The antiviral clinical candidate 6-O-butanoyl castanospermine (MDL 28,574), an alpha-glucosidase 1 inhibitor, was examined for its effect on elementary parameters of immune function. It did not affect the mitogenic response of uninfected human mononuclear leukocytes or the detection of a range of cell surface markers, with the exception of the integrin LFA-1 (CD18/CD11a), which was reduced, after cell growth in vitro. The detection of LFA-1 was also reduced on both human and murine cells after oral administration of the compound to xenochimaeric or normal mice, respectively. Altered LFA-1 expression or function may contribute to reduced cell adhesion and the observed reduction in the in vitro allogeneic response by uninfected cells, as well as the previously described prevention of cell conjugate and HIV-induced syncytium formation.
Summary The mechanism of action of 9-(l,3-dihydroxypropoxymethyl)guanine (DHPG) and phosphonoformic acid (PFA) but not 5-fluorouridinedeoxyribose (FUdR), provides selective action against cytomegalovirus (CMV)-coded events and this was used to demonstrate that the synthesis of viral DNA was continuous during the extended phase of virus growth. The synthesis de novo of viral DNA was measured by restriction enzyme analysis after exposure to [32P]orthophosphate and its interruption by DHPG or PFA resulted in a cessation in the extrusion of infective virus from treated cells. The rate of decline in infectivity appeared to correspond to the failure of cells to maintain the synthesis of late proteins once DNA synthesis was blocked. Thus, regulation of late protein synthesis appeared to be linked to synthesis de novo of viral DNA even at late stages in CMV growth. The synthesis of the polyamines spermidine and spermine, considered obligatory for CMV growth, was unaffected by early or late inhibition of viral DNA and this showed that some virus-induced events were unaffected by the restriction on virus growth by DHPG. This provided evidence that polyamine biosynthesis was a target independent of viral DNA synthesis per se, which may be important in future considerations of combined drug therapies.
SUMMARY Thirty-seven isolates of cytomegalovirus (CMV) were obtained from a group of 20 promiscuous homosexual men, either suffering from the acquired immunodeficiency syndrome (AIDS) at the time of CMV isolation, or who developed AIDS subsequently. The isolates of CMV were characterized by the method of DNA restriction analysis. All epidemiologically unrelated strains of CMV exhibited different fragment migration patterns and no one strain appeared to be associated with AIDS or any particular disease pattern in these patients. Sequential isolates of CMV were obtained from nine patients in the study group either from different sites at the same time or from the same site on different dates. In the case of seven of the men, viruses with minor differences in restriction profile were obtained, possiblyrepresenting sub-populations of an endogenous strain of CMV. In two of the patients, reinfection with different strains was apparent. We conclude that reinfections with CMV in AIDS patients can occur, but the isolation of strains exhibiting major differences in genome structure seen by restriction enzyme analysis was uncommon.
MDL 74,695, a novel dipeptide-like compound containing the ‘difluorostatone type’ transition state mimic and a potent inhibitor of the human immunodeficiency virus (HIV) proteinase, was investigated for anti-HIV activity in vitro. The compound showed selective inhibition of both HIV-1 and HIV-2 in MT-4 cells. A potent antiviral effect against a range of clinical isolates of HIV-1 cultured in human peripheral blood mononuclear cells and primary monocytes was also demonstrated. The antiviral activity of MDL 74,695 against viruses resistant to a range of reverse transcriptase inhibitors was equivalent to the wild-type. In rats MDL 74,695 (30 mg kg −1 ) was 4.9% orally bioavailable and maintained levels above the in vitro 50% inhibitory concentration (IC 50 ) for approximately 3 h. Viruses with reduced sensitivity to MDL 74,695 and saquinavir were selected in cell culture by continuous passage in increasing drug concentrations, and first appeared after 20 and 17 passages, respectively. Amino acid changes were identified at positions 48 (glycine to valine), 50 (isoleucine to valine) and 82 (valine to either isoleucine or alanine) in various combinations for MDL 74,695-resistant viruses. For saquinavir-resistant viruses changes were identified at positions 48 (glycine to valine) and 90 (leucine to methionine). Studies using MDL 74,695, saquinavir and a third proteinase inhibitor indinavir, indicated that virus selected in the presence of MDL 74,695, with amino acid exchanges at positions 48 and 82 showed cross-resistance to saquinavir. However, viruses selected in the presence of MDL 74,695 with amino acid exchanges at positions 50 and 82 showed no significant change in sensitivity to saquinavir. Likewise, viruses selected in the presence of saquinavir with amino acid exchanges at positions 48 and 90 remained sensitive to MDL 74,695. All viruses selected after growth in the presence of either MDL 74,695 or saquinavir showed little or no resistance to indinavir.
Human cytomegalovirus infection is common in the general population but generally remains silent; the risk of disease is greatest in fetuses and the newborn
The basis of azidothymidine (AZT) insensitivity in the human JM T-cell line has been investigated. It was shown that uptake of radiolabelled thymidine or AZT into cellular acid-soluble pools of JM cells was about 10-fold lower than that seen in AZT-sensitive HeLa cells. Thymidylate kinase, however, was apparently not inhibited by AZTMP in JM cells to the extent observed in most cells and, as a result, AZTTP formation proceeded at a greater rate than in HeLa cells, which exhibited accumulation primarily of AZTMP. Thus the deficit in phosphorylation in JM cells cannot solely account for the decreased AZT sensitivity. Instead, it was shown that JM cells excreted AZTMP into the culture medium and that, whereas HeLa cells continued to accumulate AZT nucleotides over time, JM cells did not. This excretion of AZTMP in JM cells also led to a failure to sustain lowered competing TTP pools. It is concluded that it may not be appropriate to use the JM cell line for testing of novel anti-HIV nucleotides designed to circumvent requirements for phosphorylation.
Abstract Previous work in this laboratory showed that polyamine biosynthesis was stimulated in fibroblasts following infection with the AD169 strain of human cytomegalovirus (HCMV) or with murine cytomegalovirus (MCMV) (Tyms et al: Biophysics Research Communications 86:312–318, 1979; Advances in Polyamine Research 4:507–517,1983). Here we compare the affect of AD169 on polyamine production in infected fibroblasts with that of the unusual Colburn strain of HCMV. The Colburn virus is unusual in that it was isolated from a 7 year old boy with encephalitis and molecular studies indicated the virus was simian like (Huang et al: Journal of Virology 26:718–723, 1978). As a consequence of CMV infection a two to ten fold increase in the spermine content of fibroblast cells is observed. Radiolabel transfer experiments show that spermine is synthesized throughout virus infection. Indeed, spermidine and spermine are specifically incorporated into the purified virions of the AD169 and Colburn strains of HCMV. Furthermore, polyamine biosynthesis is stimulated in fibroblast cells infected with a number of low passage clinical isolates of HCMV. Inhibition of polyamine biosynthesis in HCMV infection may provide a specific and novel target for antiviral chemotherapy.
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