On the basis of sequence alignments and secondary structure comparisons of the first 100 nucleotides of enterovirus and rhinovirus RNAs, chimeric constructs in which this region of poliovirus type 1 Mahoney [PV1(M)] is replaced with that of human rhinovirus type 2 (HRV2) or HRV14 have been engineered. These chimeric constructs contain the internal ribosomal entry site of either poliovirus or encephalomyocarditis virus. Independent of the internal ribosomal entry site elements, only the constructs containing either the PV1(M) or HRV2 cloverleaf sequences yielded viable viruses. The secondary structures of all three cloverleaves are quite similar. However, highly purified polioviral proteins 3CD pro and 3AB together bound to the PV1(M) and HRV2 cloverleaves, albeit with different affinities, whereas the HRV14 homolog did not interact with these proteins to any appreciable extent. These results support a mechanism of poliovirus genomic replication in which the formation of a complex between the cloverleaf structure and the 3CD pro /3AB proteins of poliovirus plays an essential role.

Proteinase 3C of poliovirus type 2 (Sabin) was expressed at 4% total protein in Escherichia coli. The protein was soluble and could be purified by a simple scheme. It was weakly active on the capsid precursor P1 (expressed in vitro), which contains two cleavage sites. The products of processing of P1 were 1ABC and 1D (VP1). The activity was insensitive to Triton X-100. Crude extracts of cells infected with poliovirus type 1 (Mahoney) gave strong processing and yielded 1AB (VPO), 1C (VP3), and 1D in the same assay system but were sensitive to detergent. 3C from cell extracts that was separated from its precursors resembled the recombinant proteinase in its activity. Recombinant 3C cleaved the peptide dansyl-Glu-Glu-Glu-Ala-Met-Glu-Gln-Gly-Ile-Thr-Asn-Lys-NH2 at the Gln-Gly bond. We conclude that 3C is merely the core of the Gln-Gly-cleaving activity which processes P1 in vivo and that there is probably a hydrophobic contact between a larger 3C precursor and its P1 substrate which allows the second processing reaction: 1ABC, 1D-* 1AB, 1C, 1D.

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 experiments 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.