Mechanism of Paramyxovirus Replication

描述(申请人提供)：副粘病毒包括许多重要的人类和动物病原体。我们使用腮腺炎病毒(MUV)的研究将揭示病毒RdRp识别核衣壳并获得隔离在核衣壳内的病毒基因组RNA的机制。目的1.P功能的分子机制。P蛋白是病毒RNA合成所必需的，是一种多结构域蛋白。我们的初步研究表明，Muv P形成一个四聚体，具有一对平行的两个亚基，另一个亚基方向相反。我们的数据还表明，与其他负链RNA病毒(NSV)的P蛋白不同，N-末端和C-末端都参与了与核衣壳的特异性结合，而其他负链RNA病毒(NSV)的P蛋白只需要C-末端区域。在目标1a中，我们将确定Muv P的N-末端结构域和C-末端结构域的晶体结构。在目标1b中，我们将检测突变的Muv P与核衣壳、单体N蛋白或L蛋白的相互作用，并将利用微型基因组系统和反向遗传学系统来检测它们对病毒转录和复制的影响。在目标1c中，将根据Muv P的N-末端结构域、齐聚结构域和C-末端结构域的晶体结构进行特定的突变。目的2.N功能的分子机制。我们已经制备了一种核衣壳样粒子(NLP)，它包含13个N亚基和一段随机RNA。MUV P及其核衣壳结合区(位于N-末端和C-末端)与NLP结合。蛋白水解性去除残基379处的C-末端区域不会破坏NLP或P的结合。在目标2a中，将用X射线结晶学解决NLP或其截断版本(N379)的三维结构。NLP晶体已经生长出来。核衣壳是如何组装的，以及在与其他病毒蛋白的相互作用中可能涉及哪些特征，这可能是从结构中得出的。病毒RNA是如何被封装的也将被揭示。在目标2b中，将确定P与Muv NLP相互作用的位置。我们将解决P或P片段在与NLP或截断NLP的络合物中的低温EM结构。在可能的情况下，P片段可以与NLP或截短的NLP共结晶，各自的结构将通过X射线结晶学进行解析。在AIM 2c中，将根据结构预测产生突变，并将检测它们对NLP组装以及与其他病毒蛋白相互作用的影响。突变对病毒转录和复制的影响也将在微型基因组系统和反向遗传学系统中进行检测。

DESCRIPTION (provided by applicant): Paramyxoviruses include many important human and animal pathogens. Our studies using mumps virus (MuV) will unveil the mechanism by which the viral RdRp recognizes the nucleocapsid and gains access to the viral genomic RNA sequestered inside the nucleocapsid. Aim 1. The molecular mechanism for P functions. The P protein is essential for viral RNA synthesis and is a multi-domain protein. Our preliminary studies have shown that MuV P forms a tetramer with a pair of two parallel subunits, and another pair in the opposite orientation. Our data also showed that both N- and C-terminal regions are involved in binding specifically to the nucleocapsid, unlike P proteins of other negative strand RNA viruses (NSV) that requires only the C-terminal region. In aim 1a, we will determine the crystal structure of the N-terminal domains and the C-terminal domains of MuV P. In aim 1b, interactions of the mutant MuV P with the nucleocapsid, monomeric N protein, or the L protein, will be examined, and their effects on viral transcription and replication will be examined using a minigenome system and a reverse genetics system. In aim 1c, specific mutations based on the crystal structure of the N-terminal domain, the oligomerization domain and the C-terminal domain of MuV P will be carried out. Aim 2. The molecular mechanism for N functions. We have previously prepared a nucleocapsid-like particle (NLP) that contains 13 N subunits and a piece of random RNA. MuV P and its nucleocapsid binding domains (both at N- and C- terminal regions) were shown to bind NLP. Proteolytic removal of the C-terminal region at residue 379 did not disrupt NLP or P binding. In aim 2a, the three dimensional structure of the NLP or its truncated version (N379) will solved by X-ray crystallography. Crystals of NLP have been grown. How the nucleocapsid is assembled and what features may be involved in interactions with other viral proteins may be derived from the structure. How the viral RNA is encapsidated will also be revealed. In aim 2b, the location of P interactions with MuV NLP will be determined. We will solve the cryoEM structure of P or P fragments in complex with NLP or truncated NLP. When possible, P fragments may be cocrystallized with NLP or truncated NLP and the respective structure will be solved by X-ray crystallography. In aim 2c, mutations will be generated based on the structure predictions, and their effects on NLP assembly and interactions with other viral proteins will be examined. Effects of mutations on viral transcriptio and replication will also be examined in a minigenome system and a reverse genetics system.