Mechanism of Paramyxovirus Replication

副粘病毒复制机制

• 批准号: 9114385
• 负责人: Biao He
• 金额: $ 57.18万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9114385
• 关键词: Address; Animals; Binding; Binding Proteins; C-terminal; Complex; Cryoelectron Microscopy; Data; Dimensions; Docking; Excision; Foundations; Funding; Genetic Transcription; Genome; Glycine decarboxylase; Health; Hendra Virus; Human; Location; Maps; Mass Spectrum Analysis; Measles virus; Messenger RNA; Molecular; Mumps virus; Mutation; N-terminal; Nipah Virus; Nucleocapsid; Nucleocapsid Proteins; Nucleotides; Paramyxovirus; Phosphoproteins; Proteins; RNA; RNA Viruses; RNA chemical synthesis; RNA-Directed RNA Polymerase; Reading; Regulation; Respiratory syncytial virus; Rest; Rhabdoviridae; Role; Structure; System; Tertiary Protein Structure; Testing; Vesicular stomatitis Indiana virus; Viral; Viral Physiology; Viral Proteins; Virus; Virus Replication; Work; X-Ray Crystallography; base; design; genome sequencing; genomic RNA; mutant; novel; parainfluenza virus; particle; pathogen; prototype; research study; reverse genetics; three dimensional structure; viral RNA

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.

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 识别核衣壳并获取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施加“六规则”以及如何通过 P 和 N 之间的相互作用来调节 mRNA 编辑。 目标 1. P 功能的分子机制。我们的初步研究表明，MuV P 与一对两个平行的亚基和另一对方向相反的亚基形成四聚体。这个方向将 N 末端 MuV P四聚体两端的P蛋白和C端区域是P的一个新颖的结构部分。我们的数据还表明，N端和C端区域都参与与核衣壳的特异性结合，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 端结构域和 C 端结构域的晶体结构。已观察到晶体命中。在目标 1b 中，P 的某些区域可能会被截短，并且将使用迷你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。在目标1c中，将基于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 交换来绘制蛋白质相互作用图。目标 2. N 功能的分子机制。我们之前制备了一个包含 13 个 N 亚基和一段随机 RNA 的核衣壳样颗粒 (NLP)。该 NLP 对应于 MuV 螺旋核衣壳的一圈。 MuV P 及其核衣壳结合域（位于 N 端和 C 端区域）被证明可以结合 NLP。蛋白水解去除残基 379 处的 C 端区域不会破坏 NLP 或 P 结合。在目标 2a 中，NLP 或其截断版本 (N379) 的三维结构将通过 X 射线晶体学来解析。 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。我们将通过 NLP 或截短的 NLP 来解析 P 或 P 片段复合物的冷冻电镜结构。如果可能，P片段可以与NLP或截短的NLP共结晶，并且将通过X射线晶体学解析各自的结构。通过质谱进行 H/D 交换将是另一种方法。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迷你基因组系统和反向遗传学系统中进行检查。