Virologie Moléculaire et Structurale, UPR-CNRS 3296, USC INRA, Centre de Recherche de Gif, CNRS, 91198 Gif sur Yvette, France.
J Mol Biol. 2011 Oct 14;413(1):209-21. doi: 10.1016/j.jmb.2011.08.008. Epub 2011 Aug 10.
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.
轮状病毒是世界范围内引起胃肠炎的主要病原体之一。在感染过程中，会形成病毒工厂（病毒质）。轮状病毒非结构蛋白 NSP5 和 NSP2 是病毒质的主要组成部分；然而，NSP5 的功能和组织仍然难以捉摸。在本报告中，我们对 NSP5 进行了结构表征。多角度激光散射、沉降速度和平衡沉降实验表明，重组全长 NSP5 在溶液中形成十聚体。Far-Western、下拉和多角度激光散射实验表明，NSP5 有两个寡聚化区域。第一个区域，残基 103-146，参与 NSP5 二聚化，而第二个区域，残基 189-198，负责 NSP5 十聚化。全长和截断形式的 NSP5 的圆二色性分析表明，十聚化区域是螺旋的，而二聚化区域涉及β-折叠。从这些圆二色性实验中，我们还表明，NSP5 原聚体包含两个α-螺旋，无序的 N 端一半和主要由β-折叠组成的 C 端一半。对 NSP5 的广泛结构表征使我们提出了其四级组织的模型。最后，在未感染的细胞中共同表达 NSP5 片段和 NSP2 表明，NSP5 十聚化区域是形成类似病毒质结构所必需的。然而，在体外，NSP5 十聚化区域部分抑制 NSP2-NSP5 相互作用。我们的 NSP5 模型表明，空间位阻阻止 NSP2 与所有 NSP5 原聚体结合。因此，一些原聚体可能自由地与其他 NSP5 结合伙伴相互作用，例如病毒 RNA 和病毒聚合酶 VP1，以执行除病毒质组织以外的功能。
