A cell-free system for the synthesis of Sindbis virus subgenomic RNA: importance of the concentration of the initiating NTP.

We describe here an in vitro system for template-dependent initiation and synthesis of a Sindbis virus (SV) subgenomic (SG) RNA transcript. The critical components of this system were (1) a minus-strand promoter-template corresponding to the region of the SV genome from nt 7441 to nt 7772 (-157 to +175 relative to the SG RNA transcription initiation site at nt 7598), and (2) a p15 fraction from cells infected with recombinant vaccinia viruses expressing the SV nonstructural proteins, P123 and nsP4 (the nsP2 coding region in P123 contained a mutation which results in more rapid than normal processing of P123). Our data indicate that the SG RNA transcript is of the expected size, of positive polarity, and is initiated at the expected site. Changing the +1 nt from A to G, U, or C resulted in decreased synthesis of the SG RNA transcript. However, in each case, increasing the concentration of the initiating NTP restored synthesis of the transcript to the wild-type level. This is the first demonstration of an in vitro synthesis of an alphavirus SG RNA transcript which is dependent on the addition of an exogenous promoter-template. As such, it will make possible new approaches for learning how the synthesis of SG RNA is regulated.