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J. Biol. Chem., Vol. 283, Issue 2, 875-888, January 11, 2008

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Picornavirus Genome Replication

IDENTIFICATION OF THE SURFACE OF THE POLIOVIRUS (PV) 3C DIMER THAT INTERACTS WITH PV 3Dpol DURING VPg URIDYLYLATION AND CONSTRUCTION OF A STRUCTURAL MODEL FOR THE PV 3C₂-3Dpol COMPLEX^*

From the Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802

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 3C₂-3Dpol complex that extrapolates well to all picornaviruses.

Received for publication, September 20, 2007 , and in revised form, November 8, 2007.

^* This work was supported in part by NIAID Research Grant AI053531 from the National Institutes of Health (to C. E. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Present address: Dept. of Biomedical Sciences, Cornell University, Ithaca, NY 14853.

³ Recipient of a Department of Homeland Security scholarship. Present address: Medical Scientist Training Program, Duke University School of Medicine, Durham, NC 27705.

⁴ Present address: Dept. of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111.

⁵ Recipient of Established Investigator Award 0340028N from the American Heart Association. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Pennsylvania State University, 201 Althouse Laboratory, University Park, PA 16802. Tel.: 814-863-8705; Fax: 814-865-7927; E-mail: cec9{at}psu.edu.

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				H. B. Pathak, H. S. Oh, I. G. Goodfellow, J. J. Arnold, and C. E. Cameron Picornavirus Genome Replication: ROLES OF PRECURSOR PROTEINS AND RATE-LIMITING STEPS IN oriI-DEPENDENT VPg URIDYLYLATION J. Biol. Chem., November 7, 2008; 283(45): 30677 - 30688. [Abstract] [Full Text] [PDF]

				C. D. Amero, J. J. Arnold, I. M. Moustafa, C. E. Cameron, and M. P. Foster Identification of the oriI-Binding Site of Poliovirus 3C Protein by Nuclear Magnetic Resonance Spectroscopy J. Virol., May 1, 2008; 82(9): 4363 - 4370. [Abstract] [Full Text] [PDF]