Sequence requirements for Sindbis virus subgenomic mRNA promoter function in cultured cells

The Sindbis virus minimal subgenomic mRNA promoter (spanning positions -19 to +5 relative to the subgenomic mRNA start site) is approximately three- t o sixfold less active than the fully active -98 to +14 promoter region. We identified two elements flanking the -19 to +5 region which increase its tr anscription to levels comparable to the -98 to +14 region. These elements s pan positions -40 to -20 and +6 to +14 and act synergistically to enhance t ranscription. Nine different virus libraries were constructed containing bl ocks of five randomized nucleotides at various positions in the -40 to +14 region. On passaging these libraries in mosquito cells, a small subset of t he viruses came to dominate the population. Sequence analysis at the popula tion level and for individual clones revealed that in general, wild-type ba ses were preferred for positions -15 to +5 of the minimal promoter. Base mu tagenesis experiments indicated that the selection of wild-type bases in th is region was primarily due to requirements for subgenomic mRNA transcripti on. Outside of the minimal promoter, the -35 to -29 region contained four p ositions which also preferred wildtype bases. However, the remaining positi ons generally preferred non-wild-type bases. On passaging of the virus libr aries on hamster cells, the -15 to +5 region again preferred the wild-type base but most of the remaining positions exhibited almost no base preferenc e. The promoter thus consists of an essential central region from -15 to +5 and discrete flanking sites that render it fully active, depending on the host environment.