Architecture et Fonction des Macromolécules Biologiques, Marseille, France.
PLoS Pathog. 2011 May;7(5):e1002030. doi: 10.1371/journal.ppat.1002030. Epub 2011 May 12.
Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs.
裂谷热病毒（RVFV）是一种布尼亚病毒，其基因组由三个单链 RNA 片段组成，通过受感染的蚊子传播，并在非洲引发大规模病毒爆发。RVFV 编码一种核蛋白（N），该蛋白将病毒 RNA 包裹起来。N 蛋白是核糖核蛋白复合物的主要成分，也是病毒聚合酶进行基因组 RNA 复制和转录所必需的。这里我们展示了 RVFV N 蛋白以六聚体形式的 1.6Å 晶体结构。环状六聚体形成了一个功能性的 RNA 结合位点，这一点通过突变实验得到了评估。电子显微镜（EM）表明，与 RNA 结合的 N 蛋白在溶液中也形成环，并且六聚体 N-RNA 复合物的单颗粒 EM 重建与晶体学 N 六聚体一致。晶体中六聚体的环状组织由 RVFV N 的 N 端的环状相互作用稳定，该 N 端形成一个伸展臂，与相邻亚基核心域中的疏水口袋结合。N 端臂的构象与之前 RVFV 的晶体结构中观察到的不同，在该结构中，它与自身核心域中的疏水口袋结合。N 端臂从分子内相互作用模式到分子间相互作用模式的转变可能是一个普遍的原则，该原则是 Bunyaviridae 家族的 N 蛋白进行多聚化和 RNA 包裹的基础。此外，N 端臂的微小结构调整将允许 RVFV N 形成更小或更大的环状寡聚体，甚至可能形成具有超螺旋亚基排列的多聚体。因此，晶体结构中观察到的亚基相互作用模式将允许在体内形成丝状核糖核蛋白capsids。N 蛋白的 RNA 结合裂缝和多聚化位点都是开发抗病毒药物的有希望的靶点。
