Recombination between Sindbis virus RNAs

The genome (49S RNA) of Sindbis virus is a positive-strand RNA of 11.7 kb that consists of two domains. The 5' two-thirds of the RNA codes for the proteins required for replication and transcription of the RNA. The 3' one-third codes for the structural proteins. The latter are translated from a 26S subgenomic RNA identical in sequence to the 3' one-third of the genome. The 26S RNA is transcribed by initiation from an internal promoter that spans the junction between the nonstructural and structural genes. We have used Sindbis virus RNAs transcribed from cloned cDNAs to demonstrate recombination between Sindbis virus RNAs in cultured cells. Several different combinations of deleted or mutationally altered RNAs gave rise to infectious recombinants. In 7 of 10 different crosses, the infectious recombinant RNAs were larger than wild-type 49S RNA. We sequenced the recombinant RNAs in the region spanning the junction between the nonstructural and structural protein genes from five different crosses. In three of the crosses, this is the only region within which recombination could have taken place to produce an infectious 49S RNA. Recombination also occurred in this region in the other two crosses. The recombinant RNAs were distinct from wild-type RNA and from each other. All contained sequence insertions derived from the parental RNAs. One contained a deletion and a rearrangement, and one also contained a stretch of 11 nucleotides not found in the Sindbis virus genome. When each of the parental RNAs contained a functional subgenomic RNA promoter, both promoters were present and functional in the recombinant RNA. Those recombinants with large sequence insertions showed evidence of evolution toward the wild-type single-junction RNA.