The goal of this proposal is to elucidate the mechanisms of Semliki Forest Virus (SFV) and Sindbis (SV) RNA replication and to determine the role of the viral nonstructural (ns) polypeptides in this process. The SFV replication complex containing template RNA and viral polymerase is capable of synthesizing in vitro 42 and 26S ssRNAs. We have recently shown that a putative SFV-encoded guanine-7-methyltransferase activity (GMT) is able to methylate the cap structure of these RNAs and is uniquely capable of methylating free GTP. We are therefore ready to begin elucidating the identity, functions and interactions among the various components required for SFV replication. We will identify, purify and characterize the protein associated with GMT activity. Identification will be carried out a) by ultraviolet light (UV) mediated cross-linking and labeling of GMT with substrates, methyl donors or analogues and by the use of photoaffinity probes, b) by screening of temperature-sensitive (ts) SFV and SV mutants as well as newly derived mutants resistant to methylation-specific inhibitors, and c) by using antisera prepared against synthetic peptides corresponding to the SFV ns proteins. We will also purify and biochemically characterize the membrane-associated GMT activity from SFV-infected cells, using techniques such as hydrophobic chromotography. We will directly test the hypothesis that the ns 86 protein is involved in regulating 26S RNA synthesis either through direct RNA binding or by binding to and modifying a viral polymerase function. This study will involve the use of a ts mutant defective in 26S RNA synthesis to study the UV light mediated crosslinkage of protein to RNA within replication complexes or the binding of proteins to each other by the use of bifunctional crosslinking reagents. We will also purify the ns 86 protein and determine its ability to bind RNAs. The identification and viral or cellular origin of the guanylyltransferase enzyme responsible for capping SFV RNA will also be determined by analysis of the formation of enzyme-guanylate intermediates. In addition to identifying the viral functions responsible for togavirus RNA replication, the studies outlined in this proposal will generate new techniques applicable to other viral systems and will provide the basis for our long range goal of the reconstitution of an active template-dependent replication system.
