9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)-adenine (DHC), a specific inhibitor of S-adenosyl-L-homocysteine (AdoHcy) hydrolase, has been used in this study to elucidate the mechanism by which DL-homocysteine (Hcy) potentiates the antiviral effects of AdoHcy hydrolase inhibitors as reported by De Clercq [Biochem. Pharmacol. 36:2567-2575 (1987)]. The potentiating effects of Hcy on the antiviral effects of DHCA were determined using murine L929 cells infected with vaccinia virus. When virus-infected cells were incubated with DHCA alone or in combination with various concentrations of Hcy, the following IC50 values (concentrations of the drug required to reduce by 50% viral plaque formation) were observed: 0.30 microM (0 mM Hcy), 0.15 microM (0.3 mM Hcy), 0.09 microM (1.0 mM Hcy), and 0.04 microM (3.0 mM Hcy). In the drug combination studies, increased cellular toxicity, compared with DHCA alone, was observed only at the highest concentration of Hcy (3.0 mM); thus, at lower concentrations Hcy increased the antiviral effectiveness [ID50 (concentration of the drug required to reduce the increase in cell number by 50%)/IC50] of DHCA. For example the following ID50/IC50 values were observed for DHCA alone or in combination with Hcy: 64 (0 mM Hcy), 113 (0.3 mM Hcy), 151 (1.0 mM Hcy), and 88 (3.0 mM Hcy). In these studies, Hcy was also observed to potentiate the increase in cellular levels of AdoHcy and the ratio of AdoHcy/S-adenosyl-L-methionine (AdoMet) in DHCA-treated cells. In earlier studies, our laboratory has shown that antiviral effects of DHCA are caused by only slight elevations in intracellular levels of AdoHcy [from 50 pmol/mg of protein (controls) to 100-200 pmol/mg of protein (drug-treated)] and slight elevations in the ratios of AdoHcy/AdoMet [from 0.05-0.1 (control) to 0.15-0.20 (drug-treated)]. Thus, in the presence of Hcy, lower concentrations of DHCA are needed to increase the intracellular concentration of AdoHcy and the AdoHcy/AdoMet ratio to levels that suppress replication of vaccinia virus. Murine L929 cells were shown to contain DHCA-sensitive and DHCA-insensitive forms of AdoHcy hydrolase. Based on the results of labeling experiments using [2,8-3H]adenosine and [35S]methionine, the elevated levels of AdoHcy were shown to arise from the reaction of [2,8-3H]adenosine and Hcy, catalyzed by the DHCA-insensitive form of AdoHcy hydrolase.