References 61 publications

Picornaviral polymerase structure, function, and fidelity modulation

Picornaviral polymerase structure, function, and fidelity modulation

Fidelity Variants and RNA Quasispecies

Hydrophobic and Charged Residues in the C-Terminal Arm of Hepatitis C Virus RNA-Dependent RNA Polymerase Regulate Initiation and Elongation

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