Receptor-initiated activation of cells and their oncogenes by herpes-family viruses.

The interaction of human cytomegalovirus (HCMV) with the cell membrane has been shown to initiate a cascade of physiologic and biochemical responses that result in the transcriptional activation of specific cellular proto-oncogenes. The cell-activation responses initiated by the virus membrane interaction appear to be important for efficient HCMV replication, as pharmacologic inhibition of cell activation responses significantly reduces the expression of immediate early viral genes and the production of infectious progeny virus. Cellular receptor proteins for other viruses have been shown to be molecules with physiologic activities. Binding of virus to these receptors may trigger the cell to initiate changes that are important for efficient viral replication. These viruses may also trigger inappropriate physiologic responses in the absence of viral replication, thereby causing more covert manifestations of viral pathology.

Cytomegalovirus-enhanced induction of chromosome aberrations in human peripheral blood lymphocytes treated with potent genotoxic agents.

Human cytomegalovirus (HCMV) has been shown to increase the frequency of chromosome aberrations, primarily chromatid-type, in human peripheral blood lymphocytes (PBLs). Because HCMV persists in most humans, pathologically activates cells, and may perturb the cell cycle, we investigated the possibility that HCMV-infected cells have a modified sensitivity to chromosome damage induced by genotoxic chemicals. Uninfected PBLs exposed to bleomycin (3 to 100 micrograms/ml) demonstrated a linear increase in the frequency of chromosome aberrations. HCMV infection of PBLs at an intensity that did not cause detectable damage followed by exposure to the same concentrations of bleomycin resulted in a significant enhancement (p less than 0.01) in the frequency of chromosome aberrations relative to the effect of bleomycin alone. A more than additive enhancement of the frequency of chromosome aberrations was also noted in HCMV-infected PBLs exposed to 4-hydroxyaminoquinoline-1-oxide (4-HAQO; 0.1 to 0.3 micrograms/ml) relative to uninfected cells treated with 4-HAQO alone. No increase in the percentage of aberrant cells or the frequency of chromosome aberrations was observed in HCMV-infected cells treated with 4-nitroquinoline-1-oxide (4-NQO) relative to similarly treated uninfected PBLs. These results suggest that HCMV can potentiate the induction of chromosome aberrations in human PBLs caused by potent DNA damaging agents.

The intellectual property landscape in the field of plasmid-based gene therapy.

As plasmid-based gene therapy products progress through clinical trials commercial entities begin to focus on the intellectual property associated with the methods and specific compositions used in these therapies. As the number of patents covering gene therapy components and methods increases it becomes increasingly difficult for a single entity to collect all of the necessary rights to be able to offer gene therapy products to consumers. The present report briefly describes the relevance of patents to product commercialization and describes certain key patents that may affect the ultimate commercialization potential of this new and exciting technology.

Multiple herpesviruses in saliva of HIV-infected individuals.

Oral infections with human herpesviruses cause increased morbidity in patients infected with HIV. In this study, multiple HHVs were often isolated from the saliva of HIV-seropositive dental patients, but their isolation rate did not differ substantially from rates reported for the general population, except for human cytomegalovirus.

Increased levels of sequence-specific DNA-binding proteins in human cytomegalovirus-infected cells.

Increased levels of active sequence-specific DNA-binding proteins, AP-1, CRE/B, and NF kappa B were observed in nuclear lysates of human cytomegalovirus-infected cells from 15 min postinfection. The activation of these cellular factors did not require infectious virus or de novo viral protein synthesis, but their abundance was significantly reduced by inhibitors of protein kinase C and/or A. These data suggest that formation of these transcription factors resulted from the virus-cell membrane interaction and/or through the action of virion structural proteins on cytoplasmic forms of these cellular factors.

Cytomegalovirus: sodium entry and development of cytomegaly in human fibroblasts.

