Abstract
In this study, we use yeast S. cerevisiae as a model to study the replication of sindbis virus (SV), which is a positive sense, single-stranded animal RNA virus belonging to alphavirus. To serve as a viral expression vector, foreign genes are often constructed under control by SV subgenomic RNA promoter, so that the expression of the given foreign genes must be the result of SV replication process. Characteristically, it is sufficient for SV to complete viral RNA replication only through its replicase complex derived from virus non-structural genes. In order to study SV replication in yeast system, we constructed a series of SV-replicase replicon in a yeast expression plasmid (pYES-X1) and used the hygromycin resistant gene as a selectable marker that was under the subgenomic RNA promoter. The generation of hygromycin-resistant colonies and the detection of virus negative strand RNA by RT-PCR were the two major assessments to evaluate SV replication in yeast system. Our results show that S1/Rep21/hyg could result in hygromycin resistant yeast colonies. To examine whether this was the outcome of SV RNA replication, we deleted the 3’UTR sequence which is required for replication, and we found S1/Rep21/hyg/△3’UTR was unexpectedly able to generate resistant colonies and negative strand of virus RNA. In contrast, S1/Rep21/hyg/△P, whose replicase was partially deleted, could not generate resistant colonies but was still able to synthesize the negative strand of virus RNA. These results suggest that there may be a reverse DNA promoter behind hygromycin resistant gene on vector that initiates the synthesis of negative strand of virus RNA. To overcome this interference, we linearized the plasmid of S1/Rep21/hyg and found that the linear form of S1/Rep21/hyg was indeed able to form resistant colonies, clearly suggesting that S1/Rep21/hyg is capable of replication in yeast. In conclusion, this study establishes a SV replication system in yeast, and hopefully this system can be extended to study the interactions between host factors and SV. So we can further realize the molecular mechanism of SV replication in the cellular levels.