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. ? ? ?
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