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Journal of Virology, March 2005, p. 3565-3577, Vol. 79, No. 6
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.6.3565-3577.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

3'-Terminal Sequence in Poliovirus Negative-Strand Templates Is the Primary cis-Acting Element Required for VPgpUpU-Primed Positive-Strand Initiation

Nidhi Sharma, Brian J. O'Donnell, and James B. Flanegan*

Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida

Received 18 June 2004/ Accepted 23 November 2004

The 5' cloverleaf in poliovirus RNA has a direct role in regulating the stability, translation, and replication of viral RNA. In this study, we investigated the role of stem a in the 5' cloverleaf in regulating the stability and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Our results showed that disrupting the duplex structure of stem a destabilized viral RNA and inhibited efficient negative-strand synthesis. Surprisingly, the duplex structure of stem a was not required for positive-strand synthesis. In contrast, altering the primary sequence at the 5'-terminal end of stem a had little or no effect on negative-strand synthesis but dramatically reduced positive-strand initiation and the formation of infectious virus. The inhibition of positive-strand synthesis observed in these reactions was most likely a consequence of nucleotide alterations in the conserved sequence at the 3' ends of negative-strand RNA templates. Previous studies suggested that VPgpUpU synthesized on the cre(2C) hairpin was required for positive-strand synthesis. Therefore, these results are consistent with a model in which preformed VPgpUpU serves as the primer for positive-strand initiation on the 3'AAUUUUGUC5' sequence at the 3' ends of negative-strand templates. Our results suggest that this sequence is the primary cis-acting element that is required for efficient VPgpUpU-primed positive-strand initiation.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610-0245. Phone: (352) 392-0688. Fax: (352) 392-2953. E-mail: Flanegan{at}ufl.edu.

Journal of Virology, March 2005, p. 3565-3577, Vol. 79, No. 6
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.6.3565-3577.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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