A possible relationship between net Na+ entry and the development of CMV-induced cytomegaly (cell enlargement) was investigated in human fibroblasts derived from skin-muscle and thyroid tissue. We found that inhibiting cellular Na+ uptake, either by pharmacological means (amiloride, an inhibitor of Na+/H+ exchange) or by replacement of extracellular Na+ (by N-methyl-D-glucamine or choline), inhibited the development of cytomegaly. Furthermore, we noted a temporal parallelism between the development of cytomegaly and enhancement of ouabain-sensitive (O-S) 86Rb+ uptake. O-S 86Rb+ uptake is a monitor for the activity of the sodium pump resident in the plasmalemma of the fibroblasts. The enhanced O-S 86Rb+ uptake reflects either an increased intracellular Na+ concentration or an increased number of sodium pump complexes per fibroblast. Amiloride inhibited the enhancement of O-S 86Rb+ uptake, as well as cytomegaly development. Addition of amiloride at selected times after infection suggested that the same phase of virus replication was sensitive to the inhibitory effect of this drug on the enhancement of O-S 86Rb+ uptake and on the development of cytomegaly. There was also a similar pattern of inhibition of O-S 86Rb+ uptake and cytomegaly with increasing concentrations of amiloride. Thus, there may be a relationship between CMV-induced Na+ entry through activation of the Na+/H+ exchanger and development of cytomegaly.

Increased frequency of specific locus mutation following human cytomegalovirus infection.

The effect of human cytomegalovirus (HCMV) infection on the frequency of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus was studied in Chinese hamster lung V79 cells. When V79 cells were infected with HCMV (strain AD169) at multiplicities of 0.1 to 50 plaque forming units (PFU) per cell the presumptive mutation frequency, as determined by the number of 6-thioguanine-resistant (TGr) colonies, was increased up to 16.8-fold (P < 0.005), depending on the multiplicity of infection. Increases in the mutation frequency at the hprt locus were also observed for other laboratory-adapted HCMV strains (C-87, Davis) and for low passage clinical isolates (82-1, 84-2). The expression time required for the maximum increase in TGr colonies was 3 days and was consistent among the HCMV strains evaluated in this study. UV-irradiation of HCMV stock up to a dose of 9.6 x 10(4) ergs/mm2 increased the mutation frequency, but further exposure to UV light or to heat (56 degrees for 30 min) significantly decreased the frequency of TGr-resistant colonies, suggesting that expression of HCMV genes was involved in the mutation process. HCMV-induced TGr cells demonstrated substantially reduced (> 96%) incorporation of [3H]hypoxanthine. PCR analysis of the hprt locus demonstrated deletions in 9 of 19 HCMV-induced TGr colonies randomly selected for further study, while 2 of 17 spontaneously developed TGr colonies demonstrated deletions. Although insertions were not detected in spontaneously developed clones, 3 of 19 HCMV-induced TGr clones had insertions in the hprt gene. Neither HCMV-specific DNA sequences nor HCMV-specific proteins were detected in the TGr clones obtained after HCMV infection. Infection of V79 cells with HCMV also increased their sensitivity to mutation with N-methyl-N'-nitro-N-nitrosoguanidine, giving a synergistic enhancement of the mutation frequency. These results indicate that HCMV infection has the capacity to induce mutations in the cellular genome and increase the sensitivity of infected cells to mutation by genotoxic chemicals. Although inactivated HCMV particles are responsible for a modest increase in the mutation frequency, expression of HCMV genes is associated with a substantial enhancement of the mutation frequency.

Human cytomegalovirus: development and progression of nuclear inclusions by primary clinical isolates and laboratory-adapted strains.

The morphogenesis of cytomegalovirus (CMV) nuclear inclusions (NIs) was investigated using unadapted clinical isolates and adapted laboratory strains. Both adapted and unadapted strains of CMVs induced NIs whose morphologic appearance was similar in human fibroblastic cells. Early NIs appeared as ring-like structures composed of dense granular and fibrillar material, while late NIs appeared to consist of multiple electron-lucent areas containing coarse granules and bounded by electron-dense fibrillar material (cellulae). Capsids and nucleocapsids were associated primarily with the electron-dense fibrillar material; however, developing nucleocapsids were most often observed at the interface of the electron-dense and -lucent areas. Although there was some variation in the rate of development and maturation of the NIs with the intensity of infection, all CMVs examined produced late NIs with similar organizational patterns consisting of cellulae. Substitution of human fibroblastic cells derived from various tissues as cellular substrate did not appreciably affect the results. Thus, the unique organization of the CMV late NI, consisting of multiple cellulae, appears to be an intrinsic feature of CMV replication since it seems to be independent of the extent of laboratory adaptation, the virus strain, the intensity of infection, or the cell type.

