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,John T Patton

doi:10.1074/jbc.m305358200

Abstract

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.

Highlights

Template Recognition and Formation of Initiation Complexes by the Replicase of a Segmented Double-stranded RNA Virus*
The RNA-dependent RNA polymerase (RdRP) alone can 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
To assess the possibility that salt inhibited dsRNA synthesis by reducing the ability of the RNA polymerase to interact with the template mRNA, rVP1 and 32P-labeled g8-3Ј60 probe were co-incubated in the presence of 0 –250 mM NaCl. rVP11⁄7probe complexes in the reaction mixtures were analyzed by electrophoretic mobility shift assays (EMSA), and their levels compared with the levels of dsRNA synthesis that occurred in replicase assays containing NaCl (Fig. 9)

Summary

The abbreviations used are

RdRP, RNA-dependent RNA polymerase; dsRNA, double strand RNA; CS, consensus sequence; EMSA, electrophoretic mobility shift assay; nt, nucleotide(s); NonV, nonviral RNA. The group A rotaviruses are the primary cause of acute dehydrating gastroenteritis in infants and young children throughout the world [8] Their genome consists of 11 segments of dsRNA, each positioned at one of the vertexes of the T ϭ 1 icosahedral core contained within the triple-layered rotavirus virion [9]. Modifications made to the mRNA that alter the panhandle structure or that convert the 3ЈCS from a single-stranded to doublestranded form inhibit dsRNA synthesis (19 –20) Both sequence and structure are critical factors influencing the ability of rotavirus mRNAs to function as templates in replication. The RdRP can bind to recognition signals in the absence of other proteins, the polymerase requires the core lattice protein to form the salt-resistant (Ϫ) strand initiation complex. Template recognition and the formation of (Ϫ) strand initiation complexes by the RdRP differ in sequence and protein requirements

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION