ANTICYTOMEGALOVIRAL ACTIVITY OF METHOTREXATE ASSOCIATED WITH PREFERENTIAL ACCUMULATION OF DRUG BY CYTOMEGALOVIRUS-INFECTED CELLS

We extend the observation that inhibitors of pyrimidine biosynthesis a re active against human cytomegalovirus by demonstrating that methotre xate (MTX) has preferential activity against cytomegalovirus replicati on, The 50% and 90% inhibitory concentrations of MTX for inhibition of cytomegaloviral DNA replication at 3 days postinfection in MRC-5 cell s were 0.05 and 0.2 mu M, respectively. No cell toxicity was observed in uninfected confluent cells at the highest concentration tested (1 m u M). Under similar conditions (3 days of treatment with 0.2 mu M MTX) , intracellular dTTP pools were diminished in cytomegalovirus-infected cells (87% decrease relative to untreated infected cells, P < 0.001) but were not reduced in uninfected cells. A potential explanation for the preferential antiviral effect of MTX was that human cytomegaloviru s-infected cells preferentially accumulated MTX. Increased intracellul ar accumulation and increased polyglutamation of MTX were observed in cytomegalovirus-infected cells compared with uninfected cells, Increas ed uptake of [H-3]MTX by cytomegalovirus-infected cells was first obse rved at 48 h postinfection, with threefold-higher accumulation within infected cells. By 96 h, accumulation had increased to approximately f ourfold in comparison with uninfected cells, The uptake of [H-3]MTX wa s saturable and was blocked by addition of unlabelled MTX. Intracellul ar MTX in infected cells was almost entirely in the polyglutamated for m, as demonstrated by thin-layer chromatography, whereas intracellular MTX was almost exclusively in the parent form in uninfected cells.