A base-specific recognition signal in the 5′ consensus sequence of rotavirus plus-strand RNAs promotes replication of the double-stranded RNA genome segments

Abstract

Replication of the segmented double-stranded (ds)RNA genome of rotavirus requires the viral RNA-dependent RNA polymerase (RdRP) to use 11 different (+)RNAs as templates for (-) strand synthesis. Complementary sequences proximal to the 5′ and 3′ termini are predicted to direct cyclization of the (+)RNAs by forming panhandle structures from which short highly conserved terminal sequences protrude as single-stranded tails. Cell-free replication assays indicate that such structural organization of the 5′- and 3′-ends is required for efficient dsRNA synthesis. Multiple specifically recognized elements exist at the 3′-end that promote dsRNA synthesis including RdRP-recruitment signals and a (-) strand initiation sequence. In contrast to the 3′-end, the role of the 5′-end has been less well defined. In this study, we determined that the 5′-end contains a base-specific recognition signal that plays an important role in the assembly of the RdRP and cofactors into a stable initiation complex for (-) strand synthesis. The 5′ recognition signal is associated with the G2 residue of the 5′-consensus sequence, a residue that shows absolute conservation among all rotavirus groups (A, B, and C) examined to date. From our results, we suggest that rotavirus (+)RNA cyclization, although likely initiated by 5′- 3′ nucleotide complementarity, may be stabilized by RdRP-dependent bridging. Given that synthesis of the (-) strand on the (+)RNA template will disrupt 5′-3′ nucleotide interactions, RdRP-dependent bridging may be the sole mechanism by which the dsRNA product can be held in the necessary cyclized conformation required for efficient multiple rounds of transcription. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 RNA Society.