Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

Astbury Centre for Structural Molecular Biology School of Molecular and Cellular Biology Faculty of Biological Sciences University of Leeds LS2 9JT Leeds United Kingdom

Australian Research Council Australia Clayton VIC 3800 Australia

Department of Biochemistry and Molecular Biology Biomedicine Discovery Institute Faculty of Medicine Nursing and Health Sciences Monash University Clayton VIC 3800 Australia

Department of Biochemistry University of Cambridge Cambridge CB2 1QW United Kingdom

Department of Chemistry Center for NanoScience Nanosystems Initiative Munich Centre for Integrated Protein Science Munich Ludwig Maximilian University of Munich D 81377 Munich Germany

Faculty of Science University of South Bohemia 370 05 Ceske Budejovice Czech Republic

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