Abstract
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.
Highlights
Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly
We showed that alanine substitutions of conserved acidic residues in the C-terminal region (CTR) (D306, D310, and E311) abrogated RV replication, while charge-preserving mutations had no detrimental effect on virus rescue in reverse genetics experiments
We determined a 3D reconstruction of NSP2-ΔC using negative-stain EM which confirmed that NSP2-ΔC remains octameric, demonstrating that the CTR does not play a role in the assembly of NSP2 into functional octamers (SI Appendix, Fig. S1)
Summary
Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. While NSP2-ΔC exhibits a reduced capacity to promote RNA–RNA interactions, it possesses enhanced RNA unwinding activity To resolve these paradoxical observations, we determined cryogenic electron microscopy (cryo-EM) structures of NSP2 and an NSP2–ribonucleoprotein (RNP) complex at global resolutions of 3.9 Å and 3.1 Å, respectively. Selective incorporation of viral genomes into nascent virions is essential for virus replication This process is highly challenging for RNA viruses with multisegmented genomes (including rotaviruses [RV]) since they must coordinate the selection and assembly of multiple distinct RNAs [1, 2]. NSP2 is a multivalent, nonspecific RNA chaperone with high nanomolar affinity for single-stranded (ss) RNA [4, 7, 8] This allows it to both act as a matchmaker of intermolecular duplexes and limit transient, nonspecific RNA–RNA interactions [4, 9]. This viral RNA chaperone plays an absolutely critical role in RV replication [10, 11]
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