TEMPLATE-DEPENDENT INITIATION OF SINDBIS VIRUS-RNA REPLICATION IN-VITRO

Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of m inus-strand RNA synthesis, Based on this observation, we have succeede d in reconstituting an in vitro system for template-dependent initiati on of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and a ssayed by the addition of exogenous template RNAs, Extracts from cells expressing P123(C>S) a protease-defective P123 polyprotein, and nsP3 synthesized a genome-length minus-sense RNA product. Replicase activit y was dependent upon addition of exogenous RNA and was specific for al phavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23(C>S), and nsP4. However, extracts from cell s expressing nsP3 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity, In addition, a P123 polyprotein contain ing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These re sults provide further evidence that processing of the polyprotein inac tivates the minus-strand initiation complex. Finally, RNA synthesis wa s detected when soluble nsP3 was added to a membrane fraction containi ng P123(C>S), thus providing a functional assay for purification of th e nsP4 RNA polymerase.