Alphaviruses constitute a group of widely distributed, significant human and animal pathogens. They differ in their ability to cause disease, but are believed to have similar life cycles, replication strategies and interactions with host cells. Sindbis virus is one of the least pathogenic among the alphaviruses. However, to date it is the most valuable source of information about fundamental issues of alphavirus replication and pathogenesis on the molecular and cellular levels. Most of the 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 alphavirus RNAs. We found that 5'UTR of Sindbis virus genome is an essential element of the promoter for negative-strand RNA synthesis that starts in the 3' end of the genome. Based on this finding, we propose a new model of initiation of Sindbis virus genome replication. Having created a collection of Sindbis virus mutants with defective nsP2 protein, we find that this nonstructural protein plays a critical role in virus replication and development of cytopathic effect caused by Sindbis virus replication in tissue culture. Accordingly, the goal of this proposal is to identify RNA and protein components required for initiation of Sindbis virus genome replication. They include both the RNA motifs on the 5' and 3' ends of viral RNA, that form the promoter, and host cell and viral proteins that bind to the promoter and form the replicative complex. We also plan to determine whether the nsP2 protein of Sindbis virus has a direct effect on cell metabolism or the decrease in cytopathic effect observed for viruses with mutated nsP2 is a result of lower levels of RNA replication. Our study will not only elucidate fundamental issues of alphavirus replication and pathogenesis on molecular and cellular levels, but will have also a positive impact on modification of viral vectors to make them more efficient tools for basic research and design of recombinant vaccines against human pathogens.