VIRAL PROTEINS IN ROTAVIRUS REPLICATION

轮状病毒，呼肠孤病毒科的成员，是主要原因 急性胃肠炎的症状 尽管致病性 这些病毒的重要性， 轮状病毒还不是很清楚。 这些病毒的基因组 由11段双链RNA（dsRNA）组成。 的 基因组的分段性质允许不同的菌株 轮状病毒在自然界中进行重组， 变体。 基因组的每个片段的复制 以不对称的方式与病毒信使RNA（mRNA;加上- 作为负链RNA的模板， dsRNA。 为了了解轮状病毒RNA复制的机制， 我们开发了一种无细胞系统， 猴轮状病毒萨尔dsRNA、mRNA和蛋白。 随后，这 系统允许我们识别、分离并提供基本描述- 轮状病毒亚病毒颗粒（SVP）的合成 病毒基因组 这些数据表明复制酶颗粒 包含可能仅部分被VP 6包围的核心，包含 两个非结构蛋白（NS 34和NS 35），并绘制模板 用于在dsRNA合成期间复制到核心中。 重点 该建议的目的是更全面地描述结构 复制酶颗粒，与复制酶相关的蛋白质的功能 这些颗粒，以及病毒mRNA的识别信号， 允许它们被复制酶颗粒复制。 具体地说， 蛋白质-蛋白质交联，免疫电镜，和 有限的蛋白水解消化将用于研究整体 复制酶颗粒的蛋白质结构。 位点特异 切割、核酸酶消化和其它方法将用于 用复制酶检查mRNA模板的方向 粒子以及模板在复制过程中如何移动。 到 建立病毒蛋白在复制、病毒 将鉴定结合RNA和核苷酸的蛋白质。 的 VP 6、NS 34、NS 35和其他病毒蛋白在轮状病毒RNA中的作用 复制，组装单壳颗粒， 复制酶颗粒，并在mRNA翻译将研究使用 无细胞系统 核心粒子会被破坏， 从子单元重新组装，以便更好地了解 轮状病毒SVP的形态发生。 负链RNA的起始 在无细胞系统中外源病毒mRNA的合成将是 优化和定量。 修饰的病毒mRNA将被 通过含有cDNA的转录载体体外合成， 添加到系统中以识别病毒mRNA上的识别信号 启动负链合成所需的。 这些 结果应提供有关 轮状病毒的复制可能有助于发展 控制由这些病毒引起的疾病的策略。

Rotaviruses, members of the family Reoviridae, are the major cause of acute gastroenteritis in young children. Despite the pathogenic importance of these viruses, the basic molecular biology of the rotaviruses is not well understood. The genome of these viruses consists of eleven segments of double-stranded RNA (dsRNA). The segmented nature of the genome allows different strains of the rotaviruses to undergo reassortment in nature producing new unique variants. The replication of each segment of the genome proceeds in an asymmetrical manner with viral messenger RNA (mRNA; plus- strand RNA) serving as the template for minus-strand RNA to produce dsRNA. To understand the mechanism of rotavirus RNA replication, we developed a cell-free system that supports the synthesis of simian rotavirus SAll dsRNA, mRNA and protein. Subsequently, this system allowed us to identify, isolate and provide a basic descrip- tion of rotavirus subviral particles (SVPs) that synthesize the viral genome. These data indicate that replicase particles contain cores that may be only partially surrounded by VP6, contain two nonstructural proteins (NS34 and NS35), and draw the template for replication into the core during dsRNA synthesis. The focus of this proposal is to characterize more completely the structure of the replicase particle, the function of proteins associated with these particles, and the recognition signals of viral mRNAs that allow their replication by replicase particles. Specifically, protein-proteins crosslinking, immunoelectron microscopy, and limited proteolytic digestion will be used to study the overall protein structure of the replicase particle. Site-specific cleavage, nuclease digestion and other methods will be used to examine the orientation of the mRNA template with replicase particles and how the template moves during replication. To establish possible function of viral proteins in replication, viral proteins that bind RNA and nucleotides will be identified. The role of VP6, NS34, NS35, and other viral proteins in rotavirus RNA replication, the assembly of single-shelled particles from replicase particles, and in mRNA translation will be studies using the cell-free system. Core particles will be disrupted and reassembled from subunits so as to understand better the morphogenesis of rotavirus SVPs. Initiation of minus-strand RNA synthesis on exogenous viral mRNAs in the cell-free system will be optimized and quantitated. Modified viral mRNAs will be synthesized in vitro by transcription vectors containing cDNAs and added to the system to identify recognition signals on viral mRNAs required for the initiation of minus-strand synthesis. These results should provide valuable information on events in the replication of the rotaviruses that may be useful for developing strategies of controlling diseases caused by these viruses.