Identification of essential amino acid residues in the functional activity of poliovirus 2A protease

Abstract

Proteolytic processing of poliovirus polyprotein is carried out by the products of two viral genes, 2A and 3C. 2A protease catalyzes cleavage of the polyprotein of type 1 poliovirus at two sites, one a cis cleavage at the 2A N-terminus and the other a trans cleavage within the 3D polymerase. In addition to polyprotein cleavage activity, 2A protease also indirectly induces cleavage of the p220 component of the cap-binding protein complex, which results in selective inhibition of host protein synthesis. Molecular genetic and biochemical analyses of 2A protease were performed to test its putative homology to small trypsin-like serine proteases and to examine the roles of individual amino acids in the reaction mechanism of 2A protease. A recombinant plasmid containing poliovirus 1 C, 1 D, and 2A gene sequences was expressed in a cell-free transcription/translation system, resulting in synthesis of a precursor protein that underwent efficient self-processing and produced mature 2A protease. To identify residues involved in the catalytic center and/or substrate-binding loops, we generated a series of 2A mutants by site-specific mutagenesis of this plasmid. Mutants were then expressed in vitro and tested for autocatalytic cis cleavage activity, trans cleavage of the 1 D/2A junction, and trans-activation of p220-specific protease. Our data suggest that the conserved His20, Asp38, and Cys109 residues recently proposed to be equivalent to the catalytic triad of known serine proteases may comprise the catalytic triad of 2A protease. Surprisingly, Asp38 could be replaced with glutamic acid and retain autocatalytic function. Other amino acid substitutions at Tyr88, Tyr89, and Thrl24 suggested that these residues lie in loops involved in substrate binding. Biochemical studies with protease inhibitors indicate that 2A protease activity is blocked by inhibitors specific for serine and cysteine proteases. Overall, the results are consistent with the hypothesis that 2A proteinase is structurally similar to the trypsin-like family of serine proteases with the substitution of cysteine 109 as the active site nucleophile.