Structural basis of rotavirus RNA chaperone displacement and RNA annealing

A multidisciplinary team from the University of Cambridge and other leading research institutions in the UK and around the world come together to reveal the auto-regulatory mechanism of the RNA chaperone-assisted RNA remodelling in rotaviruses

Accurate RNA folding is essential for virus replication. Rotaviruses are viruses infecting humans and animals. Rotavirus genome comprises 11 distinct RNAs, and successful replication requires the incorporation of all 11 RNAs into a virion. The RNA chaperone NSP2 binds viral transcripts, regulating their interactions with each other. NSP2 must release RNAs after they base pair prior to their packaging.

Using single-molecule fluorescence tools, the team led by Dr Alex Borodavka dissected the individual steps of the RNA chaperone activity of NSP2.

The team has used two-colour fluorescence cross-correlation spectroscopy (FCCS) to investigate RNA annealing between fluorescently labelled partially complementary viral transcripts, S6 and S11 (schematically shown in green and red - left panel), in the presence of the RNA chaperone NSP2, and its mutants. In the presence of NSP2, S6 and S11 formed inter-molecular contacts (right panel, cross-correlation amplitude in red), whereas in the presence of its C-terminal truncation variant (blue) there was a reduced amplitude of cross-correlation amplitude.

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To investigate the role if the C-terminal region of NSP2 in its RNA chaperone activity, the team has used structural proteomics (protein-RNA cross-linking and hydrogen-deuterium exchange mass-spectrometry) and cryo-EM to reveal that NSP2 regulates RNA unfolding and the release of the RNA using its charged C-terminal region (CTR). Remarkably, the CTR was not directly involved in RNA binding or unwinding, yet it was important for chaperone recycling and RNA release. These findings were further validated by generating several rotavirus mutants with lesions within the C-terminal region of NSP2, allowing the team to propose the model, in which the CTR plays an important auto-regulatory role in fine-tuning both the RNA helix unwinding and RNA release activities of the rotavirus RNA chaperone NSP2.

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These studies were supported by the Wellcome Trust and CeNS (Center for NanoScience, Munich), and they were carried out through a multi-disciplinary collaboration using structural biology, biophysics, mass-spectrometry and molecular virology tools developed at the Universities of Cambridge and Leeds (UK), LMU, Munich (Germany), and the University of Monash (Australia).

Reference: Bravo, J.P.K., Bartnik, K., Venditti, L., Acker, J., Gail, E.H., Colyer, A., Davidovoch, C., Lamb, D.C., Tuma, R., Calabrese, A.N. and Borodavka, A., “Structural basis of rotavirus RNA chaperone displacement and RNA annealing”, PNAS 118, 41 e2100198118 (2021). https://doi.org/10.1073/pnas.2100198118