Processing determinants required for in vitro cleavage of the poliovirus P1 precursor to capsid proteins

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Abstract
We generated defined alterations in poliovirus protein-processing substrates and assayed the effects of these alterations with an in vitro expression system. A complete cDNA copy of the poliovirus genome was inserted into a bacteriophage T7 transcription vector. Using this expression template, we produced RNA transcripts containing defined regions of the poliovirus capsid precursor polypeptide (P1) and RNA transcripts containing mutations in the P1 and P2 regions. In vitro translation of P1-derived transcripts allowed us to characterize the 3C-mediated cleavage of P1 to capsid proteins. We demonstrated that, for either posttranslational or cotranslational cleavage at any of the Q-G amino acid pairs within P1, almost the entire P1 precursor is required. We also examined the sequences involved in 2A-mediated processing of the Y-G site at the P1-P2 junction. We demonstrated that minimal sequences 3'' to the 2A coding sequences are required to generate active 2A proteinase in vitro and that two specific four-amino-acid insertions in protein 2C do not alter 2A- or 3C-mediated processing of the poliovirus polyprotein. In addition, we demonstrated that substantial deletion of P1 sequences does not alter 2A-mediated cleavage of the Y-G site at the P1-P2 junction. These results allowed us to compare the P1 sequences required for 2A-versus 3C-mediated processing of the capsid precursor, and we discuss these results in the context of the three-dimensional structure of the capsid proteins.