Rotavirus VP1 alone specifically binds to the 3' end of viral mRNA, but the interaction is not sufficient to initiate minus-strand synthesis.
Recent studies have shown that disrupted (open) rotavirus cores have an associated replicase activity which supports the synthesis of dsRNA from viral mRNA in a cell-free system (D. Chen, C. Q.-Y. Zeng, M. J. Wentz, M. Gorziglia, M. K. Estes, and R. F. Ramig, J. Virol. 68:7030-7039, 1994). To determine which of the core proteins, VP1, VP2, or VP3, recognizes the template mRNA during RNA replication, SA11 open cores were incubated with 32P-labeled RNA probes of viral and nonviral origin and the reaction mixtures were analyzed for the formation of RNA-protein complexes by gel mobility shift assay. In mixtures containing a probe representing the 3' end of SA11 gene 8 mRNA, two closely migrating RNA-protein complexes, designated s and f, were detected. The interaction between the RNA and protein of the s and f complexes was shown to be specific by competitive binding assay with tRNA and brome mosaic virus RNA. By electrophoretic analysis of RNA-protein complexes recovered from gels, VP1 was shown to be the only viral protein component of the complexes, thereby indicating that VP1 specifically recognizes the 3' end of gene 8 mRNA. Analysis of VP1 purified from open cores by glycerol gradient centrifugation verified that VP1 recognizes the 3' end of viral mRNA but also showed that in the absence of other viral proteins, VP1 lacks replicase activity. When reconstituted with VP2-rich portions of the gradient, VP1 stimulated levels of replicase activity severalfold. These data indicate that VP1 can bind to viral mRNA in the absence of any other viral proteins and suggest that VP2 must interact with the RNA-protein complex before VP1 gains replicase activity.
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA.
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Patton
D002460:Cell Line; D015347:RNA Probes / Q000378:metabolism; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D014758:Viral Core Proteins / Q000378:metabolism*
D015347:RNA Probes; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C069187:VP1 protein, Rotavirus; D014758:Viral Core Proteins; D012324:RNA-Dependent RNA Polymerase