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

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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.