MOLECULAR AND ANTIGENIC ANALYSIS OF HUMAN CALICIVIRUS

诺瓦克病毒属于杯状病毒科，是引起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，其基于这些因子的临床意义增加，因为使用了检测这些病毒的新方法。 最近的研究发现，这些病毒导致美国几乎所有（超过95%）的非细菌性胃肠炎爆发。 这种病毒是人类唯一的病原体，具有由180个拷贝的单一蛋白质（ORF 2）形成的衣壳结构。第二种蛋白质（ORF 3）目前在少量最近已被确定在病毒粒子。 对动物杯状病毒的研究已经确定了持续性感染，并且一些动物病毒最近被证明与人类杯状病毒有遗传相关性。 存在多种遗传类型的杯状病毒，其中一些代表不同的血清型，表明可能难以开发疫苗。 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物中培养，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，衣壳蛋白自发地组装成病毒样颗粒（VLP）。 通过X射线晶体学测定的重组诺瓦克病毒VLP的高分辨率3.4埃单位结构已经表明，这些颗粒表现出T=3的二十面体对称性，具有独特的结构，包括围绕32个大空洞的90个拱形壳粒。原子分辨率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性质的洞察，并允许制定关于可能调节这些途径的机制的可检验假设。 这项资助申请概述了继续使用结构，分子和生物化学方法来了解这些独特的单链RNA人类病原体的组装，表达和抗原特性的实验。 拟议研究的具体目标是继续（1）剖析调节衣壳组装和拆卸的分子相互作用，（2）了解ORF 3在基因组组装中的作用，以及（3）绘制病毒衣壳上的抗原和生物结构域。由于杯状病毒结构的独特特征，预计所获得的结果将为开发新型抗病毒药物提供基础。 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，其可允许感染性颗粒在细胞培养物中复制。

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.