THE VACCINIA VIRUS A18R DNA HELICASE IS A POSTREPLICATIVE NEGATIVE TRANSCRIPTION ELONGATION-FACTOR

Loss of vaccinia virus A18R gene function results in an aberrant trans cription profile termed promiscuous transcription, defined as transcri ption within regions of the genome which are normally transcriptionall y silent late during infection. Promiscuous transcription results in a n increase in the intracellular concentration of double-stranded RNA, which in turn results in activation of the cellular 2-5A pathway and s ubsequent RNase L-catalyzed degradation of viral and cellular RNAs. On e of three hypotheses could account for promiscuous transcription: (i) reactivation of early promoters late during infection, (ii) random tr anscription initiation, (iii) readthrough transcription from upstream promoters. Transcriptional analysis of several viral genes, presented here, argues strongly against the first two hypotheses. We have tested the readthrough hypothesis by conducting a detailed transcriptional a nalysis of a region of the vaccinia virus genome which contains three early genes (M1L, M2L, and K1L) positioned directly downstream of the intermediate gene, K2L. The results show that mutation of the A18R gen e results in increased readthrough transcription of the M1L gene origi nating from the K2L intermediate promoter. A18R mutant infection of RN ase L knockout mouse fibroblast (KO3) cells does not result in 2-5A pa thway activation, yet the virus mutant is defective in late viral gene expression and remains temperature sensitive. These results demonstra te that the A18R gene product is a negative transcription elongation f actor for postreplicative viral genes.