Translation of the pestiviral polyprotein is initiated cap independently at an internal site of the viral RNA, the internal ribosome entry site (IRES). We investigated the translation from the IRES of bovine viral diarrhea virus (BVDV) and the possible interaction of the unconventional cellular RNA-binding proteins, particularly of polypyrimidine tract-binding protein (PTB). The BVDV IRES is translationally active in rabbit reticulocyte lysate (RRL), and it is translated most efficiently at low concentrations of Mg(2+)- and K(+)-ions. In the UV cross-link assay, several proteins from RRL bind to the BVDV IRES, including proteins of 50, 65 and 72kDa, but no protein of 57kDa possibly corresponding to PTB, although PTB is endogenously present in RRL. However, the BVDV IRES can bind PTB weakly under certain conditions. Interestingly, in a functional depletion and add-back translation system, PTB does not enhance translation of BVDV, although PTB enhances translation of a picornavirus in this translation stimulation assay. These results indicate that PTB can bind the BVDV IRES RNA, but translation is independent of the action of PTB.