Effects of the nucleoside analog 2'-nor-2'-deoxyguanosine on human cytomegalovirus replication

In preparation for an attempt to elucidate some aspects of the interaction between ganciclovir and human cytomegalovirus (HCMV) DNA replication in cells infected with HCMV, we developed a dot blot DNA-DNA hybridization technique to quantify intracellular HCMV DNA replication. We studied the effect of ganciclovir on the time course of HCMV DNA replication in human fibroblasts. Ganciclovir resulted in complete cessation of the production of infectious virus, as detected by the plaque assay. However, viral DNA synthesis, as measured by dot blot DNA-DNA hybridization with cloned HCMV DNA BamHI C fragment probe, continued in the presence of ganciclovir at 10 times the 50% effective dose (i.e., 10 ,ug/ml). The continuation of viral DNA synthesis in gagnciclovir-treated cultures leads to the intranuclear accumulation of short (subgenomic) HCMV DNA fragments. These DNA fragments are neither packaged nor released into the culture medium. Furthermore, the short DNA fragments were detected only by the BamHI C probe from the center of the unique long segment of the HCMV genome. The failure of the DNA probes from the termini of HCMV genome (BamHI-Q and HindilI-M) to detect the short DNA fragments and the intranuclear localization of these fragments suggest that these short fragments may lack the signal sequences necessary for packaging and release as infectious virions. These data strongly suggest that the anti-HCMV activity of ganciclovir is due mainly to the prevention of viral DNA chain elongation which results in the intranuclear accumulation of incomplete noninfectious viral DNA fragments.9-(1,3-Dihydroxy-2-propoxymethyl)guanine (ganciclovir [GCV]) is a structural analog of deoxyguanosine with the 2'-deoxy carbon missing. GCV is anabolized to the mono-, di-, and triphosphates (2, 8, 22) in herpes simplex virus (HSV)-and human cytomegalovirus (HCMV)-infected cells. GCV triphosphate (GCV-TP) inhibits the CMV DNA polymerase in vitro. Therefore, it has been assumed that the mechanism of action of GCV involves inactivation of the viral DNA polymerase (15). Acyclovir, a related compound and a potent inhibitor of IISV DNA replication (5,18,24), causes the production of short viral DNA fragments in 16) owing to the lack of the 3'-hydroxyl group, and therefore acyclovir is considered to be an obligatory chain terminator. The incorporation of acyclovir monophosphate at the terminus of the DNA primer inactivates the viral DNA polymerase (20). This suggests that the chain termination event is a major part of the mechanism of action of acyclovir. However, because of the presence of a 3'-hydroxyl group, GCV can be incorporated within the interior of the viral DNA molecule (3, 6). Therefore, GCV is not considered an obligatory chain terminator. Reardon (19) recently showed that GCV is detected mostly as the second nucleotide from the growing end of the DNA primer. This observation led to the conclusion that the incorporation of GCV-MP at the end of the primer will allow the addition of only one more nucleotide. These in vitro studies...

Section: Discussionsupporting

confidence: 83%

Intranuclear accumulation of subgenomic noninfectious human cytomegalovirus DNA in infected cells in the presence of ganciclovir

Hamzeh

Lietman

1991

“…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.…”

mentioning

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...

“…However, few inhibitors of human cytomegalovirus (HCMV) have been reported so far (1,5,6,17). Since HCMY does not induce viral thymidine kinase, antiviral nucleoside analogs, which are dependent on conversion to their 5'-triphosphate forms by viral thymidine kinase, cannot inhibit HCMV replication.…”

mentioning

confidence: 99%

Mechanism of selective inhibition of human cytomegalovirus replication by 1-beta-D-arabinofuranosyl-5-fluorouracil

Saneyoshi¹,

Nakayama²,

Nishiyama³

et al. 1985

Four kinds of l-p-D-arabinofuranosyl-5-halogenouracil were examined for inhibition of human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) replication. 1-j3-DArabinofuraaosyl-5-fluorouracil (ara-FU) was the most effective against HCMV, whereas l-1-D-arabinofuranosyl-5-bromouracil was the most effective against HSV-1 and HSV-2. The mechanism of action of ara-FU on HCMV replication was also studied. The dTTP pool size in human embryonic fibroblasts was increased 33-fold by HCMV infection. However, treatment with ara-FU decreased the size of the dTTP pool by approximately 50%. On the other hand, l-1B-D-arabinofuranosyl-5-fluorouracil-5'-triphosphate inhibited ICMV DNA polymerase activity competitively with dTTP. These results suggest that ara-FU acts as a bifunctional inhibitor of HCMV replication. Ara-FU is phosphorylated by cellular thymidine kinase to l-(B-D-arabinofuranosyl-5-fluorouracil-5'-monopbosphate, which inhibits cellular thymidylate synthetase, which in turn decreases the dTTP pool size in infected cells. As the dTTP pool size is reduced, inhibition of viral DNA polymerase by l-(3-D-arabinofuranosyl-5-fluorouracil-5'-triphosphate becomes more efficient.A number of compounds exhibit a highly selective inhibitory effect against herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) (2). However, few inhibitors of human cytomegalovirus (HCMV) have been reported so far (1,5,6,17). Since HCMY does not induce viral thymidine kinase, antiviral nucleoside analogs, which are dependent on conversion to their 5'-triphosphate forms by viral thymidine kinase, cannot inhibit HCMV replication. As a part of our design program of antiviral nucleosides, we examined the antiherpetic activities of 1-p3-D-arabinofuranosyl-5-halogenouracils (5-halo-ara-Us) and other related compounds by using HSV-1, HSV-2, and HCMV. The analogs used here are recognized as typical model compounds of