Many RNA viruses transcribe one or more subgenomic mRNAs in addition to the replication of the genomic RNA. One mechanism for the transcription of a subgenomic mRNA is the de novo initiation of RNA synthesis at an internal promoter site on the template RNA. This necessarily depends on the specific recognition of the cis-acting promoter by the trans-acting viral transcription factors, perhaps in association with host factors. We have been studying the mechanism of Sindbis virus transcription. Sindbis virus, the type species of the alphaviruses, transcribes a single subgenomic mRNA during infection. We showed that the Sindbis virus promoter consists of a functionally-active minimal promoter that is 24 nucleotides long, and an enhancing region that stimulates transcription by 6 fold. The nucleotide positions in the minimal promoter that are important for promoter recognition in vivo were surveyed by mutagenesis studies, and a fairly detailed picture of the minimal promoter is now available. The same methods will be used to determine the precise extent and the role of the enhancing region. Given reasonably detailed knowledge of the cis-acting sequences, we need to identify the trans-acting transcription factors, especially those that bind directly to the promoter. Both in vivo and in vitro approaches are used. An in vivo genetic selection for identifying the viral transcription factors was designed: The virus promoter was mutated, and revertants were selected for the ability to recognize the mutant promoter. These revertants carry mutations in their transcription factors, and the causal mutations in two such revertants are being mapped. The isolation of additional revertants is essential, to maximize the chances that all viral transcription factors are identified, and to identify those that bind directly to the promoter. Parallel, in vitro experiments are also designed to identify the transcription factors and to characterize their promoter-binding sites, by characterizing the complexes formed between promoter oligoribonucleotides and viral and host proteins. A powerful method is to use the promoter oligoribonucleotides for affinity purification and identification of the transcription factors. The method might also be useful for purifying the transcription factors in native form for reconstituting Sindbis virus transcription in vitro. These studies will provide a detailed understanding of the promoter, the transcription factors, and the interactions between them. They also point to a novel approach for designing anti-viral drugs. Conserved cis-acting sequences or structures are required for replication and transcription by RNA viruses. It is conceivable that drugs designed to mimic these conserved features will inhibit entire groups of viruses. Our studies of Sindbis virus transcription will provide a database and the resources to test this novel approach.
