Direct cleavage of elF4G by poliovirus 2A protease is inefficient in vitro.
Previously, the purified recombinant 2A proteases (2Apro) of coxsackievirus B4 (CVB4) and human rhinovirus type 2 (HRV2) were shown to cleave synthetic peptides derived from human or rabbit elF4G as well as elF4G protein purified from rabbit reticulocytes. These results were in contrast to previous evidence which supported the view that elF4G cleavage activity in poliovirus-infected HeLa cells required a cellular factor(s) activated by poliovirus (PV) 2Apro. In the present study, recombinant PV 2Apro was shown to cleave either rabbit or human elF4G or their derived peptides in direct cleavage reactions, but cleaved the 4G-derived peptides with 100-fold lower efficiency than with a peptide derived from the poliovirus polyprotein. In these experiments, up to 25-fold molar excess of 2Apro over elF4G protein was required to cause greater than 50% cleavage. CVB4 2Apro was also tested in peptide cleavage assays under the same conditions as PV 2Apro and was found to cleave all elF4G substrates with efficiencies similar to PV 2Apro. Finally, cleavage reactions utilizing recombinant elF4G containing a G486E substitution at the cleavage site for CVB4 and HRV2 proteases resulted in drastically reduced cleavage by PV 2Apro, similar to the reduction previously seen with HRV2 and CVB4 2Apro, confirming that all three viral 2A proteases recognize the same cleavage site on elF4G. These data show that PV 2Apro can directly cleave elF4G in vitro with efficiencies similar to those of CVB 2Apro, but cleavage efficiency of elF4G is approximately 1000-fold lower than cleavage of a peptide derived from the authentic 2A cleavage site on the poliovirus polyprotein.
Bovee
M L
ML
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Lamphear
B J
BJ
Rhoads
R E
RE
Lloyd
R E
RE
D000818:Animals; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D011051:Poliomyelitis / Q000821:virology*; D017955:Poliovirus / Q000502:physiology*; D011817:Rabbits; D014764:Viral Proteins; D014779:Virus Replication / Q000502:physiology*
D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D014764:Viral Proteins; C069883:initiation factor 4F component p220; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus