Interaction of poliovirus polypeptide 3CDpro with the 5' and 3' termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding.
Poliovirus proteinase 3CDpro by itself is not an RNA-binding protein. Two cellular proteins have been purified from HeLa cells (p50 and p36) which interact with purified 3CDpro but only p36-3CDpro bind to the 5'-terminal 110 nucleotides of polioviral RNA genome, an RNA segment 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 factor EF-1 alpha, and p36 an N-terminal fragment thereof. p36, referred to as host factor, did not appear to interact with purified 3Cpro or 3Dpol. Significantly, the formation of a 3CDpro-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 cloverleaf. Competition experiments have demonstrated that the RNA-protein interactions are specific for the full-length cloverleaf. UV cross-linking studies were employed to examine the protein components of the cloverleaf ribonucleoproteins. RNA footprinting was used to determine the site on the cloverleaf where the viral and cellular factors bind. Finally, we have discovered that 3AB-3CDpro also interacts with the 3'-terminal sequence of poliovirus RNA. In contrast to the 5'-terminal cloverleaf, the 3'-terminal RNA can bind 3AB in the absence of other proteins. A model for initiation of poliovirus RNA synthesis is presented.
Harris
K S
KS
Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York, Stony Brook 11794-5222.
Xiang
W
W
Alexander
L
L
Lane
W S
WS
Paul
A V
AV
Wimmer
E
E
D000086042:3C Viral Proteases; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017955:Poliovirus / Q000254:growth & development / Q000378:metabolism*; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D017421:Sequence Analysis; D014758:Viral Core Proteins / Q000378:metabolism; D014764:Viral Proteins; D014779:Virus Replication
D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases