Dubrau Danilo, Tortorici M Alejandra, Rey Félix A, Tautz Norbert
Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany.
Institut Pasteur, Unité de Virologie Structurale, Paris, France.
PLoS Pathog. 2017 Feb 2;13(2):e1006134. doi: 10.1371/journal.ppat.1006134. eCollection 2017 Feb.
The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation.
黄病毒科病毒拥有正链RNA基因组，并表达一种单一的多聚蛋白，该多聚蛋白会被加工成功能性蛋白质。最初，非结构（NS）蛋白（它们不是病毒粒子的组成部分）形成能够进行基因组复制的复合物。后来，NS蛋白在病毒粒子形成中也发挥着关键作用。到目前为止，尚不清楚相同的蛋白质如何形成这两个过程所需的不同复合物的分子基础。对于瘟病毒而言，未切割的NS2-3对病毒粒子形态发生至关重要，而NS3是RNA复制所必需的，但在病毒组装中无功能。最近，我们鉴定出两个功能获得性突变，分别位于NS2的C末端区域和NS3的丝氨酸蛋白酶结构域（NS3残基132），这使得NS2和NS3能够在病毒粒子组装中替代未切割的NS2-3。我们在此报告瘟病毒NS3-4A的晶体结构，表明NS3残基132定位到与NS4A的C末端区域（NS4A扭结区域）相互作用的表面斑块，提示这种相互作用在病毒粒子形态发生中起关键作用。我们表明，通过在这个NS3/4A扭结界面进行丙氨酸交换来破坏这种相互作用，会导致NS3/4A复合物在病毒粒子形成中获得功能。相反，RNA复制以及因此的复制酶组装需要NS3与NS4A扭结区域之间稳定的结合。因此，我们提出在瘟病毒感染的细胞中存在两种NS3/4A复合物变体，每种变体代表RNA复制或病毒粒子形态发生所需的基本构建模块。这可以通过对复制缺陷型NS3/4A双突变体进行反式互补研究得到进一步证实，该双突变体在病毒组装中仍具有功能。我们的观察结果说明了存在替代性重叠表面，这些表面在相同蛋白质之间提供不同的相互作用，从而实现从RNA复制到病毒粒子形成的转变。
