The poliovirus 2C cis-acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes

Goodfellow, Ian; Polacek, Charlotta; Andino, Raul; Evans, David J.

doi:10.1099/vir.0.19132-0

Cited by 57 publications

(77 citation statements)

References 27 publications

(23 reference statements)

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“…Interestingly, in PV RNA, a portion of the 2C coding sequence (a cis-acting replication element, CRE) can also direct the uridylylation of VPg (Goodfellow et al 2000;Paul et al 2000). However, VPgpU/VPgpUpU made on the CRE structure apparently primes only plus-strand RNA initiation (Goodfellow et al 2003;Morasco et al 2003;Murray and Barton 2003). Interestingly, although picornavirus poly(A) is genetically coded (Dorsch-Hasler et al 1975), its length is regulated by a special mechanism, of which a portion of the 39 UTR (oriR) is a key player (van Ooij et al 2006).…”

Section: Introductionmentioning

confidence: 99%

Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Svitkin

Costa-Mattioli²,

Herdy³

et al. 2007

RNA

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Picornavirus infectivity is dependent on the RNA poly(A) tail, which binds the poly(A) binding protein (PABP). PABP was reported to stimulate viral translation and RNA synthesis. Here, we studied encephalomyocarditis virus (EMCV) and poliovirus (PV) genome expression in Krebs-2 and HeLa cell-free extracts that were drastically depleted of PABP (96%-99%). Although PABP depletion markedly diminished EMCV and PV internal ribosome entry site (IRES)-mediated translation of a polyadenylated luciferase mRNA, it displayed either no (EMCV) or slight (PV) deleterious effect on the translation of the full-length viral RNAs. Moreover, PABP-depleted extracts were fully competent in supporting EMCV and PV RNA replication and virus assembly. In contrast, removing the poly(A) tail from EMCV RNA dramatically reduced RNA synthesis and virus yields in cell-free reactions. The advantage conferred by the poly(A) tail to EMCV synthesis was more pronounced in untreated than in nuclease-treated extract, indicating that endogenous cellular mRNAs compete with the viral RNA for a component(s) of the RNA replication machinery. These results suggest that the poly(A) tail functions in picornavirus replication largely independent of PABP.

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Section: Introductionmentioning

confidence: 99%

Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Svitkin

Costa-Mattioli²,

Herdy³

et al. 2007

RNA

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“…These data provide a picture in which 2C RNA-mediated VPg uridylylation involves a complex of 3D polymerase, VPg and 2C RNA-associated 3CD. Poly(A)-mediated VPg uridylylation, however, may involve only the polymerase and VPg, at least in vitro.Recent reports (10,25,26) provide convincing evidence that 2C RNA is required for poliovirus positive-strand, but not negative-strand, synthesis. Specifically, mutations that disrupt the 2C RNA stem-loop structure were shown to block both synthesis of positive strands and accumulation of VPg-pU(pU) in an in vitro translation-replication system (10, 25).…”

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confidence: 97%

“…This specificity could be provided if an RNA sequence or structure within the picornavirus genome were the authentic RNA template for VPg uridylylation. Such RNA sequences, such as in the capsid-coding region of rhinovirus 14 (24), the 2C-coding region of poliovirus RNA (9,10,33,35), the VP2-coding region of Theiler's and Mengo viruses (20), the 2A-coding region of rhinovirus 2 (7), and the 5Ј noncoding region (NCR) of foot-and-mouth-disease virus (23), have now been described for several picornaviruses.When poliovirus protein 3CD, engineered to be proteinasedeficient and thus to remain in precursor form, is added to reactions that contain the 2C RNA, poliovirus 3D polymerase and VPg, VPg uridylylation is stimulated substantially above the rate observed in the absence of 3CD (32,33,35). Specific binding of 3CD to the 2C CRE RNA sequence (2C RNA), which contains the stem-loop structure, has been demonstrated (44).…”

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confidence: 99%

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confidence: 99%

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Allosteric Effects of Ligands and Mutations on Poliovirus RNA-Dependent RNA Polymerase

