The structure of a protein primer–polymerase complex in the initiation of genome replication

Ferrer-Orta, Cristina; Arias, Armando; Agudo, Rubén; Pérez-Luque, R.; Escarmı́s, Cristina; Domingo, Esteban; Verdaguer, N.

doi:10.1038/sj.emboj.7600971

Cited by 119 publications

(183 citation statements)

References 22 publications

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“…2); the three residues are strictly conserved in picornaviral polymerases. The critical role of Asp-338, Lys-387, and Arg-388 in uridylytation of the VPg primer protein has recently been demonstrated (14). Furthermore, the substitution of either Asp-338 or Lys-387/Arg-388 by Ala in 3D led to noninfectious FMDVs (C.E., A.A., and E.D., unpublished results).…”

Section: Discussionmentioning

confidence: 93%

“…We have previously reported the structure and interactions of the FMDV 3D polymerase with a template-primer RNA showing structural evidence of how the physiological substrates bind the large exposed active site of the picornavirus RDRPs (12,13). In addition, the structure of the complex between the polymerase 3D and its protein-primer VPg revealed the critical interactions involved in the positioning and addition of the first nucleotide (UMP) to the primer molecule, providing insights into the mechanism of initiation of RNA genome replication in picornaviruses (14).…”

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confidence: 99%

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Sequential structures provide insights into the fidelity of RNA replication

Ferrer-Orta

Arias²,

Pérez-Luque³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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141

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RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine triphosphate with different RNAs as template-primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3 -OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases.5-fluorouracil ͉ foot-and-mouth disease virus ͉ replication fidelity ͉ ribavirin ͉ RNA elongation

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Section: Discussionmentioning

confidence: 93%

mentioning

confidence: 99%

Sequential structures provide insights into the fidelity of RNA replication

Ferrer-Orta

Arias²,

Pérez-Luque³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

118

141

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show abstract

“…In both structures, there are extensive interactions between thumb and finger domains, fully enclosing the active site. Furthermore, the shape and size of the central cavity (which accommodates primer/ template) are almost identical (Ferrer-Orta et al 2006). …”

Section: Inhibition Of Polymerase Functionmentioning

confidence: 99%

“…This represents the first complete structure of a picornavirus polymerase and has the ''palm, thumb, and fingertips'' topology seen in other RdRps (O'Reilly and Kao 1998). Recently, the structure of 3Dpol complexed with the VPg primer peptide has also been published (Ferrer-Orta et al 2006). Although these FMDV structures have significantly advanced our understanding of the properties of the polymerase, there are still many aspects of its function in replication or in the uridylation of VPg that are not fully understood.…”

Section: Introductionmentioning

confidence: 99%

Selection and characterization of RNA aptamers to the RNA-dependent RNA polymerase from foot-and-mouth disease virus

Ellingham¹,

Bunka²,

Rowlands³

et al. 2006

RNA

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Foot-and-mouth disease virus causes a highly contagious disease of agricultural livestock and is of enormous economic importance. Replication of the RNA genome of the virus, via negative strand intermediates, involves an RNA-dependent RNA polymerase (3Dpol). RNA aptamers specific to this enzyme have been selected and characterized. Some of these molecules inhibit enzymatic activity in vitro, with IC 50 values of <20 nM and K i values of 18-75 nM. Two of these show similarity, both with each other and with regions of the viral genome. Furthermore, truncated versions of one of the aptamers have been used to define the parts of the molecule responsible for its inhibitory activity.

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“…1, step 3) (13,14). VPg or some precursor thereof joins the complex either along with (37,38) or after 3Dpol (39). Finally, two rounds of UMP incorporation occur without dissociation of the peptide primer.…”

mentioning

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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The structure of a protein primer–polymerase complex in the initiation of genome replication

Cited by 119 publications

References 22 publications

Sequential structures provide insights into the fidelity of RNA replication

Sequential structures provide insights into the fidelity of RNA replication

Selection and characterization of RNA aptamers to the RNA-dependent RNA polymerase from foot-and-mouth disease virus

Picornavirus Genome Replication

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