THE TRANSCRIPTION OF THE CYTOMEGALOVIRUS GENOME.

Abstract

The replication of cytomegalovirus (CMV), Towne strain, in permissively infected cells is characterized by a long eclipse phase and lengthy growth cycle which may reflect the activity of mechanisms which regulate viral gene expression at the transcriptional level. To correlate the study of transcription with other events occurring during CMV replication, experiments were first conducted to determine the time post-infection for the onset of virus maturation and virus DNA synthesis under defined conditions. The onset of virus maturation was between 2 and 3 days post-infection. The hybridization kinetics of labeled CMV DNA with DNA extracted at different times post-infection indicated that the onset of viral DNA synthesis was between 24 and 36 hours post-infection in human foreskin fibroblast cells permissively infected with CMV. The results of hybridizations of stable viral RNA accumulating at various times post-infection, and at early times in the absence of protein synthesis, with labeled CMV DNA, showed that there is temporal regulation of transcription. The transcription of the genome is restricted in the presence of an inhibitor of protein synthesis to 5-6% of the genome (immediate early RNA). There is a rapid switch of immediate early to the early phase of transcription, which extends to at least 24 hours post-infection, and consists of transcripts homologous to approximately 30% of the viral genome throughout the entire phase. After the onset of viral DNA synthesis, the transcription extends into the late phase during which transcripts homologous to approximately 42% of the genome are synthesized. Early and late RNA was analyzed for the presence of symmetric transcripts. Transcription was found to be asymmetric at early times post-infection, with only 5% symmetric transcription during the late phase. The extent to which immediate early, early, and late stable RNA is transcribed from the repeat regions of the CMV genome was determined by hybridizations of stable RNA to labeled XbaI restriction fragments Q and M, which comprise the long repeat regions of the CMV genome. The hybridization of the probes indicated the presence of RNA transcripts at immediate early times that were homologous to 15% of the repeat sequences. Early RNA contained transcripts homologous to 18.5% of the repeat regions, and late RNA was homologous to 34% of the long repeat sequences.