Boerner

Lyle

Daijogo

et al. 2005

J Virol

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Protein priming of viral RNA synthesis plays an essential role in the replication of picornavirus RNA. Both poliovirus and coxsackievirus encode a small polypeptide, VPg, which serves as a primer for addition of the first nucleotide during synthesis of both positive and negative strands. This study examined the effects on the VPg uridylylation reaction of the RNA template sequence, the origin of VPg (coxsackievirus or poliovirus), the origin of 3D polymerase (coxsackievirus or poliovirus), the presence and origin of interacting protein 3CD, and the introduction of mutations at specific regions in the poliovirus 3D polymerase. Substantial effects associated with VPg origin were traced to differences in VPg-polymerase interactions. The effects of 3CD proteins and mutations at polymerase-polymerase intermolecular Interface I were most consistent with allosteric effects on the catalytic 3D polymerase molecule. In conclusion, the efficiency and specificity of VPg uridylylation by picornavirus polymerases is greatly influenced by allosteric effects of ligand binding that are likely to be relevant during the viral replicative cycle.Picornaviruses have a single copy, positive-strand RNA genome with a small peptide, VPg (3B), covalently attached to the 5Ј-terminal nucleotide and a poly(A) segment at the 3Ј end. RNA replication occurs in the cytoplasm of infected cells, proceeding through negative-strand intermediates which, in turn, serve as templates for production of progeny positive strands. A long-standing question has been how both positiveand negative-strand syntheses are initiated. Characterization of a reaction catalyzed by poliovirus 3D polymerase, in which the tyrosine of the VPg peptide was trans-esterified (uridylylated) in the presence of a poly(A) template to form VPg-pU (pU), provided insight into the mechanism of strand initiation (34): given the ability of poliovirus 3D polymerase to uridylylate VPg, the peptide-nucleotide conjugate could serve as the protein primer for progeny RNA strand synthesis. VPg must bind directly to poliovirus polymerase, because it can serve as an enzymatic substrate; furthermore, VPg-polymerase interactions have been observed in two-hybrid experiments (42). While the binding site on the poliovirus 3D polymerase for the VPg substrate of the uridylylation reaction has not yet been characterized, the binding site for its likely proteolytic precursor, 3AB, has been identified as a distinct site on the back of the palm of the polymerase molecule via extensive alaninescanning mutagenesis (22).Although VPg uridylylation could provide a protein primer for use in either positive-or negative-strand synthesis, this reaction is not sufficient to describe the mechanism of initiation for viral RNA synthesis in infected cells. For example, the use of a poly(A) template for VPg uridylylation does not provide specificity for a particular virus. This specificity could be provided if an RNA sequence or structure within the picornavirus genome were the authentic RNA template for VPg uridylylation...

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“…3D pol uses the same mechanism and the same active site for VPg-uridylation as for processive RNA synthesis, as demonstrated by the sensitivity of the VPg-uridylation reaction to mutations of the two catalytic aspartic acids in the 3D pol active site (34). For PV it was shown that cre(2C)-templated VPg-uridylation was important for the initiation of positive-strand RNA synthesis, whereas it seemed not to be required for negative-strand RNA synthesis (20,37,38). The poly(rA) tail was proposed to act as the template for the initiation of negative-strand RNA synthesis in vivo (38,48).…”

mentioning

confidence: 99%

The Crystal Structure of Coxsackievirus B3 RNA-Dependent RNA Polymerase in Complex with Its Protein Primer VPg Confirms the Existence of a Second VPg Binding Site on Picornaviridae Polymerases

Gruez

Selisko

Roberts

et al. 2008

J Virol

119

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The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3D pol ) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3D pol at this site excludes its uridylation by the carrier 3D pol . We suggest that VPg at this position is either uridylated by another 3D pol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3D pol /VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.

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The poliovirus 2C cis-acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes

Cited by 57 publications

References 27 publications

Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Allosteric Effects of Ligands and Mutations on Poliovirus RNA-Dependent RNA Polymerase

The Crystal Structure of Coxsackievirus B3 RNA-Dependent RNA Polymerase in Complex with Its Protein Primer VPg Confirms the Existence of a Second VPg Binding Site on Picornaviridae Polymerases

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