Identification of a temperature-sensitive mutant of vaccinia virus defective in late but not intermediate gene expression

The vaccinia virus conditional-lethal temperature-sensitive ( ts) mutant tsC63 is defective in the synthesis of some but not all postreplicative proteins. Synthesis of the temporal “intermediate” class of proteins was unaffected, whereas “late” proteins were absent at the nonpermissive temperature. At the DNA level, DNA synthesis was unaffected, but telomere resolution was severely inhibited. In order to identify the defective gene responsible for this ts defect, we performed marker rescue and DNA sequencing experiments. We localized the lesion to open reading frame (ORF) A1L, which has recently been identified as one of the three intermediate genes required for the transcription of late genes (J. G. Keck, C.1. Baldick, Jr., and B. Moss, (1990). Cell 61, 801–809). S1 nuclease analysis of viral mRNA demonstrated that the ts defect in late protein synthesis was caused by a defect in the transcription of stable mRNA and therefore provides evidence for a role of the A1L gene product during in vivo transcriptional activation of late genes or stabilization of late RNA. Furthermore, the kinetics of early protein synthesis in tsC63-infected cells suggests that, in addition to its role in trans-activation of late genes, intermediate gene expression mediates suppression of early protein synthesis. The telomere resolution defect of this mutant is presumably a secondary consequence of the defect in late gene expression.