Post-transplantation pharmacologic immunosuppression puts transplant recipients at risk for development of opportunistic infections. Of these, cytomegalovirus (CMV) remains one of the most common sources of serious complications, frequently engaging the clinician in a struggle to balance graft-preserving immunosuppressive therapy with control of CMV disease. Our recent preliminary studies suggest that leflunomide, an experimental immunosuppressive agent currently in Phase I clinical trials in : transplant recipients, exhibits unique mechanisms of antiviral activity against CMV, including (traditional) drug-resistant isolates. This investigation is designed to assess the antiviral activity of leflunomide in vivo, and to resolve the molecular mechanisms underlying the antiviral activity of this agent. To determine the effectiveness of leflunomide in control of acute CMV disease, immunodeficient rats will be inoculated with CMV, treated with this agent (or otherimmunsuppressants or anti-CMV drugs), and authorized at intervals. Salivary glands, spleen, and lungs will be examined histologically for viral pathology, and assayed for viral load by plaque assay. To determine the effectiveness of leflunomide in the prevention and reversal of viral reactivation, CMV latency will be established in immunocompetent rats and leflunomide (or other agents) will be administered prior to, or following radiation-induced viral reactivation. Animals will be authorized at intervals and assayed as above. Since leflunomide, a known inhibitor of protein kinase activity, appears to interfere with formation of the viral tegument (our preliminary observations), several major components of which are phosphoproteins, molecular mechanisms underlying the antiviral activity of leflunomide will be investigated first by assay of viral structural protein phosphorylation. CMV-infected human cells incubated in the presence or absence of leflunomide will be labeled with [32P] orthophosphate, lysed, and assayed by western blot (and confirmatory immunoprecipitation) using antibodies specific for tegument or other structural proteins. Phosphorylation patterns will be assayed by autoradiography and specific proteins will be identified and quantitated by immunoblot. Phosphoamino acids in precipitated phosphoproteins will be identified by two-dimensional electrophoresis and distribution of structural proteins will be assessed by immunogold electron microscopy. Since our preliminary data indicate that leflunomide attenuates activation of host cell transcription factors which contribute to viral gene activation, but does not inhibit viral gene transcription or DNA synthesis, we will also test the hypothesis that the antiviral activity of this agent is independent of its effect upon host cell transcription factors. Nuclear extracts derived from leflunomide-treated (or untreated) CMV-infected cells will be quantitatively analyzed by electrophoretic mobility shift assay for binding of CMV-relevant host transcription factors to their respective consensus DNA binding elements. Transfection with plasmid constructs containing reporter genes driven the CMV immediate early promoter/enhancer sequence will be employed to determine the impact of leflunomide upon CMV immediate early gene expression. Preliminary data predict that results of the proposed experiments will identify leflunomide as uniquely bifunctional in its ability to both preserve graft integrity and substantially reduce viral load. In addition, data generated by this investigation will provide the rationale for the development of leflunomide derivatives and related compounds possessing greater efficacy and/or with exclusively antiviral (but not imrnunosuppressive) activity, thereby broadening the target population to include AIDS patients and other individuals susceptible to opportunistic infections.
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