title: Structural organisation of the rotavirus nonstructural protein NSP5.
abstract: Rotavirus is one of the leading agents of gastroenteritis worldwide. During infection, viral factories (viroplasms) are formed. The rotavirus nonstructural proteins NSP5 and NSP2 are the major building blocks of viroplasms; however, NSP5 function and organisation remain elusive. In this report, we present a structural characterisation of NSP5. Multi-angle laser light scattering, sedimentation velocity and equilibrium sedimentation experiments demonstrate that recombinant full-length NSP5 forms a decamer in solution. Far-Western, pull-down and multi-angle laser light scattering experiments show that NSP5 has two oligomerisation regions. The first region, residues 103-146, is involved in NSP5 dimerisation, whereas the second region, residues 189-198, is responsible for NSP5 decamerisation. Circular dichroism analyses of full-length and truncated forms of NSP5 reveal that the decamerisation region is helical, whereas the dimerisation region involves β-sheets. From these circular dichroism experiments, we also show that the NSP5 protomers contain two α-helices, a disordered N-terminal half and a C-terminal half that is primarily composed of β-sheet folds. This extensive structural characterisation of NSP5 led us to propose a model for its quaternary organisation. Finally, co-expression of NSP5 fragments and NSP2 in uninfected cells shows that the NSP5 decamerisation region is required for viroplasm-like structure formation. However, in vitro, the NSP5 decamerisation region partially inhibits the NSP2-NSP5 interaction. Our NSP5 model suggests that steric hindrance prevents NSP2 from binding to all NSP5 protomers. Some protomers may thus be free to interact with other NSP5 binding partners, such as viral RNAs and the viral polymerase VP1, to perform functions other than viroplasm organisation.
authors.affiliation: Virologie Moléculaire et Structurale, UPR-CNRS 3296, USC INRA, Centre de Recherche de Gif, CNRS, 91198 Gif sur Yvette, France. davy.martin@vms.cnrs-gif.fr
authors.forename: Davy
authors.initials: D
authors.lastname: Martin
authors.forename: Malika
authors.initials: M
authors.lastname: Ouldali
authors.forename: Julie
authors.initials: J
authors.lastname: Ménétrey
authors.forename: Didier
authors.initials: D
authors.lastname: Poncet
mesh_terms: D026481:Blotting, Far-Western; D002942:Circular Dichroism; D008958:Models, Molecular; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D014461:Ultracentrifugation; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C434828:NSP5 protein, rotavirus group A; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins