Sindbis virus is typical of alphaviruses and other RNA viruses in using an RNA-dependent RNA polymerase, called the replicase, for replication. It synthesizes a subgenomic mRNA, again using an RNA-dependent RNA polymerase called the transcriptase. The interactions between the viral replicase and transcriptase with their cognate, cis-acting recognition sequences are central to the virus life cycle, and are potentially vulnerable to inhibition by properly designed drugs. However, the mechanism of neither replication nor transcription is known. Indeed, it is not known which Sindbis proteins constitute the replicase or the transcriptase. Although potential cis-acting recognition sequences have been proposed, based on comparative sequence analysis of alphavirus RNA, there has been little direct evidence that they are biologically significant. The experiments described here are designed to identify the viral proteins that make up the transcriptase, and to identify the cis-acting transcription signal that directs the synthesis of a subgenomic mRNA. The experiments use full length cDNA clones of Sindbis virus and its defective-interfering RNAs, that can be transcribed in vitro to produce infectious transcripts. The clones allow the modification of the viral genomes using standard molecular cloging techniques, the modifications are reflected in the in vitro transcripts, that are then tested for biological activity. The transcription signal is identified by cloning the region spanning the start of the Sindbis subgenomic mRNA into a defective-interfering RNA (that normally does not produce subgenomic RNA). The resulting defective-interfering RNA produces subgenomic RNA in the presence of Sindbis helper virus. Deletions and site-specific mutagenesis are then used to map the minimal extent of the transcription signal. The viral transcriptase is identified by selecting for second-site revertants that can at lest partially overcome the effects of an insertion mutation in the transcription signal. The mutation in the transcription signal results in viruses with defective subgenomic mRNA synthesis, that make very small plaques, and give low virus yields. Revertants are easy to obtain, that make larger plaques, and that give better virus yields. Most revertants appear to have retained the insertion mutation, and have mutations elsewhere in their genomes. Among such second-site mutations are those that produce mutant transcriptases that can recognize the mutated transcription signal with some efficiency. Mapping and sequencing of such mutations will then identify the viral proteins that are involved in subgenomic mRNA synthesis. A biochemical approach is also taken,to detect direct binding of transcription signal to viral proteins. This would be used to identify the recognition site of the transcriptase.
