STRUCTURAL STUDIES OF SARS AND MHV REPLICASE PROTEINS

描述(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的疾病，由一种似乎已经跨越野生动物到人类的物种屏障的病毒引起。SARS在感染阳性链RNA冠状病毒(SARS-CoV)后的几天内发展起来，这种病毒已被证明具有高度的毒力和细胞毒性。推动SARS-CoV复制的引擎是复制酶蛋白质的大型复合体，涉及多达15种对病毒生命周期至关重要的单独蛋白质。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，实验确定的SARS-CoV蛋白的唯一结构是类凝乳蛋白酶的结构。这项提议的总体目标是确定构成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，还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-CoV的结构进行比较。MHV是一种密切相关的病毒，不会感染人类。这项建议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，用于结晶学分析。目前，我们已经克隆了SARS-CoV和MHV的18个靶蛋白中的15个，其中13个已经进行了可溶性表达，6个已经进行了亲和纯化。这项提议的第二个具体目标是结晶这些复制酶蛋白质，并使用X射线衍射技术确定它们的结构。已经对6个目标蛋白质进行了高通量机器人结晶筛选。获得了SARS冠状病毒p12结构域的晶体，衍射到6A分辨率，并测定了晶胞常数。SARS-CoV和MHV的比较所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家族。与范德比尔特大学的Mark Denison博士合作，我们将产生基于结构的假说，这些假说将在他的实验室开发的SARS-CoV和MHV反向遗传学系统中进行测试。

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.