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Journal of Virology, April 2004, p. 3271-3278, Vol. 78, No. 7
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.7.3271-3278.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cleavage of Eukaryotic Translation Initiation Factor 4GII within Foot-and-Mouth Disease Virus-Infected Cells: Identification of the L-Protease Cleavage Site In Vitro

Alessandra Gradi,^1,2, Nicole Foeger,³ Rebecca Strong,¹ Yuri V. Svitkin,² Nahum Sonenberg,² Tim Skern,³ and Graham J. Belsham^1*

Institute for Animal Health, Pirbright, Woking, Surrey GU24 0NF, United Kingdom,¹ Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Division of Biochemistry, University of Vienna, A-1030 Vienna, Austria,³ Department of Biochemistry and McGill Cancer Center, McGill University, Montréal, Québec H3G 1Y6, Canada²

Received 17 July 2003/ Accepted 23 December 2003

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.

Present address: Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, 00158 Rome, Italy.

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