NOVEL MECHANISM OF INHIBITION OF CYTOMEGALOVIRUS BY THE EXPERIMENTAL IMMUNOSUPPRESSIVE AGENT LEFLUNOMIDE1,2

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Abstract
Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) remains a major cause of morbidity and mortality among pharmacologically immunosuppressed organ transplant recipients, frequently engaging the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective against acute and chronic allograft rejection in animal models. Because a number of CMV proteins are known to be phosphorylated, we tested the hypothesis that this agent might exert inhibitory activity against CMV. Plaque assays demonstrated dramatic dose-dependent attenuation of production of multiple clinical CMV isolates in leflunomide-treated human fibroblasts and endothelial cells, common targets for CMV infection in vivo. As shown by Northern blot analysis and immunohistochemical staining, leflunomide neither interferes with transcription of immediate early or late viral genes, nor with expression of corresponding proteins. CMV-specific DNA dot blots and biochemical enzyme assays indicated that, in contrast to currently approved anti-CMV drugs, leflunomide exerts no inhibitory effect on the accumulation of viral DNA in infected cells, or on viral DNA polymerase activity. Rather, as visualized by transmission electron microscopy, this agent appears to act at a late stage in virion assembly by preventing tegument acquisition by viral nucleocapsids. Finally we have demonstrated equivalent inhibitory activity of leflunomide against multi-drug-resistant CMV isolates. These findings imply that leflunomide, an effective immunosuppressive agent, shows potential to concurrently attenuate a major complication of immunosuppression, CMV disease, by a novel mechanism of antiviral activity.