Structural basis of rotavirus RNA chaperone displacement and RNA annealing

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.

Structural rearrangements allow nucleic acid discrimination by type I-D Cascade

“…However, this region of the NTS is not resolved well enough to highlight direct contacts. Contacts between this Cas10d patch and the NTS likely stabilize the nascent R-loop at early stages of TS:NTS duplex melting and thus favor R-loop completion via kinetic partitioning 9 , 38 , 39 . While this patch of positively charged residues is somewhat analogous to the K-vise and K-rim found in type I-E and I-F Cascade complexes 17 , 33 , we observe a different path for the NTS in type I-D Cascade.…”

Liquid–liquid phase separation underpins the formation of replication factories in rotaviruses

“…To move towards a better understanding of phase separation of viroplasm‐forming proteins NSP5 and NSP2, and to directly demonstrate their capacity to drive LLPS, we analysed their propensities to undergo LLPS in vitro . Previously, N‐terminal and C‐terminal tagging of NSP5 had been shown to affect formation of viroplasm‐like inclusions (Fabbretti et al , 1999), while C‐terminal His‐tagging of NSP2 does not affect their assembly (preprint: Bravo et al , 2020). We therefore examined recombinantly expressed untagged NSP5 (Fig EV2) and a C‐terminally His‐tagged NSP2 (cHis‐NSP2, see Materials and Methods).…”

“…Given that viroplasms are viewed as sites of viral replication (Silvestri et al , 2004; Patton et al , 2006) that accumulate rotavirus transcripts where they may be remodelled by the RNA chaperone NSP2 (Borodavka et al , 2017; Bravo et al , 2018; preprint: Bravo et al, 2020), we examined how solubilisation of NSP5/NSP2 condensates would affect their RNA composition in vivo . smFISH analysis of the RV genomic segment 3 (Seg3) and segment 4 (Seg4) transcripts in MA104‐NSP5‐EGFP cells confirmed that viroplasms contained both RNAs at 4 HPI (Fig 6A, left ).…”

“…These events coincide with the viroplasmic accumulation of eleven distinct types of the RV transcripts, RNA polymerase VP1, and the core protein VP2, ultimately resulting in their correct stoichiometric co‐assembly and RNA packaging (Patton & Chen, 1999; Patton & Spencer, 2000; Silvestri et al , 2004; Lu et al , 2008; Trask et al , 2012). Thus, the highly dynamic nature of viroplasms likely reflects their multi‐faceted roles in supporting all stages of RV assembly, from the assortment of eleven distinct RV transcripts facilitated by NSP2 (Borodavka et al , 2017; Bravo et al , 2018; preprint: Bravo et al, 2021), to the final acquisition of additional protein layers to form an infectious triple‐layered particle during later stages of infection. However, the earliest events leading to the formation of NSP5/NSP2‐rich viroplasms in RV‐infected cells have remained elusive due to the lack of understanding of their nature and tools to isolate these highly dynamic cytoplasmic inclusions.…”

Structural basis for broad anti-phage immunity by DISARM

“…In contrast, DNA dissociation from DrmA(∆loop)B bound DNA was not observed. Furthermore, a second super-shift occurred at protein concentrations of 1µM or higher, likely corresponding to multiple copies of the complex binding to the DNA concurrently, as observed for other nucleoprotein complexes 26,27 .…”