Cleavage of eukaryotic translation initiation factor 4GII within foot-and-mouth disease virus-infected cells: identification of the L-protease cleavage site in vitro.
Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (L(pro)) alone, generating cleavage products similar to those induced by enterovirus and rhinovirus 2A protease (2A(pro)). By the use of a fusion protein containing residues 445 to 744 of human eIF4GII, it was demonstrated that the FMDV L(pro) specifically cleaves this protein between residues G700 and S701, immediately adjacent to the site (V699/G700) cleaved by rhinovirus 2A(pro) in vitro. The G700/S701 cleavage site does not correspond, by amino acid sequence alignment, to that cleaved in eIF4GI by the FMDV L(pro) in vitro. Knowledge of the cleavage sites and the three-dimensional structures of the FMDV L(pro) and rhinovirus 2A(pro) enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.
Gradi
Alessandra
A
Institute for Animal Health, Pirbright, Woking, Surrey GU24 0NF, United Kingdom. graham.belsham@bbsrc.ac.uk
Foeger
Nicole
N
Strong
Rebecca
R
Svitkin
Yuri V
YV
Sonenberg
Nahum
N
Skern
Tim
T
Belsham
Graham J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G; D029544:Foot-and-Mouth Disease Virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
C113920:EIF4G1 protein, human; C491300:EIF4G2 protein, human; D039603:Eukaryotic Initiation Factor-4G; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D010450:Endopeptidases; C088747:leader proteinase, foot-and-mouth disease virus