Interaction of Sindbis Virus with Cellular Processes

描述(申请人提供)：甲型病毒构成了一组广泛分布的重要的人类和动物病原体，它们的致病能力不同，但被认为具有相似的生命周期、复制策略和与宿主细胞的相互作用。辛德比斯病毒(SIN)是甲型病毒中致病性最低的病毒之一，是迄今为止关于甲型病毒复制和复制的基本问题的最有价值的信息来源 在分子和细胞水平上的发病机制。对这种病毒的大多数研究结果也适用于该属的其他成员。在我们最近的研究中，我们成功地设计了一种方法的组合，使我们能够区分SIN RNA复制过程中正链和负链RNA合成的缺陷。我们发现，病毒基因组的5‘非编码区是负链RNA合成启动子的重要元件，负链RNA合成始于3’端。基于这一发现，我们提出了一种启动SIN基因组复制的新模型。我们还产生了一组基因组RNA中有缺陷的5‘顺式作用元件的突变体，这些突变体不能在蚊子细胞中生长，然后选择了在昆虫来源的细胞中恢复生长但保留了所有原始突变的假逆变体。现在我们区分了SIN基因组5‘端的两个功能元件：5’UTR是核心启动子的元件，而51nt CSE在脊椎动物细胞中作为复制增强子发挥作用，对昆虫细胞的复制至关重要。在拟议的研究中，我们将进一步剖析负链RNA合成启动子中的RNA元件，并确定启动Sindbis病毒基因组复制所需的蛋白质。我们将分析i)病毒RNA 5‘端和3’端的RNA基序，它们构成核心启动子，以及ii)宿主细胞和病毒蛋白与核心启动子和复制增强子结合形成复制复合体。我们将探索在蚊子细胞中复制所需的SIN基因组片段，并鉴定病毒蛋白(S)或其他与该片段相互作用的核糖核酸元件。我们将利用这些结果来设计委内瑞拉和东部马脑炎病毒的新减毒株。这些病毒的基因组中将包含不可逆转的变化，这些变化将降低致病性，但保留野生型祖先的抗原结构。

DESCRIPTION (provided by applicant): Alphaviruses constitute a group of widely distributed, significant human and animal pathogens that differ in their ability to cause disease, but are believed to have similar life cycles, replication strategies and interactions with host cells. Sindbis virus (SIN), one of the least pathogenic among the alphaviruses, is to date the most valuable source of information about fundamental issues of alphavirus replication and pathogenesis on the molecular and cellular levels. Most results from studies of this virus are applicable to other members of the genus. In our recent research, we succeeded in designing a combination of methods that allow us to distinguish between the defects in plus- and minus-strand RNA synthesis during replication of SIN RNA. We found that the 5'UTR of the viral genome is an essential element of the promoter for negative-strand RNA synthesis that starts in the 3' end. Based on this finding we suggested a new model of initiation of SIN genome replication. We also generated a collection of mutants with a defective 5' cis-acting element in the genome RNA, which were unable to grow in mosquito cells, and then selected the pseudorevertants that resumed growth in the cells of insect origin, but retained all of the original mutations. Now we distinguish two functional elements in the 5' end of the SIN genome: the 5'UTR that is an element of core promoter, and the 51nt CSE that functions as a replicational enhancer in vertebrate cells and is critical for replication in insect cells. In the proposed study, we will dissect further the RNA elements in the promoter for negative-strand RNA synthesis and identify proteins required for initiation of Sindbis virus genome replication. We will analyze i) the RNA motifs on the 5' and 3' ends of the viral RNA, which form the core promoter, and ii) host cell and viral proteins that bind to the core promoter and the replicational enhancer to form the replicative complex. We will explore the segment of SIN genome, required for replication in mosquito cells, and identify viral protein(s) or another RNA element interacting with this fragment. We will use the results to design new attenuated strains of Venezuelan and eastern equine encephalitis viruses. These viruses will contain irreversible changes in their genomes that will reduce pathogenicity, but retain the antigenic structure of the wild-type ancestors.