Nucleic acid hybridization for measurement of effects of antiviral compounds on human cytomegalovirus DNA replication

166

Cytomegalovirus strains with reduced in vitro susceptibilities to ganciclovir have been recovered from patients who failed long-term ganciclovir therapy. The ganciclovir-resistant clinical isolates in this study were unable to induce ganciclovir phosphorylation in virus-infected cells. The viral DNA polymerase function appeared unaltered in one genetically pure ganciclovir-resistant strain, compared with that of its wild-type ganciclovir-sensitive counterpart. All nine of the ganciclovir-resistant strains were susceptible to foscarnet. Moreover, these strains were sensitive to inhibition both by vidarabine and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC), antiviral agents that are activated by cellular enzymes, and by (S)-1(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), which is a monophosphate nucleoside analog. The in vitro resistance to ganciclovir of the ganciclovir-resistant clinical isolates studied was attributed to the inability of the cells infected with these isolates to phosphorylate ganciclovir; the virally encoded DNA polymerase did not appear to play a role in this ganciclovir resistance.

Section: Resultsmentioning

confidence: 64%

Ganciclovir-resistant cytomegalovirus clinical isolates: mode of resistance to ganciclovir

Stanat

Reardon

Erice

et al. 1991

166

“…Nine primary isolates came from bone marrow transplant recipients (27). The inhibition of CMV DNA synthesis was performed by nucleic acid hybridization by using four BamHI restriction fragments cloned into plasmid pBR322 (9). The effect on CMV multiplication in human lung cells was determined by measuring the effect of the various analogs on viral production of late CMV antigens by enzyme-linked immunosorbent assay (Stenberg et al, in press) in the viral sensitivity assay (25,28).…”

Section: Methodsmentioning

confidence: 99%

Acyclic guanosine analogs as inhibitors of human cytomegalovirus

Wahrén¹,

Larsson²,

Rudén³

et al. 1987

The acyclic guanosine analogs R-and S-enantiomers of 9-(3,4-dihydroxybutyl)guanine [(R)-and (S)-DHBG], 9-(4-hydroxybutyl)guanine (HBG), and 9-(2-hydroxyethoxymethyl)guanine (ACV) were examined for their effects on human cytomegalovirus (CMV) replication and on CMV DNA synthesis in cell culture as well as for their ability as triphosphates to interact with CMV DNA polymerase. Production of early CMV antigens was not affected. All analogs inhibited CMV DNA synthesis and late viral antigen synthesis. Primary CMV isolates were less susceptible to all tested analogs than was the laboratory strain CMV Ad.169. The triphosphate of ACV was the most potent inhibitor of CMV DNA polymerase, with an observed Ki of 0.0076 ,uM. The corresponding Ki values of the triphosphates of (R)-DHBG, (S)-DHBG, and HBG were 3.5, 13.0 and 0.23 ,uM, respectively. All triphosphates of the analogs given above inhibited CMV DNA polymerase in a competitive manner with respect to dGTP. The triphosphates of the analogs also inhibited reactions when the synthetic template poly(dC)oligo(dG)1218 was used, whereas no inhibition was observed with poly(dA)oligo(dT)12-18None of the triphosphate analogs supported DNA synthesis in the absence of dGTP, showing that no analog was an alternative substrate to dGTP.Several nucleoside analogs are selective inhibitors of viral replication at concentrations that do not affect host cells. Two classes of nucleoside analogs have received particular attention: 5-substituted 2'-deoxypyrimidines (3) and acyclic guanosine analogs (4). They are strongly active against herpes simplex virus (HSV) types 1 and 2 (HSV-1 and HSV-2). A general property of these analogs is postulated to be a selective phosphorylation to monophosphates by the HSV-induced thymidine kinase (TK), followed by preferential inhibition of HSV-induced DNA polymerase activity by the triphosphorylated forms (3,4,8,13,20). Human cytomegalovirus (CMV) lacks a virus-encoded TK (7, 29). Therefore, one would expect the acyclic nucleoside analogs, which do not seem to be substrates for cellular TK, to remain unphosphorylated in CMV-infected cells and thus not to be inhibitors of CMV replication. Acyclic nucleoside analogs have been reported to have lower activities against CMV than against HSV or varicella zoster virus (1, 10). Therefore, it was surprising that 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) was a much more potent inhibitor of CMV multiplication in cell culture than 9-(2-hydroxyethoxymethyl)guanine (ACV) (18, 24). The triphosphates of these analogs, DHPGTP and ACVTP, were inhibitors of the partially purified CMV DNA polymerase. ACVTP had a higher affinity than DHPGTP for the CMV DNA polymerase (19). Higher amounts of DHPGTP, however, were formed in CMV-infected cells compared with those of ACVTP (2). This probably explains the better activity of DHPG, but the enzyme which mediates monophosphorylation of DHPG still has not been defined.In this study we investigated several acyclic guanosine analogs, the R-and S-enantiomers of 9-(3,4-dihydroxybutyl...

“…The amount of viral DNA synthesized in HCMV-infected cells was measured by DNA-DNA dot-blot hybridization procedures patterned after those of Gadler (19). In brief, subconfluent monolayer cultures of HFF cells were infected with HCMV at a multiplicity of infection of 0.5 PFU per cell in MEM(E) supplemented with 5% fetal bovine serum.…”

Section: * Corresponding Authormentioning

confidence: 99%

Pyrrolo[2,3-d]pyrimidine nucleosides as inhibitors of human cytomegalovirus

Turk¹,

Shipman²,

Nassiri³

et al. 1987

128

188

Seven arabinosyl, 2'-deoxyribosyl, and ribosyl pyrrolo[2,3-dlpyrimidines were evaluated in vitro for activity against human cytomegalovirus and for cytotoxicity in primary and established cell lines of human origin. The parent ribosyl analogs exhibited little antiviral selectivity owing to high cytotoxicity. In contrast, aratubercidin, ara-toyocamycin, ara-sangivamycin, and deoxysangivamycin exhibited selectivity between antiviral effect (measured by plaque or titer reduction or both) and cytotoxicity (measured microscopically and by incorporation of radioactive precursors into DNA, RNA, and protein). The selectivity (in vitro therapeutic indexes) for these four compounds ranged from 2 to 40. The two sangivamycin analogs were the most potent and selective. Ara-sangivamycin, for example, inhibited virus replication 105-fold at a concentration (10 ,uM) which produced only partial inhibition of cell growth and labeled precursor incorporation. The four arabinosyl and deoxyribosyl nucleosides appeared to act by inhibition of viral DNA synthesis as quantitated by DNA-DNA dot blot hybridization. These four analogs also were tested for activity against two strains of type 1 herpes simplex virus by a plaque reduction assay. Unexpectedly, all compounds inhibited herpes simplex virus to a lesser extent than human cytomegalovirus.