Thymidine-kinase in cytomegalovirus infected cells

201

We studied the effect of a novel purine acyclic nucleoside, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), on human cytomegalovirus (HCMV) replication. The susceptibility of HCMV to this drug was monitored in cell culture by plaque reduction assay. HCMV replication of various strains was inhibited to the extent of 50% by 1 to 5 microM DHPG. DHPG was highly specific in its anti-HCMV activity, since at concentrations as high as 100 microM it did not exert any detectable inhibitory effect on uninfected cell macromolecular synthesis and cell growth. At concentrations of 2 to 4 microM, the drug inhibited the synthesis of six virus-specific polypeptides with molecular weights of 200,000 (VP200), 150,000 (VP150), 67,000 (VP67), 54,000 (VP54), 32,000 (VP32), and 27,000 (VP27) up to 96 h after infection. HCMV DNA synthesis was also considerably suppressed at concentrations of 2 to 4 microM DHPG. Upon removal of the inhibitor, however, viral DNA synthesis resumed and infectious virus reappeared, indicating that this inhibition was a virostatic reversible-type inhibition.

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confidence: 99%

“…A virus-encoded thymidine kinase (TK) is required to activate ACV in infected cells (5). ACV has been found to have limited anti-human cytomegalovirus (HCMV) activity (4,11,13) since HCMV does not encode virus-specific TK enzyme in infected cells (6,16).…”

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confidence: 99%

Effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine on human cytomegalovirus replication in vitro

Mar¹,

Cheng²,

Huang³

1983

201

“…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.…”

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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...

“…This is intriguing since MCMV (and HCMV), unlike vaccinia, lacks its own virus-specified TK (4,12,14,25,34) and has been reported to inhibit cellular TK (25), and one might anticipate that the low level of cellular TK present could result in low antiviral activity. A difference in substrate specificity between HSV TK and cellular TK has been proposed as a major explanation of acyclovir's specific activity against HSV (13).…”

Section: -mentioning

confidence: 99%

Activity of trifluorothymidine against cytomegalovirus

Wingard¹,

Stuart²,

Saral³

et al. 1981

Trifluorothymidine (TFT) was tested for antiviral activity against mouse cytomegalovirus (MCMV) and human cytomegalovirus (HCMV) Trifluorothymidine (TFT) is a thymidine analog developed in the search for fluorinated pyrimidines with antineoplastic activity (16). After phosphorylation by thymidine kinase (TK), TFT has been shown to inhibit deoxyribonucleic acid (DNA) polymerase (32), to be incorporated into DNA (15, 16), and to inhibit thymidylate synthetase (31). Antiviral activity against bacteriophage T4 (16), vaccinia virus (1, 33), and herpes simplex virus (HSV) type 1 (1, 6, 9) has been demonstrated. This communication reports the antiviral activity of TFT against mouse cytomegalovirus (MCMV) and human cytomegalovirus (HCMV). MATERIALS AND METHODSVirus. The Smith strain of MCMV (ATCC MCMV assays were performed by adsorbing 0.1-ml portions of serially diluted samples in quadruplicate onto confluent MEF monolayers in TC-24 (Costar) 16-mm wells. After 1 h, unadsorbed virus was aspirated, the monolayers were overlaid with 0.9% methylcellulose containing minimal essential medium (with Earle salts), 5% NCS, 15 mM N-2-hydroxyethylpiperazine-N-2'-ethanesulfonic acid (HEPES) buffer, and gentamicin (35 ug/ml), and the cultures were incubated at 37°C in 5% CO2. After 6 days the overlays were removed, and the monolayers were fixed in absolute ethanol before staining with 0.5% methylene blue and counting plaques. HCMV assays were performed by adsorbing 0.025-ml portions of serially diluted samples onto HFF monolayers in TC96 (Costar) 6.4-mm wells.After 1 h, unadsorbed virus was aspirated, medium was added, and the monolayers were incubated for 25 days and examined twice weekly for the presence of cytopathic effect. The 50% inhibitory dose (ID5o)