Template recognition and formation of initiation complexes by the replicase of a segmented double-stranded RNA virus.
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.
Tortorici
M Alejandra
MA
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Broering
Teresa J
TJ
Nibert
Max L
ML
Patton
John T
JT
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D016679:Genome, Viral; D007313:Insecta; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012328:RNA Viruses / Q000737:chemistry* / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D012401:Rotavirus / Q000235:genetics; D012492:Salts / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic
D012333:RNA, Messenger; D012492:Salts; D008345:Manganese; D012965:Sodium Chloride; D012313:RNA; D012324:RNA-Dependent RNA Polymerase; D008274:Magnesium