Interaction of Sindbis Virus with Cellular Processes

描述（由申请方提供）：甲病毒是一组广泛分布的重要人类和动物病原体，其致病能力不同，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。辛德毕斯病毒（SIN）是甲病毒中致病性最低的病毒之一，是迄今为止关于甲病毒复制和传播的基本问题的最有价值的信息来源。 分子和细胞水平上的发病机制。对该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组合，使我们能够区分SIN RNA复制过程中正链和负链RNA合成中的缺陷。我们发现病毒基因组的5'UTR是启动子的基本元件，用于从3'端开始的负链RNA合成。基于这一发现，我们提出了一种新的SIN基因组复制起始模型。我们还产生了一系列在基因组RNA中具有缺陷的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，然后选择在昆虫来源的细胞中恢复生长但保留所有原始突变的假回复突变体。现在，我们区分SIN基因组5'端的两个功能元件：作为核心启动子元件的5'UTR，以及在脊椎动物细胞中作为复制增强子并且对昆虫细胞中的复制至关重要的51nt CSE。在拟议的研究中，我们将进一步解剖负链RNA合成启动子中的RNA元件，并确定辛德毕斯病毒基因组复制启动所需的蛋白质。我们将分析i）病毒RNA的5'和3'末端上的RNA基序，其形成核心启动子，和ii）结合核心启动子和复制增强子以形成复制复合物的宿主细胞和病毒蛋白。我们将探索SIN基因组的片段，在蚊子细胞中复制所需的，并确定病毒蛋白（S）或另一种RNA元件与此片段相互作用。我们将利用这些结果设计委内瑞拉和东部马脑炎病毒的新减毒株。这些病毒将在其基因组中包含不可逆的变化，这将降低致病性，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.