Structural basis of rotavirus RNA chaperone displacement and RNA annealing

Abstract

Rotavirus genomes are distributed between 11 distinct RNA segments, all of which are essential for virus replication. Stoichiometric genome segment selection and assembly is achieved through a series of sequence-specific, intersegment RNA-RNA interactions that are facilitated by the rotavirus RNA chaperone protein NSP2. The C-terminal region (CTR) of NSP2 has been proposed to play a role in rotavirus replication, although its mechanistic contribution to the RNA chaperone activity of NSP2 remained unknown. Here, we use single-molecule fluorescence assays to directly demonstrate that the CTR is required for promoting RNA-RNA interactions and that it limits the RNA unwinding activity of NSP2. Unexpectedly, hydrogen-deuterium exchange-mass spectrometry and UV-crosslinking data indicate that the CTR does not interact with RNA. However, removal of the CTR reduced the RNA release activity of NSP2, suggesting that the CTR is important for chaperone recycling. To further interrogate the role of the CTR, we determined cryo-EM structures of NSP2 and its ribonucleoprotein complexes. These reveal that although the CTR is ampholytic in nature, it harbours a highly conserved acidic patch that is poised towards bound RNA. Using a reverse genetics approach, we demonstrate that rotavirus mutants harbouring triple alanine mutations within the acidic patch failed to replicate, while mutations that preserve the charge of the CTR successfully restored viral replication. Together, our data suggest that the CTR reduces the accumulation of kinetically trapped NSP2-RNA complexes by accelerating RNA dissociation via charge repulsion, thus promoting efficient intermolecular RNA-RNA interactions during segment assembly.

Reovirus Nonstructural Protein σNS Recruits Viral RNA to Replication Organelles

“…Rotavirus NSP2, which has been hypothesized to be a functional homolog of σNS ( 26 ), also binds RNA using electrostatic interactions and, analogous to avian reovirus σNS, chaperones rotavirus mRNAs ( 25 ). NSP2 regulates RNA-binding using residues that electrostatically repulse RNA ( 27 ). While we have identified residues required for σNS to bind RNA, questions about the regulation and specificity of RNA binding remain.…”

Biomolecular condensate phase diagrams with a combinatorial microdroplet platform

“…NSP2 from Rotavirus A (strain RF) was produced as a C-terminal His 6 -tagged protein (NSP2-His 6 ) in BL21(DE3) Escherichia coli transformed with a pET-28b-NSP2 construct, as previously described 56,57 . Expression was carried out at 24 °C in Luria Bertani (LB) media supplemented with 1% (v/v) glucose, and cultures were induced with 0.5 mM IPTG once they reached optical density (OD 600 ) of 0.6, and harvested 14 h post-induction.…”

Engineering a Vaccine Platform using Rotavirus A to Express SARS-CoV-2 Spike Epitopes

“…For viruses of the Reoviridae family, genome packaging is a tightly orchestrated and controlled process, so increasing the RV genome size may affect packaging efficiency [30,[77][78][79]. To examine whether our mutations may have affected genome packaging, RNA was extracted from equal volume of purified viruses and analysed by urea-PAGE and silver staining.…”