Transcriptional and translational analysis of a strongly expressed early region of the vaccinia virus genome

Abstract

A transcriptional and translational map was obtained for a 7-kilobase pair EcoRI fragment of vaccinia virus DNA containing 4% of the viral genome. This particular region contains a cluster of early genes which are transcribed in viral cores in vitro and in infected cells before uncoating of the viral DNA. Expression of this region was characterized by using vaccinia virus DNA sequences cloned in phage and plasmid vectors. Polypeptides encoded on the 7-kilobase pair fragment were identified by cell-free translation of viral mRNA selected by hybridization to immobilized DNA fragments. Early RNA programmed the synthesis of six proteins having apparent molecular weights of 63,000, 38,000, 37,000, 25,000, 15,000, and 13,000. The same result was obtained with RNA synthesized in vitro. A new species of 40 kilodaltons was synthesized in response to late RNA. Of the six "early" polypeptides, only the 13-kilodalton component was synthesized from late mRNA. RNA derived from the 7-kilobase pair region was analyzed by a variety of methods including hybridization blot, in vitro recapping, and S1 nuclease techniques. A surprisingly complex pattern of early transcription was found, indicating the existence of families of overlapping RNA species which share common 5'-proximal sequences. In addition, larger RNAs were identified spanning two or more adjacent genes. These RNAs appear to possess a common 5' terminus with transcripts derived from the first gene and are coterminal at the 3' end with RNAs from the downstream gene. Late RNA encoding the 40-kilodalton protein was shown to be heterogeneous in size. A single 5' terminus but no unique 3' terminus was identified for this class of transcripts. RNA species synthesized by cores in vitro were of similar size to authentic transcripts isolated from infected cells at early times.