We previously have described a model of in vitro herpes simplex virus (HSV) latency in which latent infection was (i) established with human leukocyte interferon (IFN-alpha) in combination with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) or 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir); (ii) maintained after termination of combined inhibitor treatment by incubation at 40.5 degrees, and (iii) reactivated by either reducing the incubation temperature to 37 degrees or by superinfecting at the elevated temperature with human cytomegalovirus (HCMV). We now report the use of this system to examine the transcriptional activity of the HSV genome during establishment, maintenance, and reactivation of HSV latency in vitro. Numerous species of virus-specific polyadenylated RNAs were present during the first 3 days of combined BVDU and IFN-alpha treatment of HSV type 1 (HSV-1)-infected human fetus lung fibroblast cells. However, after 7 days of combined inhibitor treatment, only a very small quantity of virus-specific RNA could be detected utilizing the short unique region of the HSV-1 genome as probe. After terminating combined BVDU and IFN-alpha treatment and increasing the temperature from 37 to 40.5 degrees on day 7 after infection, virus-specific RNA was undetectable by RNA blot hybridization analysis; however, a small amount of HSV-specific RNA was detected in 2% of the cells by in situ hybridization. The HSV-1 transcriptional products produced after HCMV superinfection in the presence of selected inhibitors of macromolecular synthesis also were examined and demonstrated that the efficient activation of HSV-1 immediate-early gene transcription required the expression of not only immediate-early HCMV gene product(s), but also at least a subset of early-late gene products.
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