ASSEMBLY AND FUNCTION OF PARAMYXOVIRUS NUCLEOCAPSIDS

其目的是为了了解其基本的生化机制。 病毒组装和复制。这项提案将重点放在 仙台病毒核衣壳蛋白，一个有用的实验室模型 医学上重要的副粘病毒家族。建议的实验是 旨在确定每种核衣壳蛋白的功能，以及 探索蛋白质结构与功能的关系。三 具体的问题将会得到解答。[I]病毒基因组RNA是怎样的 病毒核蛋白NP？这一初始组装步骤是关键 病毒形态发生中的事件。[II]病毒蛋白L和P是如何结合的 与被包裹的病毒基因组一起形成功能RNA聚合酶 复杂？将这些额外的蛋白质组装到核糖核蛋白上 核衣壳的核心必须先于病毒RNA的合成。[III] 核衣壳复合体如何发挥合成病毒RNA的功能？ 完整核衣壳的酶活性负责这两个 病毒基因表达和病毒基因组复制。 这项提议将检验每个核衣壳蛋白都是 多功能，每个功能都可以映射到一个离散的蛋白质上 域。每种蛋白质在组装和酶中的单独作用 核衣壳复合体的活性将通过生成 代表各自的VDNA克隆中的靶向缺失或点突变 蛋白。突变的蛋白质将被评估参与的能力。 在蛋白质中：RNA或蛋白质：对核衣壳重要的蛋白质相互作用 组装，以及在病毒RNA合成的酶步骤中。功能性 突变的后果将通过体外重组来评估 组装或转录过程，并通过利用一种系统 外源RNA的反向遗传包装与表达 最近在这个实验室里开发出来的。这一组合 生化和反向遗传技术将允许绘制 个体活动到离散的蛋白质区域，对 每种核衣壳成分的功能表征和一种 全面了解病毒复制所需的交互作用。 所获得的知识将有助于制定有效的战略 副粘病毒病的治疗或预防。

The objective is to understand the fundamental biochemical mechanisms of virus assembly and replication. This proposal will focus on the nucleocapsid proteins of Sendai virus, a useful laboratory model of the medically important paramyxovirus family. The proposed experiments are aimed at determining the functions of each nucleocapsid protein, and exploring the relationship of protein structure to function. Three specific questions will be addressed. [i] How is virus genome RNA encapsidated by viral nucleoprotein NP? This initial assembly step is a key event in virus morphogenesis. [ii] How do viral proteins L and P combine with the encapsidated virus genome to form the functional RNA polymerase complex? Assembly of these additional proteins onto the ribonucleoprotein core of the nucleocapsid necessarily precedes viral RNA synthesis. [iii] How does the nucleocapsid complex function to synthesize viral RNA? Enzymatic activities of the complete nucleocapsid are responsible both for viral gene expression and for viral genome replication. This proposal will test the hypotheses that each nucleocapsid protein is multifunctional and that each function can be mapped to a discrete protein domain. Individual roles of each protein in assembly and enzymatic activity of the nucleocapsid complex will be dissected by generating targeted deletions or point mutations in vDNA clones representing each protein. Mutated proteins will be evaluated for the ability to participate in protein:RNA or protein:protein interactions important for nucleocapsid assembly, and in the enzymatic steps of viral RNA synthesis. Functional consequences of mutation will be evaluated both by in vitro reconstitution of assembly or transcription processes, and by exploiting a system for reverse-genetic encapsidation and expression of foreign RNA which has recently been developed in this laboratory. This combination of biochemical and reverse-genetic techniques will permit the mapping of individual activities to discrete protein regions, contributing both to the functional characterization of each nucleocapsid component and to an overall understanding of the interactions required for virus replication. The knowledge gained will be useful in devising effective strategies for the treatment or prevention of paramyxovirus diseases.