POLY(RC) BINDING-PROTEIN-2 BINDS TO STEM-LOOP-IV OF THE POLIOVIRUS RNA 5'-NONCODING REGION - IDENTIFICATION BY AUTOMATED LIQUID-CHROMATOGRAPHY TANDEM MASS-SPECTROMETRY

The 5' noncoding region of poliovirus RNA contains an internal ribosom e entry site (IRES) for cap-independent initiation of translation, Uti lization of the IRES requires the participation of one or more cellula r proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined, In this report, we identify a cellular RNA binding protein isolated from the ribosomal s alt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES, The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tand em mass spectrometry. The sequence obtained matched that of poly(rC) b inding protein 2 (PCBP2), previously identified as an RNA binding prot ein from human cells, PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an exp ression plasmid to produce a histidine-tagged fusion protein, Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis, The closely related PCBP 1 showed no stable interaction with the RNA, Stem-loop IV RNA containi ng a three nucleotide insertion that abrogates translation activity an d virus viability was unable to bind PCBP2.