INTERACTION OF POLYPYRIMIDINE TRACT BINDING-PROTEIN WITH THE ENCEPHALOMYOCARDITIS VIRUS MESSENGER-RNA INTERNAL RIBOSOMAL ENTRY SITE

Translation of encephalomyocarditis virus (EMCV) mRNA occurs in a cap- independent manner, requiring instead a cis-acting element termed the internal ribosomal entry site (IRES). Binding of a 57-kDa ribosome-ass ociated protein (p57) to the EMCV IRES has been found to correlate wit h cap-independent translation. p57 has recently been reported to be ve ry similar, if not identical, to the polypyrimidine tract binding prot ein (pPTB), a spliceosome-associated factor possibly involved in U2 sn RNP/pre-mRNA complex formation or 3'-splice-site recognition. The inte raction between purified pPTB and the EMCV IRES was characterized in t his study using nitrocellulose filter binding and UV cross-linking ass ays. pPTB bound the EMCV IRES with high affinity (K(d) = 40 nM at 25-d egrees-C, pH 5.5, 80 mM ionic strength). pPTB also bound strongly to R NA fragments containing either the 5'-end, 3'-end, or an internal stem -loop of the IRES. The binding properties of 16 RNA variants derived f rom the IRES revealed however that purified pPTB bound with less speci ficity than pPTB in a mixture of cytoplasmic HeLa cell polypeptides. T he addition of HeLa extract to purified pPTB increased the binding spe cificity, suggesting that factors within the extract alter the binding specificity of pPTB. The binding of pPTB to the full-length IRES and three IRES-derived fragments was studied in detail. Complex formation was optimal at low pH and was driven entirely by entropy. As many as f our ion pairs are formed upon binding, with electrostatic interactions accounting for approximately 35% of the total free energy of complex formation.