Mechanism of Paramyxovirus Replication

描述(申请人提供)：我们使用腮腺炎病毒(MUV)，一种副粘病毒的研究，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。它还将讨论核衣壳如何强加“六规则”，以及P和N之间的相互作用如何调节mRNA编辑。目的1.P功能的分子机制。我们的初步研究表明，Muv P形成一个四聚体，其中一对有两个平行的亚基，另一对具有相反的取向。该方向将N端子 而Muv P四聚体两端的C末端区域是P的一个新的结构部分。我们的数据还表明，N末端和C末端区域都参与了与核衣壳的特异性结合，而不像其他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-端结构域和C-端结构域的晶体结构。在目标1b中，P的某些区域可能被截断，它们对病毒转录和复制的影响将使用微型基因组系统和反向遗传学系统进行检测。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异性突变分析。对于这些突变体，将检查与N蛋白的相互作用，并将使用微型基因组系统和反向遗传学系统检查它们对病毒转录和复制的影响。另一种方法包括通过质谱学的H/D交换来绘制蛋白质相互作用的图谱。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。这个NLP对应于Muv的螺旋核衣壳的一个转角。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。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。质谱法H/D交换将是一种替代方法。在AIM 2c中，将产生突变来改变N和P之间的相互作用，并将检测它们对NLP组装和蛋白质结合的影响。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): Our studies using mumps virus (MuV), a paramyxovirus, will unveil the mechanism by which the viral RdRp recognizes the nucleocapsid and gains access to the viral genomic RNA sequestered inside the nucleocapsid. It will also address how the "rule of six" is imposed by the nucleocapsid and how mRNA editing is regulated by interactions between P and N. Aim 1. The molecular mechanism for P functions. Our preliminary studies have shown that MuV P forms a tetramer with one pair of two parallel subunits, and another pair in the opposite orientation. This orientation that places the N-terminal and C-terminal regions on both ends of the MuV P tetramer is a novel structure moiety of P. Our data also showed that both N- and C-terminal regions are involved in binding specifically to the nucleocapsid, unlike P proteins of other NSVs 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. Crystal hits have been observed. In aim 1b, certain regions of P may be truncated and their effects on viral transcription and replication will be examined using a mini-genome system and a reverse genetics system. In aim 1c, specific mutational analysis 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. For these mutants, interactions with the N proteins will be examined and their effects on viral transcription and replication will also be examined using a mini-genome system and a reverse genetics system. Alternative approaches include H/D exchange by mass spectrometry to map protein interactions. 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. This NLP corresponds to one turn of the helical nucleocapsid of MuV. 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 be solved by X-ray crystallography. Crystals of NLP have been grown. 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. H/D exchange by mass spectrometry will be an alternative approach. In aim 2c, mutations will be generated to alter interactions between N and P, and their effects on NLP assembly and protein binding will be examined. Effects of mutations on viral transcription and replication will also be examined in a mini- genome system and a reverse genetics system.