A “Slide-back” Mechanism for the Initiation of Protein-primed RNA Synthesis by the RNA Polymerase of Poliovirus

Paul, Aniko V.; Jiang, Yin; Mugavero, JoAnn; Rieder, Elizabeth; Liu, Ying; Wimmer, Eckard

doi:10.1074/jbc.m307441200

Cited by 82 publications

(100 citation statements)

References 41 publications

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“…There is consensus that initiation requires formation of a ribonucleoprotein complex at oriI and that this complex adds two uridylate residues to a tyrosine of VPg or some precursor thereof (61,63). The prevailing model suggests that the 3AB protein serves as the source of VPg, which, as a processed peptide, is used as the primer for initiation (74).…”

Section: Discussionmentioning

confidence: 99%

Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants

Pathak

Goodfellow

et al. 2009

J Virol

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A poliovirus (PV) mutant (termed GG), which is incapable of producing 3AB, VPg, and 3CD proteins due to a defective cleavage site between the 3B and 3C proteins, replicated, producing 3BC-linked RNA rather than the VPg-linked RNA produced by the wild type (WT). GG PV RNA is quasi-infectious. The yield of infectious GG PV relative to replicated RNA is reduced by almost 5 logs relative to that of WT PV. Proteolytic activity required for polyprotein processing is normal for the GG mutant. 3BC-linked RNA can be encapsidated as efficiently as VPg-linked RNA. However, a step after genome replication but preceding virus assembly that is dependent on 3CD and/or 3AB proteins limits production of infectious GG PV. This step may involve release of replicated genomes from replication complexes. A pseudorevertant (termed EG) partially restored cleavage at the 3B-3C cleavage site. The reduced rate of formation of 3AB and 3CD caused corresponding reductions in the observed rate of genome replication and infectious virus production by EG PV without impacting the final yield of replicated RNA or infectious virus relative to that of WT PV. Using EG PV, we showed that genome replication and encapsidation were distinct steps in the multiplication cycle. Ectopic expression of 3CD protein reversed the genome replication phenotype without alleviating the infectious-virus production phenotype. This is the first report of a trans-complementable function for 3CD for any picornavirus. This observation supports an interaction between 3CD protein and viral and/or host factors that is critical for genome replication, perhaps formation of replication complexes.

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Section: Discussionmentioning

confidence: 99%

Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants

Pathak

Goodfellow

et al. 2009

J Virol

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“…While no detailed studies of slippage rates in RNA-dependent RNA polymerases have been carried out, there is precedent for active-site slippage by poliovirus 3D pol . The double uridylylation reaction that generates the VPgpUpU primer used for viral genome replication is templated by a pair of adenosines in the cis-acting replication element 2C cre , but the reaction itself uses a slide-back mechanism where the same adenosine templates both incorporation events (46). The molecular details of slippage across a longer 8-to 10-bp homopolymer duplex in the polymerase during poly(A) tail synthesis are likely to be somewhat different from those needed for a simple single-basepair slippage step during VPg uridylylation, but similar templateshifting mechanisms in the active site are probably used by both events.…”

Section: Discussionmentioning

confidence: 99%

Structural Features of a Picornavirus Polymerase Involved in the Polyadenylation of Viral RNA

Kempf

Kelly²,

Springer

et al. 2013

J Virol

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Picornaviruses have 3= polyadenylated RNA genomes, but the mechanisms by which these genomes are polyadenylated during viral replication remain obscure. Based on prior studies, we proposed a model wherein the poliovirus RNA-dependent RNA polymerase (3D pol ) uses a reiterative transcription mechanism while replicating the poly(A) and poly(U) portions of viral RNA templates. To further test this model, we examined whether mutations in 3D pol influenced the polyadenylation of virion RNA. We identified nine alanine substitution mutations in 3D pol that resulted in shorter or longer 3= poly(A) tails in virion RNA. These mutations could disrupt structural features of 3D pol required for the recruitment of a cellular poly(A) polymerase; however, the structural orientation of these residues suggests a direct role of 3D pol in the polyadenylation of RNA genomes. Reaction mixtures containing purified 3D pol and a template RNA with a defined poly(U) sequence provided data consistent with a template-dependent reiterative transcription mechanism for polyadenylation. The phylogenetically conserved structural features of 3D pol involved in the polyadenylation of virion RNA include a thumb domain alpha helix that is positioned in the minor groove of the double-stranded RNA product and lysine and arginine residues that interact with the phosphates of both the RNA template and product strands.P icornaviruses, like many other positive-strand RNA viruses, have RNA genomes with variable-length 3= poly(A) tails (1). The poly(A) tails of picornaviruses are important for viability (2), with the length of the poly(A) tails influencing the magnitudes of both viral mRNA translation and RNA replication (3, 4). RNA sequences and structures within the 3= nontranslated region are reported to influence both the length of poly(A) tails in picornaviral RNA (5) and the efficiency of virus replication (6, 7). Viral RNA-dependent RNA polymerases (3D pol s) are predicted to catalyze the polyadenylation of picornaviral RNA in a template-dependent manner during viral RNA replication (8); however, the mechanisms by which RNA genomes are polyadenylated during viral RNA replication remain obscure. In particular, there is little understanding of the mechanisms regulating the length of poly(A) tails synthesized during RNA replication. The RNA-dependent RNA polymerases of negative-strand RNA viruses reiteratively transcribe a small poly(U) sequence within intergenic regions of the viral RNA genome to produce long poly(A) tails on viral mRNAs (9). In a similar manner, the poliovirus RNA-dependent RNA polymerase can reiteratively transcribe poly(A) and poly(U) sequences during viral RNA replication, producing poly(A) and poly(U) sequences longer than those in the respective template RNAs (8).Viral RNA-dependent RNA polymerases are well studied at the structural level (10-12), yet there have been no reports describing the structural features of viral polymerases involved in the polyadenylation of viral RNA. The RNA-dependent RNA polymerase of picornaviruses a...

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“…Finally, two rounds of UMP incorporation occur without dissociation of the peptide primer. Both uridylate residues are templated by a single adenylate residue by using a slideback mechanism (40).…”

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confidence: 99%

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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A “Slide-back” Mechanism for the Initiation of Protein-primed RNA Synthesis by the RNA Polymerase of Poliovirus

Cited by 82 publications

References 41 publications

Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants

Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants

Structural Features of a Picornavirus Polymerase Involved in the Polyadenylation of Viral RNA

Picornavirus Genome Replication

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