title: Analysis of long-term human cytomegalovirus latency in vitro.
abstract: Human leukocyte interferon (IFN-alpha) and acyclovir (ACV) have been used to establish human cytomegalovirus (HCMV) latency in infected human embryo lung fibroblast (HEL-F) cells. HCMV latency was maintained for a short interval (less than 9 days) after removal of inhibitors by increasing the incubation temperature. We now report a model system in which HCMV latency has been dramatically extended. HEL-F cells pretreated with IFN-alpha (200 IU/ml) and ACV (300 microM) were infected with a low MOI of HCMV, and treated for 23 days with the same inhibitor combination at 37 degrees. Infectious HCMV and virus antigens were undetectable at the time of inhibitor removal and remained undetectable during continued incubation at 40.5 degrees. A minimum of 0.4% of the cell population, however, contained a virus genome that could be reactivated at the time of inhibitor removal; this value declined to 0.0005% after 77 days at 40.5 degrees. HCMV reactivation was achieved by maintaining the infected cells at 37 degrees after inhibitor removal or by decreasing the incubation temperature to 37 degrees at any time during maintenance at 40.5 degrees. The HCMV genome was analyzed by blot hybridization in latently infected cells 23 days after inhibitor treatment at 37 degrees or 4 days after inhibitor removal at 40.5 degrees. Although many HCMV-unique DNA genomic sequences were retained in HEL-F cultures after 23 days of inhibitor treatment, a significant reduction in retained HCMV sequences occurred after inhibitor removal and temperature shift to 40.5 degrees. The XbaI HCMV DNA fragments retained in the latently infected HEL-F cultures were present at a copy number of at least 0.5 copies per haploid cell genome equivalent.
mesh_terms: D000212:Acyclovir / Q000494:pharmacology; D002478:Cells, Cultured; D002874:Chromosome Mapping; D003587:Cytomegalovirus / Q000254:growth & development*; D003586:Cytomegalovirus Infections / Q000503:physiopathology*; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007370:Interferon Type I / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014779:Virus Replication / Q000187:drug effects
chemical_list: D004279:DNA, Viral; D007370:Interferon Type I; D004262:DNA Restriction Enzymes; D000212:Acyclovir