ROTAVIRUS AND NORWALK VIRUS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们研究项目的主要目的是了解具有医学重要性的病毒的结构-功能关系，如轮状病毒(NIH AI36040)、杯状病毒(NIH P01 AI57788-01，与M.K.Estes博士合作)、正源病毒(NIH RO1 AI32539，与T.Dermody博士合作)和流感病毒(与安德鲁·赖斯博士合作，赠款提交正在进行中)。多年来，我们一直在使用NCRR资助的NCMI设施，我们将继续使用这一出色的设施。世界一流的高分辨率成像和图像处理设施非常适合我们的项目。 轮状病毒：轮状病毒是幼儿和动物中严重的、危及生命的胃肠炎的主要原因。轮状病毒是大的(1000°)、复杂的二十面体组件。这种病毒一直是广泛的生化、遗传和结构研究的对象，因为它与医学相关，具有有趣的结构复杂性，以及独特的复制和形态发生策略。我们将使用NCMI CRYO-EM设备继续深入了解轮状病毒进入细胞、内源性转录、病毒质形成、基因组复制和亚纳米分辨率包装的结构基础。我们的重点将特别放在人类菌株上。虽然其中一些研究涉及轮状病毒颗粒(直径1000？)，但其他研究与病毒质形成、涉及NSP2、NSP5、VP1、VP2和NSP6等轮状病毒非结构蛋白的基因组复制/包装有关。在受感染的细胞中，这些蛋白质在病毒质形成、基因组复制/封装过程中协同工作。为了获得对这些轮状病毒蛋白如何发挥作用的结构洞察，高分辨率的低温电子显微镜分析可能是唯一的选择。 杯状病毒：杯状病毒分为四个属，是重要的人类和兽医病原体，有可能导致人畜共患病2。在这些单链RNA病毒中，与衣壳相关的功能，如组装、抗原性和受体相互作用，都编码在形成二十面体衣壳的单一蛋白质中。我们有兴趣获得关于杯状病毒科成员的结构信息。我们将继续使用NCMI设备来研究杯状病毒-受体以及杯状病毒-抗体的相互作用。此外，我们计划使用NCMI提供的这些资源来表征具有解旋酶/NTPase活性的诺如病毒p41蛋白。诺如病毒是人类流行性腹泻的病原体。 正形呼肠孤病毒：建议的研究目标是以哺乳动物呼肠孤病毒为实验系统，确定无包膜病毒内化、分解和膜穿透的机制。我们将继续使用NCMI设施进一步研究呼肠孤病毒拆解和膜穿透的结构基础，方法是确定呼肠孤病毒连续拆解中间产物的结构，以及在持续呼肠孤病毒感染(PI病毒)期间选择的呼肠孤病毒突变体的结构，这些突变体显示在内体酸化(氯化铵)4和蛋白降解抑制剂存在的情况下体外拆解和生长的动力学增强。 流感病毒NS1蛋白：最近出现的高致病性禽流感病毒(H5N1)，它们的流行性和泛发性，以及它们与致命的人类感染的关联，已经引起了重大的全球卫生问题。一些研究强调了非结构蛋白NS1在提高这些毒株的致病性和毒力方面的重要性。NS1是宿主体内抗病毒I型干扰素反应的拮抗剂。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Major thrust of our research projects is to understand structure-function relationships in medically important viruses such as rotavirus (NIH AI36040), caliciviruses (NIH P01 AI57788-01 in collaboration with Dr. M.K. Estes), orthoreoviruses (NIH RO1 AI32539 in collaboration with Dr. T. Dermody), and influenza viruses (in collaboration with Dr. Andrew Rice, grant submission in progress). We have been using NCRR funded NCMI facility over years, and we will continue to use this excellent facility. The world-class high resolution imaging and image processing facilities are excellently suited for our projects. Rotavirus: Rotavirus is the major cause of severe, life-threatening gastroenteritis in young children and animals. Rotaviruses are large (1000 ¿), complex, icosahedral assemblies. This virus has been the subject of extensive biochemical, genetic and structural studies because of its medical relevance, intriguing structural complexity, and unique strategies of replication and morphogenesis. We will continue further understanding of the structural basis of rotavirus cell entry, endogenous transcription, viroplasm formation, genome replication and packaging at sub-nanometer resolution using the NCMI cryo-EM facility. Our focus will be particularly on human strains. While some of these studies involve rotavirus particles (1000 ¿ diameter), others that are related to viroplasm formation, genome replication/packaging involving rotavirus non-structural proteins such as NSP2, NSP5, VP1, VP2 and NSP6. In infected cells, these proteins work in concert during viroplasm formation, genome replication/encapsidation. To obtain structural insights into how these rotavirus proteins function, high-resolution cryo-EM analysis is perhaps the only option. Caliciviruses: Caliciviruses, grouped into four genera, are important human and veterinary pathogens with a potential for zoonosis2. In these ssRNA viruses, capsid related functions such as assembly, antigenicity, and receptor interactions are encoded in a single protein that forms an icosahedral capsid. We are interested in obtaining structural information about members of the Caliciviridae. We will continue to use NCMI facility to investigate calicivirus-receptor, and calicivirus-antibody interactions. In addition, we plan to use these resources available at NCMI for characterizing norovirus p41 protein that exhibits helicase/NTPase activities. Noroviruses are causative agents of epidemic diarrhea in humans 3. Orthoreoviruses: The goal of the proposed research is to determine mechanisms of non-enveloped virus internalization, disassembly, and membrane penetration using mammalian reovirus as an experimental system. We will continue to use NCMI facilities to further investigate structural basis for reovirus disassembly and membrane penetration by determining structures of sequential reovirus disassembly intermediates, and that of reovirus mutants selected during persistent reovirus infection (PI viruses), which demonstrate enhanced in vitro kinetics of disassembly and grow in the presence of inhibitors of endosomal acidification (ammonium chloride)4 and proteolysis. Influenza virus NS1 protein: Recent emergence of highly pathogenic avian (H5N1) influenza viruses, their epizootic and panzootic nature, and their association with lethal human infections have raised significant global health concerns. Several studies have underscored the importance of non-structural protein NS1 in the increased pathogenicity and virulence of these strains. NS1, is an antagonist of antiviral type-I interferon (IFN) response in the host.