Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro

Franco, David; Pathak, Harsh B.; Cameron, Craig Ε.; Rombaut, Bartholomeus; Wimmer, Eckard; Paul, Aniko V.

doi:10.1186/1743-422x-2-86

Cited by 26 publications

(19 citation statements)

References 54 publications

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“…The D64A mutant was dead and could not be studied, but it is likely that this mutant belongs to this group given its close proximity to several members of this class. By using a cell-free system for PV synthesis, Paul and co-workers (64,65) have shown that addition of 3C(D) to these extracts stimulates a post-replication step in PV synthesis by greater than 100-fold. Currently, it is thought that 3CD stimulates maturation of the PV virion (64,65).…”

Section: Discussionmentioning

confidence: 99%

“…By using a cell-free system for PV synthesis, Paul and co-workers (64,65) have shown that addition of 3C(D) to these extracts stimulates a post-replication step in PV synthesis by greater than 100-fold. Currently, it is thought that 3CD stimulates maturation of the PV virion (64,65). Similarly, it is possible that these amino acid substitutions alter the kinetics and/or specificity of capsid precursor processing, leading to problems with virion assembly and/or maturation.…”

Section: Discussionmentioning

confidence: 99%

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Picornavirus Genome Replication

Shen¹,

Reitman²,

Zhao

et al. 2008

Journal of Biological Chemistry

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Picornaviruses have a peptide termed VPg covalently linked to the 5-end of the genome. Attachment of VPg to the genome occurs in at least two steps. First, Tyr-3 of VPg, or some precursor thereof, is used as a primer by the viral RNA-dependent RNA polymerase, 3Dpol, to produce VPg-pUpU. Second, VPg-pUpU is used as a primer to produce full-length genomic RNA. Production of VPg-pUpU is templated by a single adenylate residue located in the loop of an RNA stem-loop structure termed oriI by using a slide-back mechanism. Recruitment of 3Dpol to and its stability on oriI have been suggested to require an interaction between the back of the thumb subdomain of 3Dpol and an undefined region of the 3C domain of viral protein 3CD. We have performed surface acidic-to-alanine-scanning mutagenesis of 3C to identify the surface of 3C with which 3Dpol interacts. This analysis identified numerous viable poliovirus mutants with reduced growth kinetics that correlated to reduced kinetics of RNA synthesis that was attributable to a change in VPg-pUpU production. Importantly, these 3C derivatives were all capable of binding to oriI as well as wild-type 3C. Synthetic lethality was observed for these mutants when placed in the context of a poliovirus mutant containing 3Dpol-R455A, a residue on the back of the thumb required for VPg uridylylation. These data were used to guide molecular docking of the structures for a poliovirus 3C dimer and 3Dpol, leading to a structural model for the 3C 2 -3Dpol complex that extrapolates well to all picornaviruses.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Picornavirus Genome Replication

Shen¹,

Reitman²,

Zhao

et al. 2008

Journal of Biological Chemistry

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“…The crystal structure of 3CD pro revealed that the 3C/3D cleavage site in the polypeptide is on the opposite side from the catalytic site of the 3C pro protein (86). The RNA binding activity of 3CD pro appears to be also important for virus maturation (48). …”

Section: Factors Involved In the Initiation Of Protein-primed Rna mentioning

confidence: 99%

Initiation of protein-primed picornavirus RNA synthesis

Paul

Wimmer

2015

Virus Research

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Plus strand RNA viruses use different mechanisms to initiate the synthesis of their RNA chains. The Picornaviridae family constitutes a large group of plus strand RNA viruses that possess a small terminal protein (VPg) covalently linked to the 5’-end of their genomes. The RNA polymerases of these viruses use VPg as primer for both minus and plus strand RNA synthesis. In the first step of the initiation reaction the RNA polymerase links a UMP to the hydroxyl group of a tyrosine in VPg using as template a cis-replicating element (cre) positioned in different regions of the viral genome. In this review we will summarize what is known about the intiation reaction of protein-primed RNA synthesis by the RNA polymerases of the Picornaviridae. As an example we will use the RNA polymerase of poliovirus, the prototype of Picornaviridae. We will also discuss models of how these nucleotidylylated protein primers might be used, together with viral and cellular replication proteins and other cis-replicating RNA elements, during minus and plus strand RNA synthesis.

