The leader proteinase (L-pro) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-beta (IFN-beta) antagonist that disrupts the integrity of transcription factor nuclear factor kappa B (NF-kappa B). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-alpha 1/beta expression caused by L-pro was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-alpha/beta. Furthermore, overexpression of L-pro significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L-pro mutants indicated that the ability to process eIF-4G of L-pro is not required for suppressing dsRNA-induced activation of the IFN-alpha 1/beta promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-kappa B, L-pro also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection. (C) 2010 Elsevier Inc. All rights reserved.