Mechanism of Paramyxovirus Replication

副粘病毒复制机制

• 批准号: 8709986
• 负责人: Biao He
• 金额: $ 55.99万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 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 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8709986
• 关键词: Address; Animals; Binding; C-terminal; Complex; Cryoelectron Microscopy; Data; Dimensions; Docking; Enzymes; Excision; Genetic Transcription; Genome; Genomics; Glycine decarboxylase; Hendra Virus; Human; Infection; Lead; Location; Maps; Measles virus; Methyltransferase; 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; Respiratory syncytial virus; Rest; Role; Structure; System; Tertiary Protein Structure; Viral; Viral Physiology; Viral Proteins; Virus; X-Ray Crystallography; base; design; genome sequencing; insight; mutant; novel; parainfluenza virus; particle; pathogen; positional cloning; public health relevance; research study; three dimensional structure; viral RNA

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.

性状（由申请方提供）：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。目标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，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。我们的数据还表明，N-和C-末端区域都参与特异性结合核衣壳，不像其他负链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，将检查突变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。在目标lc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。目标二。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。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，NLP或其截短版本（N379）的三维结构将通过X射线晶体学解决。NLP晶体已经生长。核衣壳如何组装以及与其他病毒蛋白质相互作用的特征可能来自结构。病毒RNA是如何被糖苷化的也将被揭示。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，并通过X射线晶体学解析相应的结构。在目标2c中，将基于结构预测生成突变，并将检查它们对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微型基因组系统和反向遗传学系统中进行研究。