ABSTRACT
Transcription of the subgenomic mRNA of alphaviruses initiates at an internal site, called the promoter, which is highly conserved. To determine the functional significance of this conservation, we used an approach that randomizes positions -13 to -9 of the promoter to generate a library containing all possible sequences within this region, including the wild-type sequence. Viruses in the mixed population with more-efficient promoters were selected for during passaging in mammalian (BHK-21) cells. Results from early passage populations indicate that a large number of different promoters are functionally active. Analysis of eight individual viruses found that although each contained a promoter with different degrees of sequence identity to the wild-type sequence, all eight viruses produced progeny. This suggests that the mechanism for transcription allows for a diversity of sequences to serve as promoters. Further passaging of the viral library led to a population consensus sequence that increasingly resembled the wild-type sequence, despite the fact that these promoters are not constrained by the need to 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 sequences closely similar to it seem to be optimal for promoter function in BHK-21 cells.
