Mechanism of Paramyxovirus Replication

描述（由申请人提供）：副粘病毒包括许多重要的人类和动物病原体。我们对腮腺炎病毒（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函数的分子机制。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与一对平行的两个亚基形成一个四聚体，另一对在相反的方向。我们的数据还表明，与其他负链RNA病毒（NSV）的P蛋白只需要c端区域不同，N端和c端区域都参与特异性地与核衣壳结合。在目标1a中，我们将确定MuV P的N端结构域和c端结构域的晶体结构。在目标1b中，将研究突变MuV P与核衣壳、单体N蛋白或L蛋白的相互作用，并使用微基因组系统和反向遗传学系统研究它们对病毒转录和复制的影响。在aim 1c中，将根据MuV P的n端结构域、寡聚化结构域和c端结构域的晶体结构进行特异性突变。目标2。N函数的分子机制。我们已经制备了一种核衣壳样颗粒（NLP），它包含13n个亚基和一段随机RNA。MuV P及其核衣壳结合域（在N端和C端区域）被证明与NLP结合。残基379 c端区域的蛋白水解去除不会破坏NLP或P的结合。在目标2a中，NLP或其截断版本（N379）的三维结构将通过x射线晶体学解决。NLP晶体已经生长出来。核衣壳如何组装以及与其他病毒蛋白相互作用可能涉及的特征可以从结构中得出。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。我们将用NLP或截断的NLP来求解P或P片段的低温电镜结构。在可能的情况下，P片段可以与NLP或截断的NLP共晶，并通过x射线晶体学解析各自的结构。在目标2c中，将根据结构预测产生突变，并检查其对NLP组装和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小基因组系统和反向遗传系统中进行研究。

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.