VIRAL PROTEINS IN ROTAVIRUS REPLICATION

轮状病毒，呼肠孤病毒科的成员，是引起严重腹泻的主要原因。 婴儿和幼儿肠胃炎，估计 每年死亡百万人。尽管它们很重要， 这些分段的双链（ds）RNA病毒的复制周期。 关于轮状病毒分子生物学的其他信息 将有助于开发疫苗和确定 抑制病毒在受感染个体中的复制。以前的研究表明 轮状病毒基因组的复制是不对称的， 信使RNA作为负链合成的模板， 产生dsRNA。获得的证据表明基因组复制 只有在包装（分类）所有11种 病毒mRNA转化为复制中间体（RI）。从被感染的细胞， 三种不同类型的RI的特征在于， 蛋白质组成、结构和相关的复制酶活性（mRNA 双链RNA）。RI由结构蛋白和非结构蛋白组成， 两种类型的RI可能具有复制酶活性。总之，这些研究 表明轮状病毒RNA复制和形态发生是同时发生的 在感染的细胞质内含物（病毒颗粒）中发生的过程 细胞该项目的长期目标是表征轮状病毒RNA 复制，包括描述蛋白质和mRNA的组装 并定义与之相关的蛋白质的结构和功能 dsRNA的合成。该项目的具体目标如下： (i)表征非结构蛋白的结构-功能 与RI有关。特别是，特异性，功能结构域 以及RNA结合蛋白N553、N535和NS 34的相关活性 将被审查。(ii)分析单壳的形成 粒子和R1。病毒蛋白质参与了这些 将使用瞬时牛痘病毒表达来研究中间体 系统(iii)为了表征内部的结构和功能， 衣壳蛋白将检查VP 1的RNA结合和聚合酶 将绘制VP 2和VP 6的活性和功能结构域。(iv)到 分析病毒mRNA的保守末端的二级结构， 识别信号。假定的二级结构将由 RNA酶敏感性试验，并将进行功能检测。（v）。发展 轮状病毒感染性RNA系统。 将采用两种方法， 基因操纵轮状病毒的基因组， 牛痘病毒系统和外源mRNP系统。这两个系统将 用于尝试将质粒衍生的mRNA引入复制 轮状病毒的周期

Rotaviruses, members of the family Reoviridae, are a major cause of severe gastroenteritis in infants and young children, causing an estimated one million deaths per year. Despite their importance, little is known about the replication cycle of these segmented, double-stranded (ds)RNA viruses. Additional information about the-molecular biology of the rotaviruses would be useful for developing vaccines and for identifying approaches to inhibit virus replication in infected individuals. Previous studies showed that the rotavirus genome is replicated asymmetrically with viral messenger (m)RNA acting as the template for minus-strand synthesis to produce dsRNA. Evidence was obtained that suggests genome replication initiates only after the packaging (assortment) of all eleven species of viral mRNAs into replication intermediates (RIs). From infected cells, three distinct types of RIs have been characterized with respect to protein composition, structure and associated replicase activity (mRNA dsRNA). The RIs are composed of structural and nonstructural proteins, and two types of RIs probably have replicase activity. Together, such studies indicated that rotavirus RNA replication and morphogenesis are concurrent processes that occur in cytoplasmic inclusions (viroplasms) in infected cells. The long-term goal of the project is to characterize rotavirus RNA replication which includes describing the assembly of proteins and mRNAs into RIs and defining the structure and function of proteins involved with the synthesis of dsRNAs. The Specific Aims of the project are as follows: (i) To characterize the structure-function of nonstructural proteins associated with RIs. In particular, the specificity, functional domains and associated activities of the RNA-binding proteins N553, N535, and NS34 will be examined. (ii) To analyze the formation of single-shelled particles and Rls. Viral proteins involved in the assembly of these intermediates will be studied using a transient vaccinia virus expression system. (iii) To characterize the structure and function of the inner capsid proteins. VP1 will be examined for RNA-binding and polymerase activity and the functional domains of VP2 and VP6 will be mapped. (iv) To analyze the conserved termini of viral mRNAs for secondary structures and recognition signals. Putative secondary structures will be examined by RNase sensitivity assay and will be tested for function. (v). To develop a rotavirus infectious RNA system. Two approaches will be used to genetically manipulate the genome of the rotaviruses, a recombinant vaccinia virus system and an exogenous mRNP system. Both systems will be used in attempts to introduce plasmid-derived mRNAs into the replication cycle of the rotaviruses.