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The cleavage of poliovirus precursor polypeptides occurs at specific amino acid pairs that are recognized by viral proteinases. Most of the polio-specific cleavages occur at glutamine-glycine (Q-G) pairs that are recognized by the viral-encoded proteinase 3C (formerly called P3-7c). In order to carry out a defined molecular genetic study of the enzymatic activity of protein 3C, we have made cDNA clones of the poliovirus genome. The cDNA region corresponding to protein 3C was inserted into an inducible bacterial expression vector. This recombinant plasmid (called pIN-III-C3-7c) utilizes the bacterial lipoprotein promoter to direct the synthesis of a precursor polypeptide that contains the amino acid sequence of protein 3C as well as the amino- and carboxy-terminal Q-G cleavage signals. These signals have been previously shown to allow autocatalytic production of protein 3C in bacteria transformed with plasmid pIN-III-C3-7c. We have taken advantage of the autocatalytic cleavage of 3C in a bacterial expression system to study the effects of site-specific mutagenesis on its proteolytic activity. One mutation that we have introduced into the cDNA region encoding 3C is a single amino acid insertion near the carboxy-terminal Q-G cleavage site. The mutant recombinant plasmid (designated pIN-III-C3-mu 10) directs the synthesis of a bacterial-polio precursor polypeptide that is like the wild-type construct (pIN-III-C3-7c). However, unlike the wild-type precursor, the mutant precursor cannot undergo autocatalytic cleavage to generate the mature proteinase 3C. Rather, the precursor is able to carry out cleavage at the amino-terminal Q-G site but not at the carboxy-terminal site. Thus, we have generated an altered poliovirus proteinase that is still able to carry out at least part of its cleavage activities but is unable to be a suitable substrate for self-cleavage at its carboxy-terminal Q-G pair.

We used a synthetic double-stranded oligonucleotide to introduce amino acid substitutions into the proteinase 3C region of a poliovirus type 1 cDNA clone. The six different mutant viruses recovered exhibited a small-plaque phenotype when assayed on HeLa cells. Further investigation revealed that all the mutations (with the exception of one) yielded P3 region proteins that displayed altered mobility in sodium dodecyl sulfatepolyacrylamide gel electrophoresis. A conservative Val-* Ala change at amino acid 54 of the proteinase resulted in a virus that was deficient in the production of the mature viral RNA polymerase 3D. Although this mutant achieved less than one-half of the wild-type levels of RNA synthesis during the course of infection, it still grew to nearly wild-type titers.

Cross-species proteolytic processing was demonstrated by the 3C proteinases of human rhinovirus 14 and coxsackievirus B3 on poliovirus-specific polypeptide precursors. Chimeric picornavirus cDNA genomes were constructed in a T7 transcription vector in which the poliovirus 3C coding region was substituted with the corresponding allele from human rhinovirus 14 or coxsackievirus B3. In vitro translation and processing of the polypeptides encoded by the chimeric genomes demonstrated that the proteolytic processing of poliovirus P2 region (nonstructural) proteins could be functionally substituted by the heterologous proteinases. In contrast, the 3C proteinase activities expressed from the chimeric genomes were incapable of recognizing the poliovirus-specific processing sites within the capsid precursor. Since the amino acid sequences flanking and inclusive of the P2 region cleavage sites of the three viruses are not stringently conserved, these results provide evidence for the existence of common conformational determinants necessary for 3C-mediated processing.

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