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 2Apro 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 2Apro. We introduced single-amino-acid substitutions in the 2Apro-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 2Apro. This is consistent with the proposal that 2Apro is structurally related to a subclass of trypsinlike serine proteinases. Moreover, 2Apro 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 2Apro-catalyzed proteolytic cleavage.
Hellen
C U
CU
Department of Microbiology, State University of New York, Stony Brook 11794-8621.
Fäcke
M
M
Kräusslich
H G
HG
Lee
C K
CK
Wimmer
E
E
D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism*; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014158:Transcription, Genetic; D014162:Transfection
D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D011498:Protein Precursors; C069883:initiation factor 4F component p220; D010450:Endopeptidases