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 minus-strand RNA synthesis, Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed 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 activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23(C>S), and nsP4. However, extracts from cells expressing nsP3 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity, In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP3 was added to a membrane fraction containing P123(C>S), thus providing a functional assay for purification of the nsP4 RNA polymerase.