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J. Biol. Chem., Vol. 282, Issue 22, 16202-16213, June 1, 2007
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Picornavirus Genome Replication
ASSEMBLY AND ORGANIZATION OF THE VPg URIDYLYLATION RIBONUCLEOPROTEIN (INITIATION) COMPLEX*
From the Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802
All picornaviruses have a protein, VPg, covalently linked to the 5'-ends of their genomes. Uridylylated VPg (VPg-pUpU) is thought to serve as the protein primer for RNA synthesis. VPg-pUpU can be produced in vitro by the viral polymerase, 3Dpol, in a reaction in which a single adenylate residue of a stem-loop structure, termed oriI, templates processive incorporation of UMP into VPg by using a "slide-back" mechanism. This reaction is greatly stimulated by viral precursor protein 3CD or its processed derivative, 3C; both contain RNA-binding and protease activities. We show that the 3C domain encodes specificity for oriI, and the 3D domain enhances the overall affinity for oriI. Thus, 3C(D) stimulation exhibits an RNA length dependence. By using a minimal system to evaluate the mechanism of VPg uridylylation, we show that the active complex contains polymerase, oriI, and 3C(D) at stoichiometry of 1:1:2. Dimerization of 3C(D) is supported by physical and structural data. Polymerase recruitment to and retention in this complex require a protein-protein interaction between the polymerase and 3C(D). Physical and functional data for this interaction are provided for three picornaviruses. VPg association with this complex is weak, suggesting that formation of a complex containing all necessary components of the reaction is rate-limiting for the reaction. We suggest that assembly of this complex in vivo would be facilitated by use of precursor proteins instead of processed proteins. These data provide a glimpse into the organization of the ribonucleoprotein complex that catalyzes this key step in picornavirus genome replication.
Received for publication, November 15, 2006 , and in revised form, March 27, 2007.
* This work was supported, in part, by NIAID, National Institutes of Health, Grant AI053531 (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 Present address: Dept. of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
2 Recipient of American Heart Association Established Investigator Award 0340028N. 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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