Purification and characterization of poliovirus polypeptide 3CD, a proteinase and a precursor for RNA polymerase

A cDNA clone encoding the 3CD proteinase (3CDP"') of poliovirus type 2 (Sabin), the precursor to proteinase 3CPr ' and RNA polymerase 3DP"', was expressed in bacteria by using a T7 expression system. Site-specific mutagenesis of the 3C/3D cleavage site was performed to generate active proteolytic precursors impaired in their ability to process themselves to 3CPr ' and 3DP'. Of these mutations, the exchange of the Thr residue at the P4 position of the 3C/3D cleavage site for a Lys residue (3CDPr ' T181K) resulted in a mutant polypeptide exhibiting the smallest amount of autoprocessing. This mutant was purified to 86 % homogeneity and used for subsequent proteolytic studies. Purified 3CDPl°M (M designates the cleavage site mutant 3CDPr ' T181K) was capable of cleaving the P1 capsid precursor, a peptide representing the 2BC cleavage site, and the 2BC precursor polypeptide. Purified 3CDPmM demonstrated the same detergent sensitivity in processing experi-ments with the capsid precursor as was observed by using P1 and crude extracts of poliovirus-infected HeLa cell lysates. Purified 3CDP1°M did not have any detectable RNA polymerase activity, whereas 3D°"', separated from 3CDPIOM by gel filtration in the last step of purification, did. We conclude that 3CDP ° can process both structural and nonstructural precursors of the poliovirus polyprotein and that it is active against a synthetic peptide substrate. Moreover, cleavage of 3CD to 3D"° ' is needed to activate the 3D RNA polymerase. One of the most intriguing phenomena in picornaviru