Temperature-sensitive RNA-mutants of Sindbis virus and Semliki Forest virus, two members of the alphavirus group of Togaviruses, will be analyzed for their ability to continue minus-strand RNA synthesis after shift up to non-permissive temperature. Recent studies have identified one Sindbis virus mutant that was unable to continue minus-strand synthesis at non-permissive temperature, and at least one Sindbis virus mutant that may be responsible for the normal temporal cessation of minus-strand synthesis seen in a alphavirus infected cells. We propose to confirm these findings by investigation of these mutants. We will confirm the role of the """"""""regulatory"""""""" mutant by analysis of infected cells at permissive and non-permissive temperature for their presence of newly synthesized minus-strand RNA and for the requirement for continued protein synthesis for minus-strand cessation. We will determine if 26S RNA synthesis is correlated with the ability to synthesize minus-strand RNA. We will determine if more than one complementation group plays a role in this cessation. We propose to identify and characterize the Sindbis virus nonstructural proteins and to attempt to assign one of these proteins as the required viral component in the minus-strand polymerase that is temperature-sensitive in Tsll, the mutant unable to continue minus-strand synthesis at the non-permissive temperature. We will initiate studies to determine how the cessation of the function of the minus-strand polymerase may occur and to understand the interaction of the nonstructural proteins with each other.
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