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STRUCTURAL STUDIES OF SARS AND MHV REPLICASE PROTEINS

SARS 和 MHV 复制酶蛋白的结构研究

基本信息

批准号：
6953799
负责人：
L WAYNE SCHULTZ
金额：
$ 32万
依托单位：
HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-30 至 2006-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6953799
关键词：
Escherichia coli SARS virus X ray crystallography molecular cloning murine hepatitis virus protein purification protein structure function replicase virus protein

项目摘要

DESCRIPTION (provided by applicant): Severe Acute Respiratory Syndrome (SARS) has recently emerged as a highly infectious and deadly disease and is caused by a virus that appears to have crossed the species barrier from wild animal to human. SARS develops within days after infection by a positive-stranded RNA coronavirus (SARS-CoV), which has proven to be highly virulent and cytotoxic. The engine driving the replication of the SARS-CoV is a large complex of replicase proteins that involves up to 15 individual proteins essential for the viral life cycle. Many of these proteins have no assigned function or sequence homology to other known proteins. Currently, the only experimentally determined structure of a SARS-CoV protein is that of the chymotrypsin-like protease. The overall goal of this proposal is to determine the structures of individual proteins that form the SARS-CoV replicase complex and to use the structures as a foundation to understand their biological functions. In addition, the structures of homologous replicase proteins from the mouse hepatitis virus (MHV), a closely related virus that does not infect humans, will be determined and compared to those of the SARS-CoV. The first specific aim of this proposal is to clone, express and purify replicase proteins of the SARS-CoV and MHV for crystallographic analysis. Currently, we have clones for 15 of 18 target proteins from SARS-CoV and MHV, of which 13 have been expressed solubly and 6 have been affinity purified. The second specific aim of this proposal is to crystallize these replicase proteins and determine their structures using the technique of X-ray diffraction. High-throughput robotic crystallization screens have been performed on 6 target proteins. Crystals of the p12 domain of the SARS-CoV were obtained, diffracted to 6 A resolution, and cell constants determined. The information generated by the comparison of SARS-CoV and MHV will be instrumental in understanding biological differences such as species specificity (human vs. animal) and virulence (cytotoxicity and infectivity) in these viruses and may be applicable to other virus families. In collaboration with Dr. Mark Denison of Vanderbilt University, we will generate structure based hypotheses that will be tested in SARS-CoV and MHV reverse genetics systems developed in his laboratory.

描述（由申请人提供）：严重急性呼吸系统综合症（SARS）是最近出现的一种高度传染性和致命性疾病，它是由一种病毒引起的，这种病毒似乎跨越了物种屏障，从野生动物传染给人类。SARS在被正链RNA冠状病毒（SARS- cov）感染后几天内发展，这种病毒已被证明具有很强的毒性和细胞毒性。驱动sars冠状病毒复制的引擎是一个巨大的复制酶蛋白质复合体，它涉及病毒生命周期所必需的多达15个单独的蛋白质。这些蛋白质中的许多与其他已知蛋白质没有指定的功能或序列同源性。目前，唯一通过实验确定的sars冠状病毒蛋白结构是凝乳胰蛋白酶样蛋白酶的结构。本提案的总体目标是确定形成SARS-CoV复制酶复合体的单个蛋白质的结构，并将这些结构作为了解其生物学功能的基础。此外，将确定小鼠肝炎病毒（MHV）的同源复制酶蛋白的结构，并将其与sars冠状病毒进行比较。MHV是一种密切相关的病毒，不会感染人类。本方案的第一个具体目的是克隆、表达和纯化SARS-CoV和MHV的复制酶蛋白，用于晶体学分析。目前，我们已经克隆了来自SARS-CoV和MHV的18个靶蛋白中的15个，其中13个已被可溶性表达，6个已被亲和纯化。本建议的第二个具体目标是结晶这些复制酶蛋白并使用x射线衍射技术确定它们的结构。高通量机器人结晶筛选已在6个目标蛋白上进行。获得了SARS-CoV的p12结构域晶体，衍射到6 A分辨率，并确定了细胞常数。SARS-CoV和MHV比较产生的信息将有助于了解这些病毒的物种特异性（人与动物）和毒力（细胞毒性和传染性）等生物学差异，并可能适用于其他病毒科。我们将与范德比尔特大学（Vanderbilt University）的马克·丹尼森（Mark Denison）博士合作，生成基于结构的假设，并在他的实验室开发的SARS-CoV和MHV反向遗传系统中进行测试。