Analysis of Long-Term Human Cytomegalovirus Latency in vitro

Abstract

Human leukocyte interferon (IFN-α) 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-α (200 IU/ml) and ACV (300 µM) were infected with a low MOI of HCMV, and treated for 23 days with the same inhibitor combination at 37 °. Infectious HCMV and virus antigens were undetectable at the time of inhibitor removal and remained undetectable during continued incubation at 40.5 °. 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°. HCMV reactivation was achieved by maintaining the infected cells at 37° after inhibitor removal or by decreasing the incubation temperature to 37° at any time during maintenance at 40.5°. The HCMV genome was analyzed by blot hybridization in latently infected cells 23 days after inhibitor treatment at 37 ° or 4 days after inhibitor removal at 40.5 °. 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 °. The Xbal 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.

Maintenance of Marek's Disease Herpesvirus Latency in vitro by a Factor Found in Conditioned Medium

“…Establishment and maintenance of herpes simplex virus (HSV) and human cytomegalovirus latency in vitro with the use of drugs alone or in combination with IFN, and with or without changes in the incubation temperature, have been reported (Wigdahl et al, t 982, 1984;Rapp, 1984;Shiraki & Rapp, 1986;Scheck et al, 1986;Cockley & Rapp, 1986). However, there are conflicting reports on the effect of IFN and other lymphokines on herpesvirus latency.…”

A human cytomegalovirus function inhibits replication of herpes simplex virus

“…Nick-translated probe DNA was prepared by a modification (27) of the protocol designed by Rigby et al (33). The reaction mixture was described previously (7). Specific activities of 3 x 108 to 4 x 10' cpm/4g of DNA were achieved.…”

Mechanisms associated with the generation of biologically active human immunodeficiency virus type 1 particles from defective proviruses.

“…Recombination between viral DNAs may also occur in cells upon transfection, as has been documented in other systems (25,26,28). In addition, several types of nongenetic interactions may occur between defective viral genomes, which include phenotypic mixing, genotypic mixing, interference, enhancement, and complementation (29). It is well established that two viral RNA genomes are packaged in a retroviral particle.…”