Structural Studies on Noroviruses

诺如病毒的结构研究

• 批准号: 7774781
• 负责人: Bidadi Venkataram Prasad
• 金额: $ 29.17万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-15 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7774781
• 关键词: Accounting; Acute; Animals; Antiviral Agents; Bacterial Gastroenteritis; Blood Group Antigens; Caliciviridae; Calicivirus; Capsid; Capsid Proteins; Complex; Cysteine; Epidemic; Eukaryotic Initiation Factors; Exhibits; Experimental Designs; Family; Family Picornaviridae; Fingers; Funding; Genome; Genotype; Goals; Grant; Human; Link; Molecular; Molecular Target; N-terminal; Norovirus; Norwalk virus; Pattern; Pattern Recognition; Peptide Hydrolases; Peptide Initiation Factors; Polymerase; Polyproteins; Proteins; Proteolytic Processing; RNA-Directed RNA Polymerase; Ribosomes; Roentgen Rays; Role; Sequence Analysis; Serine Protease; Set protein; Site; Staging; Structural Protein; Structure; Substrate Specificity; System; Therapeutic; Time; Translation Initiation; Viral; Viral Genome; Virus; Virus Replication; Walkers; base; design; drug discovery; helicase; inhibitor/antagonist; insight; novel; nucleoside triphosphatase; pathogen; prevent; programs; protein function; receptor; small molecule; viral RNA

This project focuses on continuing our X-ray crystallographic studies on Norovirus capsids and some ofthe non-structural proteins encoded by the norovirus genome that are critical for norovirus replication. Based on the substantial progress made in the previous funding period, the new specific aims in this proposal are designed to further our understanding of the structural basis of various virus related functions that could be helpful in the design of experimental strategies in Project 2, and identify novel targets for developing effective antiviral drugs for noroviruses in conjunction with proposed studies in Project 1. In Specific Aim 1, our focus will be to further investigate the structural basis of genotype-specific recognition of HBGA by noroviruses. In Specific Aim 2, our goal is to delineate the structural determinants of substrate specificity in the NV protease, making use of a novel system that we discovered during the current grant period. In addition, we will perform structural analysis on small molecule inhibitors identified in Project 1 in complex with the protease. These structures will facilitate the design of inhibitors with increased potency. In Specific Aim 3, we will determine the structure of a norovirus VPg. Several studies on both animal andtlhuman caliciviruses have strongly implicated this protein as a "cap substitute" for translation initiation of the viral RNA and in a primer function during genome replication. We have expressed and purified VPg ,''VPg-Pro, VPg-Pro-Pol and obtained crystals of VPg-Pro demonstrating the feasibility of obtaining the structure of this important protein and setting the stage for structural studies to understand the molecular basis of its interaction with ribosomal initiation factors and viral polymerase, and the mechanism of uridylylation. In Specific aim 4, we will determine the structure ofthe p41 of Norwalk virus. p41 protein is an NTPase/helicase analogous to picornavirus 2C protein. Based on sequence analysis, p41 exhibits motifs such as A, B, B', C and Arg finger found in SF3-2 helicases. Although, the function of this protein is unclear, one hypothesis is that it provides a mechanism to prevent viral ssRNA from annealing during the strand synthesis by the viral polymerase. We have been able to express and purify this protein to homogeneity.

这个项目的重点是继续我们对诺如病毒衣壳和一些诺如病毒衣壳的X射线结晶学研究。 诺沃克病毒基因组编码的对诺沃克病毒复制至关重要的非结构蛋白。基于 在上一个供资期间取得的实质性进展，这项提案的新的具体目标是 旨在加深我们对各种病毒相关功能的结构基础的理解，这些功能可能是 帮助设计项目2中的实验策略，并确定有效开发的新目标 诺如病毒的抗病毒药物与项目1中拟议的研究相结合。在具体目标1中，我们的重点是 将进一步研究诺如病毒对HBGA的基因型特异性识别的结构基础。在……里面 具体目标2，我们的目标是描述NV蛋白酶底物专一性的结构决定因素， 使用了我们在当前赠款期间发现的一种新系统。此外，我们还将表演 项目1中鉴定的小分子抑制剂与酶的复合体的结构分析。这些 结构将有助于设计具有更高效力的抑制剂。在具体目标3中，我们将确定 诺如病毒VPG的结构。对动物和人类杯状病毒的几项研究都有很强的说服力 暗示该蛋白是病毒RNA翻译启动的“帽子替代品”，并具有引子功能。 在基因组复制过程中。我们表达和纯化了VPG、VPG-Pro、VPG-Pro-Pol，并获得了 VPG-Pro的晶体证明了获得这一重要蛋白质结构和 为结构研究了解其与核糖体相互作用的分子基础奠定了基础 启动因子和病毒聚合酶，以及尿苷基化的机制。在具体目标4中，我们将 确定诺瓦克病毒p41的结构。P41蛋白是一种NTPase/解旋酶，与 小核糖核酸病毒2C蛋白。根据序列分析，p41具有A、B、B‘、C和Arg Finger等基序 存在于SF3-2解旋酶中。尽管这种蛋白质的功能尚不清楚，但有一种假设是，它提供了 一种防止病毒单链RNA在病毒聚合酶合成链过程中退火的机制。我们 已经能够表达和纯化这种蛋白质，使其达到同质。