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J. Biol. Chem., Vol. 278, Issue 45, 43951-43960, November 7, 2003

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

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

From the Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, New York 11790

Poliovirus RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction can be reproduced in vitro with an assay that utilizes two purified viral proteins, RNA polymerase 3D^pol and viral protein 3CD^pro, synthetic VPg, UTP, and Mg²⁺. The template for the reaction is either poliovirus RNA or transcripts of a small RNA hairpin, termed cre(2C), located in the coding sequence of protein 2C^ATPase. The products of the reaction are VPgpU and VPgpUpU, the primers used by 3D^pol for RNA synthesis. With mutant template RNAs in this assay we determined the precise initiation site. Our results indicate that 1) 3D^pol does not possess strict specificity toward the nucleotide it links to VPg, 2) A-5 of the conserved ¹GXXXAAAXXXXXXA¹⁴ sequence in the loop is the template nucleotide for the linkage of both the first and second UMPs to VPg, 3) VPgpUpU is synthesized by a "slide-back" mechanism, and 4) A-6 provides specificity to the reaction during the slide-back step and also modulates the uridylylation reaction. In additional experiments we determined the effect of mutations in the ⁵AAA⁷ sequence of cre(2C) on viral growth, RNA replication, and on the activity of the 2C^ATPase protein. Furthermore, we observed that the spacing between G-1 and A-5 and the size of the loop affect the yield but not the nature of the VPg-linked products.

Received for publication, July 11, 2003 , and in revised form, August 20, 2003.

^* This work was supported by an NIAID, National Institutes of Health Grant R37 AIO15122-30. 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.

Present address: United States Dept. of Agriculture, Agricultural Research Service, Plum Island Animal Disease Center, Greenport, NY 11944.

To whom correspondence should be addressed. Tel.: 631-632-9777; Fax: 631-632-8891; E-mail: apaul{at}ms.cc.sunysb.edu.

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