MOLECULAR AND ANTIGENIC ANALYSIS OF HUMAN CALICIVIRUS

诺瓦克病毒属于杯状病毒科，是人类流行性非细菌性肠胃炎的主要病因。诺瓦克病毒代表了一种新出现的病毒，其基础是随着检测这些病毒的新方法的使用，这些药物的临床意义日益增加。最近的研究发现，在美国，几乎所有（超过95%）的非细菌性胃肠炎爆发都是由这些病毒引起的。这种病毒完全是一种人类病原体，其衣壳结构由180个拷贝的单一蛋白质（orf2）组成。最近在病毒粒子中发现了少量存在的第二种蛋白质（orf3）。对动物杯状病毒的研究已经确定了持续性感染，最近一些动物病毒已被证明与人类杯状病毒有遗传关系。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，这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中培养，但其基因组的克隆和表达导致发现衣壳蛋白在使用杆状病毒系统表达时自发组装成病毒样颗粒（vlp）。重组诺瓦克病毒VLPs的高分辨率3.4埃单位结构，通过x射线晶体学确定，表明这些颗粒具有T=3二十面体对称，具有独特的结构，包括90个拱形帽体周围的32个大空洞。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本质的见解，并允许制定关于可能调节这些途径的机制的可测试假设。该资助申请概述了继续使用结构，分子和生化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orf3在基因组封装中的作用，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必要的基础。全长和亚基因组rna的表达系统也将在哺乳动物细胞系统中建立，可能允许在细胞培养中复制感染性颗粒。

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.