4. with well-defined antipolyomavirus activity (-)-Epicatechin gallate (23,37), the main treatment is reduction of immunosuppression at the expense of an increased risk of rejection (21). The active metabolite of the immunomodulatory drug leflunomide, A771726 (LEF-A), inhibitis mitochondrial dihydroorotate dehydrogenase (10), leading to pyrimidine depletion and cytostasis, particularly in activated lymphocytes (7). Tyrosine kinase (29), cyclooxygenase (18), and NF-B signaling (15) may also be affected at higher concentrations. Leflunomide has demonstrated antiviral (-)-Epicatechin gallate activity toward human immunodeficiency virus 1 (HIV-1) (38) and herpesviruses (25,44) and is now also used in treatment of PVAN (2,3,6,9,12,13,24,26,30,34,39,41,47), although its Rabbit polyclonal to TLE4 clinical efficacy has not been formally tested in controlled trials. For herpesviruses, the antiviral effect is attributed to impaired nucleocapsid tegumentation (25,44). Since BKV lacks tegument, the putative antiviral effect must be different. Two (-)-Epicatechin gallate previous BKV studies performed with WI-38 and Vero cells concluded that leflunomide inhibits BKV replication (14,24), but the detailed mechanism was not investigated. Here we report on effects of LEF-A on the BKV replication cycle in primary human renal proximal tubule epithelial cells (RPTECs). To examine the effect of LEF-A on BKV progeny production in RPTECs, LEF-A at 2.5 to 30 g/ml was added 2 h postinfection (h.p.i.) and extracellular BKV loads were measured by quantitative PCR (qPCR) 72 h.p.i. (5). LEF-A reduced BKV loads in a concentration-dependent manner (Fig.1A). At 10 g/ml (37 M) and 30 g/ml (111 M), the BKV load was about 1 log (92%) and 2 logs (99%) reduced, respectively. Next, assessing cytotoxicity in BKV-infected cells, we found that LEF-A at 10 g/ml reduced cellular DNA replication (BrdU incorporation) (5) by about 50% and mitochondrial metabolic activity (WST-1 cleavage) (5) by 40% 72 h.p.i. (Fig.1B). LEF-A at 30 g/ml reduced cellular DNA replication by 75% and mitochondrial metabolic activity by 47%. The overall metabolic activity (resazurin reduction) was not affected by LEF-A concentrations up to 25 g/ml. In uninfected cells, similar results were obtained. The LEF-A 90% inhibitory concentration (IC90), 10 g/ml, was used to determine the influence on subsequent steps in the BKV life cycle. == FIG. 1. == Effect of LEF-A titration on BKV load and RPTEC cytotoxicity. RPTECs (Lonza) (passage 4) were seeded in 24- or 96-well plates (-)-Epicatechin gallate and supernatant infected with BKV-Dunlop at 50% confluency from Vero cells (multiplicity of infection [MOI] of 1 1) or left uninfected. At 2 h.p.i., virus or supernatant was removed, cells were washed, and medium with increasing LEF-A concentrations (A771726; Calbiochem) or without LEF-A was added. (A) Supernatants were harvested 72 h.p.i., and extracellular BKV loads were measured by qPCR with primers and probe targeting the LT-ag gene (5). Data are presented as Geq/ml (Geq = genome equivalents). (B) The cytotoxicity of LEF-A was monitored 72 h.p.i. by measuring cellular DNA replication by cell proliferation enzyme-linked immunosorbent assay (ELISA), BrdU (Roche Applied Science) (5), mitochondrial metabolic activity with cell proliferation reagent WST-1 (Roche Applied Science) (5), and total cellular metabolic activity (monotoring mithochondrial, microsomal, and cytosolic enzymes) with the resazurin-based assay TOX-8 (Sigma-Aldrich). For all three assays, colorimetric measurements were performed as described by the manufacturer. Absorbance for untreated cells was set as 100%. To study the effect on viral entry, LEF-A was added: (i) 2 h before, (ii) together with, or (iii) 2 h after BKV infection. Comparing extracellular BKV loads 72 h.p.i. (Fig.2A) or large T-antigen (LT-ag) mRNA expression 24 h.p.i. by reverse transcription (RT)-qPCR (5).