INTERACTION OF POLIOVIRUS POLYPEPTIDE 3CD(PRO) WITH THE 5'-TERMINI AND 3'-TERMINI OF THE POLIOVIRUS GENOME - IDENTIFICATION OF VIRAL AND CELLULAR COFACTORS NEEDED FOR EFFICIENT BINDING

Poliovirus proteinase 3CD(pro) by itself is not an RNA-binding protein . Two cellular proteins have been purified from HeLa cells (p50 and p3 6) which interact with purified 3CD(pro) but only p36-3CD(pro) bind to the 5'-terminal 110 nucleotides of polioviral RNA genome, an RNA segm ent whose secondary structure resembles a cloverleaf. The identity of these factors was determined by microsequencing tryptic digests of the purified proteins. Host protein p50 is the eukaryotic elongation fact or EF-1 alpha, and p36 an N-terminal fragment thereof. p36, referred t o as host factor, did not appear to interact with purified 3C(pro) or 3D(pol). Significantly, the formation of a 3CD(pro)-cloverleaf complex was also observed in the presence of purified poliovirus polypeptide 3AB, the precursor of VPg. 3AB by itself does not stably bind to the c loverleaf. Competition experiments have demonstrated that the RNA-prot ein interactions are specific for the full-length cloverleaf. UV cross -linking studies were employed to examine the protein components of th e cloverleaf ribonucleoproteins. RNA footprinting was used to determin e the site on the cloverleaf where the viral and cellular factors bind . Finally, we have discovered that 3AB-3CD(pro) also interacts with th e 3'-terminal sequence of poliovirus RNA. In contrast to the 5'-termin al cloverleaf, the 3'-terminal RNA can bind 3AB in the absence of othe r proteins. A model for initiation of poliovirus RNA synthesis is pres ented.