尼帕病毒(NiV)是一种高致病性副粘病毒,经常引起人类致命的脑炎。NiV基因组被核衣壳(N)蛋白包封。RNA合成由病毒RNA依赖性RNA聚合酶(RdRP)介导,由与同型四聚体磷蛋白(P)复合的聚合酶(L)蛋白组成。聚合酶沿其模板的前进需要在高度调节的过程中迭代溶解和P和N原聚体之间的瞬时相互作用的重新形成,但人们对此知之甚少。这项研究将功能和生化NiV聚合酶测定法应用于该问题。我们在C端P-X结构域(P-XD)上定位了三个不同的蛋白质界面,形成三角棱镜并接合L,C端N尾,和球状N核,分别。使用NiVL和N尾结合缺陷型突变体进行的反式互补分析显示,P四聚体中只有一个XD与L结合,而三个必须可用于N-结合以获得有效的聚合酶活性。N-tail复合物与P-XD的溶解是通过N核与该XD的α-1/2面之间的瞬时相互作用而协调的,但不是P四聚体的未被占据的XD,创建一个计时器,用于协调聚合酶前进。
目的:单病毒包括主要的人类病原体,例如埃博拉病毒,狂犬病病毒,呼吸道合胞病毒,麻疹病毒,尼帕病毒(NiV)对于复制和转录,它们的聚合酶复合物必须协商一个蛋白质衣壳化的RNA基因组,随着聚合酶沿模板前进,这需要蛋白质-蛋白质界面的高度协调的连续形成和分辨率。病毒P蛋白在这个过程中起着核心作用,但是对确保聚合酶迁移的分子机制知之甚少。研究NiV聚合酶复合物,我们应用功能和生化测定来绘制NiVPXD中三个不同的界面,并确定XD与核衣壳核之间的瞬时相互作用是协调聚合酶进展的工具。这些结果定义了调节副粘病毒聚合酶动力学的保守分子原理,并为广谱聚合酶抑制剂的结构指导开发提供了有希望的药物靶标。
Nipah virus (NiV) is a highly pathogenic paramyxovirus causing frequently lethal encephalitis in humans. The NiV genome is encapsidated by the nucleocapsid (N) protein. RNA synthesis is mediated by the viral RNA-dependent RNA
polymerase (RdRP), consisting of the
polymerase (L) protein complexed with the homo-tetrameric phosphoprotein (P). The advance of the
polymerase along its template requires iterative dissolution and reformation of transient interactions between P and N protomers in a highly regulated process that remains poorly understood. This study applied functional and biochemical NiV
polymerase assays to the problem. We mapped three distinct protein interfaces on the C-terminal P-X domain (P-XD), which form a triangular prism and engage L, the C-terminal N tail, and the globular N core, respectively. Transcomplementation assays using NiV L and N-tail binding-deficient mutants revealed that only one XD of a P tetramer binds to L, whereas three must be available for N-binding for efficient polymerase activity. The dissolution of the N-tail complex with P-XD was coordinated by a transient interaction between N-core and the α-1/2 face of this XD but not unoccupied XDs of the P tetramer, creating a timer for coordinated polymerase advance.
OBJECTIVE: Mononegaviruses comprise major human pathogens such as the Ebola virus, rabies virus, respiratory syncytial virus, measles virus, and Nipah virus (NiV). For replication and transcription, their polymerase complexes must negotiate a protein-encapsidated RNA genome, which requires the highly coordinated continuous formation and resolution of protein-protein interfaces as the polymerase advances along the template. The viral P protein assumes a central role in this process, but the molecular mechanism of ensuring polymerase mobility is poorly understood. Studying NiV
polymerase complexes, we applied functional and biochemical assays to map three distinct interfaces in the NiV P XD and identified transient interactions between XD and the nucleocapsid core as instrumental in coordinating polymerase advance. These results define a conserved molecular principle regulating paramyxovirus
polymerase dynamics and illuminate a promising druggable target for the structure-guided development of broad-spectrum polymerase inhibitors.