Inhibition of human cytomegalovirus replication by 9-(1,3-dihydroxy-2-propoxymethyl)guanine alone and in combination with human interferons

Abstract

The inhibitory action of 9-(1,3-dihydroxy-2-propoxymethyl)guanine on the replication of human cytomegalovirus was studied. Three laboratory strains (AD-169, Towne, and Davis) and three early passage (<10) clinical isolates were all inhibited in yield inhibition assays. In cultures infected with AD-169, virus yields could be inhibited if the drug was added as late as 3 days after the replication cycle had begun. The effects of the drug were' fully reversible during the first 4 days of the viral replication cycle. Viral infectivity and viral DNA synthesis were reduced more than viral protein synthesis. Synergistic antiviral effects were observed with betacysteine, and to a lesser extent, with beta-serine recombinant interferons, but only over a narrow range of dose combinations.The novel guanosine analog, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), is active in vitro against human cytomegalovirus (CMV) (1, 13, 24). Acycloguanosine and other nucleoside analogs have limited activity against human CMV (3,5,12,16,19,23 various concentrations was then added to the tissue cultures. When a multiplicity of infection (MOI) of 0.1 was used, the virus was allow'ed to replicate for 5 days; replication was continued for multiple cycles (10 to 12 days) if the MOI was <0.1. Infected cultures were harvested by scraping the cells into the supernatant, pooling duplicate wells, sonicating to disrupt the cells, and then freezing the cells at -70°C. For some experiments, the supernatant and intracellular virus were harvested separately. Samples were assayed for PFU of virus on HEL cells (21). Inhibition was determined by comparing the yields from drug-treated virus-infected cultures with those from untreated virus-infected cultures and was expressed as a percentage of control yields. For plaque reduction assays, the drug was incorporated into the semisolid overlay. For experiments in which the effects of IFNs and DHPG in combination were tested, cells were first treated overnight with IFN and then infected with CMV. Both drugs were included in the maintenance medium, either DMEM or DMEM with 1% agarose, during virus replication. To evaluate drug interactions, we calculated a combination index (CI) by the formula derived by Spector et al. (28): CI = ln(A) + ln(B) -ln(AB) -ln(VC), where A and B are the mean virus yields in the presence of antiviral agents A and B, respectively, AB is the virus yield in cultures treated with both agents, and VC is the virus yield in untreated controls. The CI ± standard error (SE) was calculated for each drug combination with data from independent experiments. When the CI was within 2 SEs of 0, the combined drug effects were additive. When the CI was greater than ±2 SE, the effects were synergistic; when the CI was less than ±2 SE, the effects were antagonistic. For plaque reduction assays, the drug interactions were assessed by the formula [(A)(B)]i [(AB)(VC)]. When the numerical result was >1, the effects were synergistic; when the numerical result was equal to 1, the effects were additive; and wh...

A microtiter virus yield reduction assay for the evaluation of antiviral compounds against human cytomegalovirus and herpes simplex virus

“…In commonly reported versions of this assay (Cheng et al, 1983;Rasmussen et al, 1984;Turk et al, 1987;Snoeck et al, 1988) the primary drug-treated cultures are grown in 25 cm2 fIasks, petri dishes, or 24-well tissue culture plates. Culture lysates are prepared, diluted in test tubes, and virus titrated in 6-, 24, or 96-well plates.…”

Treatment of cytomegalovirus retinitis with DHPG in a patient with AIDS

“…DHPG, also referred to as ganciclovir, 9-(2-hydroxy-l-(hydroxymethyl)-ethoxymethyl)guanine, BW B759 U, and dihydroxypropoxymethylguanine, is an acyclic nucleoside analogue of guanosine and has shown potent in vitro activity against human cytomegalovirus (Rasmussen et al, 1984). The antiviral activity is the result of incorporation of the agent into replicating viral DNA and subsequent inhibition of viral DNA replication.…”

Rapid determination of human cytomegalovirus susceptibility to ganciclovir directly from clinical specimen primocultures

“…Cytomegalovirus (CMV) resistance to ganciclovir (GCV) has been described in the clinical setting (6), and about 8% of patients with AIDS who take this drug for more than 3 months excrete GCV-resistant virus (5). The existing CMV susceptibility determination assays-plaque reduction assay (2), DNA hybridization assay (4), late antigen synthesis reduction assay (10), yield reduction assay (13), or in situ enzyme-linked immunosorbent assay (16)-require the isolation of CMV from clinical specimens. The isolate must then be passaged several times in cell culture to increase the virus titer and/or to obtain cell-free virus before testing.…”