Internal ribosomal entry site scanning of the poliovirus polyprotein: implications for proteolytic processing.
Based on previous studies of dicistronic polioviruses carrying two internal ribosomal entry sites (IRESes), we performed a novel experiment of IRES scanning through a polypeptide by inserting sequentially the IRES of encephalomyocarditis virus into the open reading frame (ORF) of the poliovirus polyprotein at selected 3Cpro-specific Q*G cleavage sites. No cytopathic effects were observed after transfection of HeLa cells with any of the dicistronic constructs, and no virus was recovered. In vitro translation of the dicistronic RNA transcripts in HeLa cell-free extracts revealed that multiple defects in the processing of the P2-P3 domain of the polyprotein is the primary reason for the lethal phenotypes. Surprisingly, the interruption of 3Cpro-catalyzed cleavages downstream of 2C interfered with the 2Apro-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 virus that appeared to be genetically stable over several passages. The results of these experiments, which are generally applicable to analyses of viral polyproteins or multidomain polypeptides, suggest that processing of the P2-P3 domain by 3C-3CDpro is rapid and accurate only in the context of the unperturbed P2-P3 precursor; this is consistent with cleavages occurring in cis. Moreover, an intact 2C-3A precursor is not required for viral proliferation.
School of Medicine, State University of New York at Stony Brook, Stony Brook, New York, 11794, USA.
A V
AV
Paul
J
J
Mugavero
A
A
Molla
E
E
Wimmer
D000086042:3C Viral Proteases; D020121:5' Untranslated Regions / Q000235:genetics*; D000595:Amino Acid Sequence; D002474:Cell-Free System; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003588:Cytopathogenic Effect, Viral; D004686:Encephalomyocarditis virus / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics; D017955:Poliovirus / Q000201:enzymology / Q000235:genetics*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D020121:5' Untranslated Regions; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D010446:Peptide Fragments; D011498:Protein Precursors; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; C057400:picornain 2A, Picornavirus