EVOLUTION OF THE SINDBIS-VIRUS SUBGENOMIC MESSENGER-RNA PROMOTER IN CULTURED-CELLS

Transcription of the subgenomic mRNA of alphaviruses initiates at an i nternal site, called the promoter, which is highly conserved. To deter mine the functional significance of this conservation, we used an appr oach that randomizes positions -13 to -9 of the promoter to generate a library containing all possible sequences within this region, includi ng the wild-type sequence. Viruses in the mixed population with more-e fficient promoters were selected for during passaging in mammalian (BH K-21) cells, Results from early passage populations indicate that a la rge number of different promoters are functionally active. Analysis of eight individual viruses found that although each contained a promote r with different degrees of sequence identity to the wild-type sequenc e, all eight viruses produced progeny. This suggests that the mechanis m for transcription allows for a diversity of sequences to serve as pr omoters, Further passaging of the viral library led to a population co nsensus sequence that increasingly resembled the wild-type sequence, d espite the fact that these promoters are not constrained by the need t o encode the carboxyl terminus of the nsP4 protein, Thus, conservation of the region of the promoter from -13 to -9 is in large part due to selection for promoter function, and the wild-type sequence and sequen ces closely similar to it seem to be optimal for promoter function in BHK-21 cells.