Mechanism of Paramyxovirus Replication

描述（由申请人提供）：我们使用腮腺炎病毒（MuV）（一种副粘病毒）进行的研究将揭示病毒RdRp识别核衣壳并接近核衣壳内隔离的病毒基因组RNA的机制。它还将解决“六规则”是如何由核衣壳强加的，以及mRNA编辑如何通过P和N之间的相互作用进行调节。目标1. P功能的分子机制。我们的初步研究表明，MuVP形成一个四聚体，其中一对两个平行的亚基，另一对在相反的方向。这种将N-末端 MuV P四聚体两端的C端区域是P的一个新结构部分。我们的数据还表明，N端和C端区域都参与特异性结合核衣壳，而不像其他NSV的P蛋白只需要C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，P的某些区域可以被截短，并且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来检查。在目标1c中，将进行基于MuV P的N-末端结构域、寡聚化结构域和C-末端结构域的晶体结构的特异性突变分析。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。替代方法包括通过质谱法进行H/D交换以绘制蛋白质相互作用。目标2. N功能的分子机制。我们之前已经制备了一个核衣壳样颗粒（NLP），它包含13个N亚基和一段随机RNA。该NLP对应于MuV的螺旋核衣壳的一圈。MuVP及其核衣壳结合结构域（在N-和C-末端区域）显示结合NLP。在残基379处的C-末端区域的蛋白水解去除不破坏NLP或P结合。在目标2a中，将通过X射线晶体学解析NLP或其截短版本（N379）的三维结构。NLP晶体已经生长。在目标2b中，将确定P与MuV NLP相互作用的位置。我们将解决与NLP或截短的NLP复合的P或P片段的cryoEM结构。在可能的情况下，P片段可以与NLP或截短的NLP共结晶，并且通过X射线晶体学解析相应的结构。通过质谱法的H/D交换将是一种替代方法。在目标2c中，将产生突变以改变N和P之间的相互作用，并将检查它们对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研究。

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.