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“…3CD is an RNA-binding protein that binds to the cis-acting element required for initiation of genome replication, possibly recruiting 3Dpol to this location (30 -32). Finally, 3CD appears to function after RNA synthesis as well in a step related to virion maturation that does not require 3CD protease activity (33). To determine to what extent RNA synthesis was attenuated for Glu-297 PV, mutations encoding this change were engineered into the PV subgenomic replicon.…”

Section: Enzymementioning

confidence: 99%

Structure-Function Relationships of the Viral RNA-dependent RNA Polymerase

Korneeva

Cameron

2007

Journal of Biological Chemistry

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Studies of the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV), 3Dpol, have shown that Asn-297 permits this enzyme to distinguish ribose from 2 -deoxyribose. All animal RNA viruses have Asn at the structurally homologous position of their polymerases, suggesting a conserved function for this residue. However, all prokaryotic RNA viruses have Glu at this position. In the presence of Mg 2؉ , the apparent affinity of Glu-297 3Dpol for 2 -deoxyribonucleotides was decreased by 6-fold relative to wild type without a substantial difference in the fidelity of 2 -dNMP incorporation. The fidelity of ribonucleotide misincorporation for Glu-297 3Dpol was reduced by 14-fold relative to wild type. A 4-to 11-fold reduction in the rate of ribonucleotide incorporation was observed. Glu-297 PV was unable to grow in HeLa cells due to a replication defect equivalent to that observed for a mutant PV encoding an inactive polymerase. Evaluation of the protein-(VPg)-primed initiation reaction showed that only half of the Glu-297 3Dpol initiation complexes were capable of producing VPg-pUpU product and that the overall yield of uridylylated VPg products was reduced by 20-fold relative to wild-type enzyme, a circumstance attributable to a reduced affinity for UTP. These studies identify the first RdRp derivative with a mutator phenotype and provide a mechanistic basis for the elevated mutation frequency of RNA phage relative to animal RNA viruses observed in culture. Although protein-primed initiation and RNA-primed elongation complexes employ the same polymerase active site, the functional differences reported here imply significant structural differences between these complexes.RNA viruses cause a variety of acute and chronic diseases in humans: common cold, summer flu, hepatitis, severe acute respiratory syndrome, and liver cancer, to name a few (1-5). The genomes of these viruses are transcribed and replicated by a virus-encoded RNA-dependent RNA polymerase (RdRp) 2 (4, 6, 7). Like other viral polymerases, the RdRp represents an important target for antiviral drug development (8 -11). The RdRp from poliovirus (PV), 3Dpol, has emerged as an important model for understanding the structure, function, and mechanism of this class of nucleic acid polymerases (12-15).PV 3Dpol will incorporate a ribonucleotide (rNMP) with an incorrect base at a frequency of ϳ10 Ϫ7 to 10 Ϫ4 (16, 17). However, this enzyme is much more tolerant of nucleotides with an incorrect sugar configuration. Both 2Ј-and 3Ј-deoxynucleotides (dNMPs) are incorporated at a frequency of ϳ10 Ϫ2 (15). It is known that incorporation of more than one incorrect ribonucleotide per PV genome decreases the specific infectivity of the RNA (16, 18). Whether or not 2Ј-dNMP incorporation has an effect on viral RNA infectivity is not known.Several factors likely limit 2Ј-dNMP incorporation into the genomes of RNA viruses of eukaryotes. First, dNTP pools in cells are thought to be low, in the 5-30 M range (19). Low dNTP levels are maintained by regulating the activity and localizat...

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Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro

Cited by 26 publications

References 54 publications

Picornavirus Genome Replication

Picornavirus Genome Replication

Initiation of protein-primed picornavirus RNA synthesis

Structure-Function Relationships of the Viral RNA-dependent RNA Polymerase

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