Mutagenesis of the conserved 51-nucleotide region of Sindbis virus.

We have constructed 25 site-specific mutations in a domain of 51 nucleotides in Sindbis virus that is highly conserved among all alphaviruses sequenced to date. These 51 nucleotides are capable of forming two hairpin structures and are found from nucleotides 155 to 205 in Sindbis virus within the region encoding nsP1. Of the mutations, 21 were silent and did not lead to a change in the amino acid sequence encoded. These silent mutations changed not only the linear sequence but also the stability of the hairpins in most cases. Two double mutants that were constructed led to the replacement of one base pair by another so that the linear sequence was altered but the nature of the hairpins was not. All of the mutants with silent mutations were viable, but 19 of the 21 mutants were severely impaired for growth in both chicken and mosquito cells. Compared with the parental virus, they grew slowly and produced virus at rates of 10(-1) to 10(-4) times the parental rate. Surprisingly, however, the plaques produced by these mutants were indistinguishable from those produced by the parental virus. Two of the silent mutations, found within the first hairpin structure, produced virus at a faster rate than the parental virus. It is clear that the exact sequence of this region is important for some aspect of virus replication. We suggest that one or more proteins, either virus encoded or cellular, bind to the hairpin structures in a sequence-specific fashion in a step that promotes replication of the viral RNA. Of the mutations that resulted in a change of coding, only one of four was viable, suggesting that the amino acid sequence encoded in this domain is essential for virus replication.