Modulation of the frequency of human cytomegalovirus-induced chromosome aberrations by camptothecin.

The effects of selected DNA repair inhibitors on the frequency of human cytomegalovirus (HCMV)-induced chromosome aberrations were evaluated in human peripheral blood lymphocytes (PBLs). Treatment of HCMV-infected PBLs with camptothecin (0.05 to 0.3 micrograms/ml), an inhibitor of topoisomerase I, for 30 hr resulted in a significant (P less than 0.01) synergistic enhancement of the frequency of HCMV-induced chromosome damage. On the other hand, a significant increase in the frequency of chromosome damage was not noted for infected PBLs treated with either 3-aminobenzamide (3-AB; 3 to 30 micrograms/ml), an inhibitor of poly(ADP-ribose) polymerase, or novobiocin (3 to 30 micrograms/ml), an inhibitor of topoisomerase II or excision repair processes, for 30 hr. Chromatid-type breaks and exchanges were the predominant type of chromosome aberrations observed in the HCMV-infected cells treated with camptothecin, suggesting that HCMV infection is associated with the induction of single-strand DNA breaks. Furthermore, these findings suggest that HCMV infection does not inflict direct DNA damage which is repaired through 3-AB- or novobiocin-sensitive pathways.

Activation of cellular oncogenes by clinical isolates and laboratory strains of human cytomegalovirus.

The effect on cellular (c) oncogene RNA levels was investigated after infection of permissive cells with cell culture adapted strains (AD-169, C-87, Davis) and unadapted clinical isolates (82-1, 84-2, 85-1) of human cytomegalovirus (HCMV). The results indicate that both adapted and unadapted strains of HCMV induce substantial increases in c-oncogene RNA levels for fos, jun, and myc measured by Northern blot hybridization. Elimination of immediate early (IE) protein synthesis between 0 and 3 hrs or reduction of virus infectivity (99.99%) by UV-irradiation did not reduce the increase in c-oncogene RNA levels. Inhibition of viral and cellular protein synthesis by cycloheximide resulted in a high abundance (superinduction) of specific RNAs which hybridized to c-oncogene probes after infection with either adapted or unadapted strains of HCMV. These data suggest that IE viral gene expression is not essential for activation of c-oncogenes. Inhibition of DNA-dependent RNA synthesis by blocking RNA elongation with actinomycin-D or by inhibiting the activity of RNA polymerase II with alpha-amanitin significantly reduced the increase in c-oncogene RNA levels, suggesting that activation of cellular genes by HCMV is controlled at the level of transcription. Activation of c-oncogenes by HCMV may be particularly important because their protein products appear to be involved in initiation and regulation of viral and cellular gene expression.

Human cytomegalovirus infection increases the number of ouabain-binding sites in human fibroblasts.

Infection of permissive human embryo fibroblasts (MRC-5) with human cytomegalovirus (HCMV) increased the number of copies of the Na+,K(+)-ATPase (the Na+ pump) in the plasma membrane, measured as ouabain-binding sites. The increase was preceded by cell enlargement by about 24 hr, becoming significant between 48 and 72 hr after infection. Reduction in Na+ or Cl- concentration in the culture media immediately after infection partially prevented the increase in the number of ouabain-binding sites. The effect was reversible upon restoring Cl- or Na+ to the incubation medium, but withdrawal of either ion at 24 or 48 hr PE failed to prevent the increase in the number of binding sites. These results suggest that the processes that resulted in the increase of copies of the Na+,K(+)-ATPase required both Na+ and Cl- during the first 24 hr PE. Amiloride and ethylisopropylamiloride, two inhibitors of Na+ transport mechanisms of the plasma membrane have been previously shown to reduce the amount of virus yields and to prevent the onset of cytomegaly (Fons et al., 1991, Proc. Soc. Exp. Biol. Med. 196, 89-96). We show here that these agents partially block the increase in ouabain-binding sites caused by HCMV infection.