VIRAL PROTEINS IN ROTAVIRUS REPLICATION

轮状病毒是呼肠孤病毒科的成员，是主要原因 幼儿的急性胃肠炎。尽管有致病性 这些病毒的重要性，基础分子生物学 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。这个 基因组的节段性允许不同的菌株 轮状病毒在自然界中进行重新分类，产生新的独特 变种。基因组的每一段的复制都在进行 以不对称的方式与病毒信使RNA(mRNA；加上- 链RNA)用作生产负链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，支持合成 猴轮状病毒SALL dsRNA、mRNA和蛋白质。随后，这 系统允许我们识别、隔离并提供基本描述- 轮状病毒亚病毒颗粒(SVP)的作用 病毒基因组。这些数据表明复制酶颗粒 包含可能仅部分被VP6包围核心，包含 两个非结构蛋白(NS34和NS35)，并绘制模板 用于在dsRNA合成过程中复制到核心。焦点 这一建议的目的是更全面地描述结构 复制酶颗粒中，与之相关的蛋白质的功能 这些颗粒以及病毒mRNAs的识别信号 允许它们通过复制酶颗粒进行复制。具体来说， 蛋白质-蛋白质交联、免疫电子显微镜和 有限的蛋白质分解将被用来研究整体 复制酶颗粒的蛋白质结构。特定于站点 将采用卵裂、核酸酶消化等方法 用复制酶检测mRNA模板的方向 颗粒以及模板在复制过程中如何移动。至 建立病毒蛋白在复制中的可能功能 结合RNA和核苷酸的蛋白质将被识别出来。这个 VP6、NS34、NS35等病毒蛋白在轮状病毒RNA中的作用 复制，单壳粒子的组装 复制酶颗粒，并在信使核糖核酸的翻译将研究使用 无细胞系统。核心粒子将被打乱， 从子单元重组，以便更好地理解 轮状病毒SVPs的形态发生。负链RNA的起始 在无细胞系统中合成外源病毒mRNAs将是 优化和量化。修改后的病毒mRNAs将是 由含有cDNA和cDNA的转录载体体外合成 添加到系统以识别病毒mRNAs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对开发 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.