INTERNAL RIBOSOMAL ENTRY SITE SCANNING OF THE POLIOVIRUS POLYPROTEIN - IMPLICATIONS FOR PROTEOLYTIC PROCESSING

Based on previous studies of dicistronic polioviruses carrying two int ernal ribosomal entry sites (IRESes), we performed a novel experiment of IRES scanning through a polypeptide by inserting sequentially the I RES of encephalomyocarditis virus into the open reading frame (ORF) of the poliovirus polyprotein at selected 3C(pro)-specific QG cleavage sites. No cytopathic effects were observed after transfection of HeLa cells with any of the dicistronic constructs, and no virus was recover ed, in vitro translation of the dicistronic RNA transcripts in HeLa ce ll-free extracts revealed that multiple defects in the processing of t he P2-P3 domain of the polyprotein is the primary reason for the letha l phenotypes. Surprisingly, the interruption of 3C(pro)-catatyzed clea vages downstream of 2C interfered with the 2A(prc)-catalyzed, primary cleavage between P1 and P2. In contrast, insertion of a foreign coding sequence (V3 loop of human immunodeficiency virus type 1 gp120) into the ORF of the polyprotein at the 2C-3A junction yielded a viable viru s that appeared to be genetically stable over several passages. The re sults of these experiments, which are generally applicable to analyses of Viral polyproteins or multidomain polypeptides, suggest that proce ssing of the P2-P3 domain by 3C-3CD(pro) is rapid end accurate only in the context of the unperturbed P2-P3 precursor; this is consistent wi th cleavages occurring in cis. Moreover, an intact 2C-3A precursor is not required for viral proliferation. (C) 1998 Academic Press.