Poliovirus
 cis
 -Acting Replication Element-Dependent VPg Uridylylation Lowers the
 
 K
 m
 
 of the Initiating Nucleoside Triphosphate for Viral RNA Replication

Steil, Benjamin P.; Barton, David J.

doi:10.1128/jvi.00427-08

Cited by 17 publications

(29 citation statements)

References 52 publications

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“…As demonstrated by previously described experiments in our lab (42), both VPg and VPgpUpU OH can prime the initiation of negative-strand RNA synthesis. Therefore, we examined whether the VPg-linked poly(U) sequences from VPgpUpU OH -primed and VPg-primed negative-strand RNA synthesis were qualitatively different.…”

Section: Resultsmentioning

confidence: 74%

“…3A, lanes 1 to 3 versus lanes 4 to 6, respectively). KO CRE RNA templates fail to make VPgpUpU OH primers (29), and low UTP concentrations favor VPgpUpU OH priming over VPg priming (42). Consequently, we compared wild-type and KO CRE RNA templates under two reactions conditions, low and high UTP concentrations (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…PV RNA-dependent RNA polymerase primes the initiation of negative-strand RNA synthesis with VPg and VPgpUpU OH on the 3Ј poly(A) tails of input positive-strand RNA templates (40,42). In order to determine the size of VPg-linked poly(U) at the 5Ј terminus of negative-strand RNA, [␣-32 P]UTP was incorporated into PV negative-strand RNA as it was synthesized within PV RNA replication complexes (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Poly(A) at the 3′ End of Positive-Strand RNA and VPg-Linked Poly(U) at the 5′ End of Negative-Strand RNA Are Reciprocal Templates during Replication of Poliovirus RNA

Steil¹,

Kempf²,

Barton

2010

J Virol

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A 3 poly(A) tail is a common feature of picornavirus RNA genomes and the RNA genomes of many other positive-strand RNA viruses. We examined the manner in which the homopolymeric poly(A) and poly(U) portions of poliovirus (PV) positive-and negative-strand RNAs were used as reciprocal templates during RNA replication. Poly(A) sequences at the 3 end of viral positive-strand RNA were transcribed into VPg-linked poly(U) products at the 5 end of negative-strand RNA during PV RNA replication. Subsequently, VPg-linked poly(U) sequences at the 5 ends of negative-strand RNA templates were transcribed into poly(A) sequences at the 3 ends of positive-strand RNAs. The homopolymeric poly(A) and poly(U) portions of PV RNA products of replication were heterogeneous in length and frequently longer than the corresponding homopolymeric sequences of the respective viral RNA templates. The data support a model of PV RNA replication wherein reiterative transcription of homopolymeric templates ensures the synthesis of long 3 poly(A) tails on progeny RNA genomes.Many positive-strand RNA viruses (e.g., members of the Picornavirales, Nidovirales, Togaviridae, Caliciviridae, and Astroviridae) have long poly(A) sequences at the 3Ј termini of their RNA genomes (16,25), yet the mechanisms by which 3Ј poly(A) sequences are derived during viral replication are unclear. Picornavirus RNA genomes, including that of poliovirus (PV), have a covalently linked 5Ј-terminal protein called VPg (viral protein, genome linked), a 5Ј untranslated region (UTR), a single large open reading frame, a 3Ј UTR, and a poly(A) tail of variable length (ϳ20 to 150 adenosine residues) (1). RNA polymerases encoded by viruses in the order Picornavirales utilize viral proteins (and their nucleotidylylated intermediates) to prime the initiation of RNA replication at the 3Ј termini of viral RNA templates (28,29,31,34). In this mechanism, the viral protein VPg becomes covalently linked to the 5Ј ends of both positive-and negative-strand RNAs during viral RNA replication (30, 32). PV, which is commonly studied to elucidate mechanisms of picornavirus replication, is viable when the 3Ј UTR of the genome is deleted (12, 44); however, the 3Ј poly(A) tail is essential for RNA replication (33, 39). The length of the 3Ј poly(A) tail required for virus viability and for efficient negative-strand RNA synthesis has been examined in some detail (35,45). PV RNAs with 3Ј poly(A) tails less than 9 bases long support less than 1% of wild-type negative-strand RNA synthesis, whereas poly(A) tails Ն20 bases long support wild-type levels of negative-strand RNA synthesis (35).In this investigation, we programmed PV RNAs with defined 3Ј 84-, 51-, and 32-base poly(A) sequences [designated poly(A) (84) , poly(A) (51) , and poly(A) (32) , respectively] into cell-free reactions that faithfully reconstitute all of the metabolic steps of viral mRNA translation (11,22,23) and viral RNA replication (5, 7, 27). A significant advantage of this experimental system is the ability to study one cycle of seq...

