CHARACTERIZATION OF POLIOVIRUS-2A PROTEINASE BY MUTATIONAL ANALYSIS - RESIDUES REQUIRED FOR AUTOCATALYTIC ACTIVITY ARE ESSENTIAL FOR INDUCTION OF CLEAVAGE OF EUKARYOTIC INITIATION FACTOR-4F POLYPEPTIDE P220

The poliovirus proteinase 2A is autocatalytically released from the poliovirus polyprotein by cotranslational cleavage at its own amino terminus, resulting in separation of structural and nonstructural protein precursors. Cleavage is a prerequisite for further processing of the structural protein precursor and consequently for poliovirus encapsidation. A second function of 2A(pro) is in the rapid shutoff of host cell protein synthesis that occurs upon infection with poliovirus. This is associated with proteolytic cleavage of the p220 component of eukaryotic initiation factor eIF-4F, which is induced but not directly catalyzed by 2A(pro). We introduced single-amino-acid substitutions in the 2A(pro)-coding region of larger poliovirus precursors that were subsequently translated in vitro and thus demonstrated that His-20, Asp-38, and Cys-109 (which constitute the putative catalytic triad) are essential for, and that His-117 is an important determinant of, the autocatalytic activity of 2A(pro). This is consistent with the proposal that 2A(pro) is structurally related to a subclass of trypsinlike serine proteinases. Moreover, 2A(pro) containing a Cys109Ser substitution retained a small but significant autocatalytic activity. Cleavage of p220 was not induced by those mutants that had reduced proteolytic activity, indicating that the cellular factor that cleaves p220 is probably activated by 2A(pro)-catalyzed proteolytic cleavage.