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J. Biol. Chem., Vol. 278, Issue 35, 32673-32682, August 29, 2003

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Template Recognition and Formation of Initiation Complexes by the Replicase of a Segmented Double-stranded RNA Virus^*

M. Alejandra Tortorici , Teresa J. Broering , Max L. Nibert  and John T. Patton  ¶

From the Laboratory of Infectious Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892 and the Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

Replication of the segmented double-stranded (ds) RNA genome of viruses belonging to the Reoviridae family requires the RNA-dependent RNA polymerase (RdRP) to use 10–12 different mRNAs as templates for (–) strand synthesis. Rotavirus serves as a model system for study of this process, since its RdRP (VP1) is catalytically active and can specifically recognize template mRNAs in vitro. Here, we have analyzed the requirements for template recognition by the rotavirus RdRP and compared those to the requirements for formation of (–) strand initiation complexes. The results show that multiple functionally independent recognition signals are present at the 3'-end of viral mRNAs, some positioned in nonconserved regions upstream of the highly conserved 3'-terminal consensus sequence. We also found that RdRP recognition signals are distinct from cis-acting signals that promote (–) strand synthesis, because deletions of portions of the 3'-consensus sequence that caused viral mRNAs to be poorly replicated in vitro did not necessarily prevent efficient recognition of the RNA by the RdRP. Although the RdRP alone can specifically bind to viral mRNAs, our analysis reveals that this interaction is not sufficient to generate initiation complexes, even in the presence of nucleotides and divalent cations. Rather, the formation of initiation complexes also requires the core lattice protein (VP2), a virion component that forms a T = 1 icosahedral shell that encapsidates the segmented dsRNA genome. The essential role that the core lattice protein has in (–) strand initiation provides a mechanism for the coordination of genome replication and virion assembly.

Received for publication, May 22, 2003

^* 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.

^¶ To whom correspondence should be addressed: Laboratory of Infectious Diseases, NIAID, National Institutes of Health, 50 South Dr., MSC 8026, Rm. 6314, Bethesda, MD 20892. Tel.: 301-594-1615; Fax: 301-496-8312; E-mail: jpatton{at}niaid.nih.gov.

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