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Section: Resultsmentioning

confidence: 74%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Poly(A) at the 3′ End of Positive-Strand RNA and VPg-Linked Poly(U) at the 5′ End of Negative-Strand RNA Are Reciprocal Templates during Replication of Poliovirus RNA

Steil¹,

Kempf²,

Barton

2010

J Virol

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“…3C). Poliovirion RNA has poly(A) tails ranging in size from 20 to 120 bases [A (20) to A (120) ] long, with a classic bell curve distribution around a mean of ϳ50 bases (Fig. 3B, lane 8, and C).…”

Section: Resultsmentioning

confidence: 99%

“…Poliovirus RNA translation, polyprotein processing, and RNA replication were assayed in reaction mixtures containing cytoplasmic extracts from uninfected HeLa cells as previously described (18)(19)(20). Radiolabeled proteins from reaction mixtures containing [ 35 S]methionine were fractionated by SDS-PAGE and detected by phosphorimaging.…”

Section: Methodsmentioning

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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Unmasking the information encoded as structural motifs of viral RNA genomes: a potential antiviral target

Romero-López

Berzal‐Herranz

2013

Reviews in Medical Virology

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RNA viruses show enormous capacity to evolve and adapt to new cellular and molecular contexts, a consequence of mutations arising from errors made by viral RNA-dependent RNA polymerase during replication. Sequence variation must occur, however, without compromising functions essential for the completion of the viral cycle. RNA viruses are safeguarded in this respect by their genome carrying conserved information that does not code only for proteins but also for the formation of structurally conserved RNA domains that directly perform these critical functions. Functional RNA domains can interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. They are therefore potential targets for novel therapeutic strategies. This review summarises our knowledge of the functional RNA domains of human RNA viruses and examines the achievements made in the design of antiviral compounds that interfere with their folding and therefore their function.

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Poliovirus cis -Acting Replication Element-Dependent VPg Uridylylation Lowers the K _m of the Initiating Nucleoside Triphosphate for Viral RNA Replication

Cited by 17 publications

References 52 publications

Poly(A) at the 3′ End of Positive-Strand RNA and VPg-Linked Poly(U) at the 5′ End of Negative-Strand RNA Are Reciprocal Templates during Replication of Poliovirus RNA

Poly(A) at the 3′ End of Positive-Strand RNA and VPg-Linked Poly(U) at the 5′ End of Negative-Strand RNA Are Reciprocal Templates during Replication of Poliovirus RNA

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

Unmasking the information encoded as structural motifs of viral RNA genomes: a potential antiviral target

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Poliovirus cis -Acting Replication Element-Dependent VPg Uridylylation Lowers the K m of the Initiating Nucleoside Triphosphate for Viral RNA Replication

Cited by 17 publications

References 52 publications

Poly(A) at the 3′ End of Positive-Strand RNA and VPg-Linked Poly(U) at the 5′ End of Negative-Strand RNA Are Reciprocal Templates during Replication of Poliovirus RNA

Poly(A) at the 3′ End of Positive-Strand RNA and VPg-Linked Poly(U) at the 5′ End of Negative-Strand RNA Are Reciprocal Templates during Replication of Poliovirus RNA

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

Unmasking the information encoded as structural motifs of viral RNA genomes: a potential antiviral target

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Poliovirus cis -Acting Replication Element-Dependent VPg Uridylylation Lowers the K _m of the Initiating Nucleoside Triphosphate for Viral RNA Replication