Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 ,ug/mI) for 3 or 24 h, conditions known to result in accumulation of HCMV immediate-early and early mRNA, was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5°C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. However, replication of adenovirus, another DNA virus, was not restricted in these cells under the same conditions. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with [3H]thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection. In addition, superinfection with vesicular stomatitis virus ruled out a role for interferon in restriction of HSV replication in this system.

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.

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Epithelial chips of human foreskin or cervix infected in vitro with one strain of human papillomavirus type 11 (HPV-11) and subsequently transplanted to the renal capsule of athymic mice will yield, after 3 months of in vivo incubation, epithelial cysts that are morphologically transformed and appear identical in every way to human condylomata. These cysts synthesize virus RNA, DNA, proteins, and infectious virions. The cysts can be utilized as a source of virus for continued passage of infection. The original HPV-11 infecting material was a cell-free saline extract of pooled human vulvar condylomata. DNAs of other HPV types were not detected in this material by either dot blot or Southern blot analyses. The copy number of HPV-11 virus genomes per cell genome in experimental condylomata ranged from about 200 to 1,000, a range expected for episomal papillomavirus DNA. Analyses of cloned HPV-11 DNA (pBT-1) from experimental lesions demonstrated that the size and restriction endonuclease map of the HPV-11-Hershey isolate closely matched that of the prototype. A few nucleotide changes that were detected during analyses of pBT-1 DNA resulted either in no amino acid change or a conservative change of amino acid. Physical characterization of the cloned experimental HPV-11 DNA as well as HPV typing in clinical lesions and experimental cysts are presented.