Use of coupled transcription and translation to study mRNA production by vaccinia cores

Abstract

VACCINIA is a double-stranded DNA virus which replicates in the cytoplasm of animal cells. Soon after infection, the outer layers of the virion are removed to yield core particles¹ which make mRNA that is transcribed, capped² and polyadenylated³ by enzymes present in the cores. Later, the cores break down, DNA synthesis begins, and ‘late’ genes are expressed⁴. Cores can be made in vitro by treating virions with non-ionic detergent and mercapto-ethanol. When incubated with nucleoside triphosphates, these particles produce mRNA⁵ which can be translated in cell-free systems to yield authentic vaccinia early proteins^3,6,7. It has recently been suggested that this mRNA may be derived from large primary transcripts⁸ which are processed in a manner similar to that proposed for cellular mRNA^9,10. I have obtained evidence that early vaccinia mRNA is made by monocistronic transcription in vitro, by comparing the relative sensitivity of the synthesis of individual messages to ultraviolet irradiation. Production of mRNA was assayed indirectly using a coupled cell-free transcription–translation system in which protein synthesis is dependent on added vaccinia cores.