Abstract
This report concerns the stable viral RNA sequences that accumulate in HEp-2 cells infected with herpes simplex virus type 1. By hybridizing labeled total DNA and restriction endonuclease DNA fragments with excess unlabeled total nuclear and cytoplasmic RNA, we determined the genetic complexity of the RNA and we mapped the regions on the physical map of herpes simplex virus type 1 DNA that are homologous to the RNA. Our results show the following. (i) The viral RNAs accumulating in the nucleus and cytoplasm of cells infected and maintained in the presence of inhibitory concentrations of either cycloheximide or emetine were homologous to 33 and 12% of viral DNA, respectively. All of the fragments tested contained sequences homologous to nuclear RNA. However, only the fragments mapping between 0.00 and 0.18, and 0.53 and 1.00 map units contained sequences homologous to cytoplasmic RNA. (ii) The viral RNAs that accumulate in the nucleus and cytoplasm of cells infected and maintained in the presence of inhibitory concentrations of phoaphonoacetic acid were homologous to 39 and 26% of viral DNA, respectively. In this instance all of the fragments except those mapping between 0.42 and 0.53 map units contained sequences homologous to cytoplasmic RNA. (iii) The viral RNAs that accumulate in the nucleus and cytoplasm 8 h after infection were homologous to greater than 50 and 41%, respectively. All of the fragments tested contained sequences homologous to cytoplasmic RNA. (iv) The viral RNAs that accumulate in the nucleus and cytoplasm of cells infected and maintained in the presence of canavanine are homologous to 33 and 19% of viral DNA, respectively. All of the fragments tested contained sequences homologous to both nuclear and cytoplasmic RNAs. Our results indicate the following. First, there are at least three phases of transcription of viral DNA. Phase 1 does not require the synthesis of host cell or viral proteins. Phase 2 requires the synthesis of viral proteins made before the initiation of viral DNA synthesis. Phase 3 appears to be related to the initiation of viral DNA synthesis. Second, both the extent of transcription and the accumulation of viral RNA in the cytoplasm are tightly regulated. The genetic complexity of total RNA accumulating in infected cells increased in each successive phase. Moreover, the genetic complexity of nuclear RNA was invariably higher than that of cytoplasmic RNA in each phase. Lastly, the results of the studies on viral RNA accumulating in canavanine-treated cells reinforce the hypothesis made previously that more than one polypeptide in each of the alpha and beta polypeptide groups is involved in the transcription preceding the transitions from alpha to beta and beta to gamma polypeptide synthesis, respectively, and that canavanine selectively inactivated subsets of these polypeptides permitting only partial transitions from alpha to beta and beta to gamma to occur.