MOLECULAR AND ANTIGENIC ANALYSIS OF HUMAN CALICIVIRUS

诺沃克病毒属于杯状病毒科，是人类流行性非细菌性胃肠炎的主要病原。诺沃克病毒代表了一种新兴的病毒，其基础是这些试剂的临床意义越来越大，因为检测这些病毒的新方法正在被利用。最近的研究发现，这些病毒导致了美国几乎所有(超过95%)的非细菌性胃肠炎的爆发。这种病毒只是一种人类病原体，具有由180个拷贝的单一蛋白(ORF2)形成的衣壳结构。最近在病毒粒子中发现了第二种少量存在的蛋白质(ORF3)。对动物杯状病毒的研究已经确定了持续性感染，最近发现一些动物病毒与人类杯状病毒有遗传关系。杯状病毒存在多种遗传类型，其中一些代表不同的血清型，这表明可能很难开发疫苗。相反，需要设计其他抗病毒策略。诺瓦克病毒尚未在细胞培养中培养，但其基因组的克隆和表达导致发现，当使用杆状病毒系统表达时，衣壳蛋白自发组装成病毒样颗粒(VLP)。由X射线结晶学测定的重组诺瓦克病毒VLP的高分辨率3.4埃单位结构表明，这些颗粒具有T=3二十面体对称性，具有独特的结构，包括围绕32个大空洞的90个拱形胶囊。原子分辨结构与生化分析一起提供了对控制组装、拆卸和受体识别的化学相互作用的本质的洞察，并允许就可能调节这些途径的机制提出可检验的假说。这项拨款申请概述了继续使用结构、分子和生化方法了解这些独特的单链RNA人类病原体的组装、表达和抗原性的实验。这些研究的具体目的是继续(1)剖析调控衣壳组装和拆解的分子相互作用，(2)了解ORF 3在基因组封装中的作用，以及(3)绘制病毒衣壳上的抗原域和生物域。由于杯状病毒结构的独特特点，预计所获得的结果将为开发新型抗病毒药物提供所需的基础。还将在哺乳动物细胞系统中建立全长和亚基因组RNA的表达系统，以允许在细胞培养中复制感染颗粒。

Norwalk virus, belonging to the family of Caliciviridae, is the major cause of epidemic non-bacterial gastroenteritis in humans. Norwalk virus represents an emerging virus based on an increased clinical significance of these agents being recognized as new methods to detect these viruses are utilized. Recent studies have found these viruses cause almost all (greater than 95 percent) outbreaks of nonbacterial gastroenteritis in the United States. This virus, which is exclusively a human pathogen, has a capsid structure formed by 180 copies of a single protein (ORF 2). A second protein (ORF 3) present in small amounts has recently been identified in virions. Studies of animal caliciviruses have identified persistent infections and some animal viruses have recently been shown to be genetically related to human caliciviruses. Multiple genetic types of caliciviruses exist and some of these represent different serotypes indicating it may be difficult to develop vaccines. Instead, other antiviral strategies need to be devised. Norwalk virus has not yet been cultivated in cell culture, but the cloning and expression of its genome resulted in the discovery that the capsid protein spontaneously assembles into virus-like particles (VLPs) when expressed using the baculovirus system. A high resolution 3.4 Angstrom units structure of the recombinant Norwalk virus VLPs, determined by X-ray crystallography, has shown that these particles exhibit T=3 icosahedral symmetry with a distinctive architecture that includes 90 arch-like capsomeres surrounding 32 large hollows. The atomic resolution structure together with biochemical analyses have provided insight into the nature of the chemical interactions that govern the assembly, disassembly, and receptor recognition, and allowed the formulation of testable hypotheses about mechanisms that may regulate these pathways. This grant application outlines experiments to continue to use structural, molecular, and biochemical approaches to understand the assembly, expression and antigenic properties of these unique singlestranded RNA human pathogens. The specific aims of the proposed studies are to continue (1) to dissect the molecular interactions that regulate capsid assembly and disassembly, (2) to understand the role of ORF 3 in genome encapsidation, and (3) to map antigenic and biologic domains on the virus capsid. Because of the unique features of the calicivirus structure, it is anticipated that the results obtained will provide the foundation needed to develop new types of antivirals. Expression systems of full-length and subgenomic RNAs also will be established in mammalian cell systems that may permit replication of infectious particles in cell culture.