Protection of cap-dependent protein synthesis in vivo and in vitro with an eIF4G-1 variant highly resistant to cleavage by Coxsackievirus 2A protease

The shutoff of host protein synthesis by certain picornaviruses is mediated, at least in part, by proteolytic cleavage of eIF4G-1. Previously, we developed a cleavage site variant of eIF4G-1, termed eIF4G-1(SM), that was 100-fold more resistant to in vitro cleavage by Coxsackievirus 2A protease (2A(...

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Bibliographic Details
Published in:The Journal of biological chemistry 2003-02, Vol.278 (7), p.4449-4457
Main Authors: Zhao, Xiaohong, Lamphear, Barry J, Xiong, Dingding, Knowlton, Kirk, Rhoads, Robert E
Format: Article
Language:English
Subjects:
Coxsackievirus Infections - genetics
Coxsackievirus Infections - metabolism
Cysteine Endopeptidases - metabolism
Eukaryotic Initiation Factor-4G
Gene Expression Regulation
Green Fluorescent Proteins
HeLa Cells
Humans
Luminescent Proteins
Peptide Fragments - genetics
Peptide Fragments - metabolism
Peptide Initiation Factors - genetics
Peptide Initiation Factors - metabolism
Protein Biosynthesis
Proteins - genetics
Ribosomes - metabolism
Ribosomes - virology
Transfection
Viral Proteins
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Summary:The shutoff of host protein synthesis by certain picornaviruses is mediated, at least in part, by proteolytic cleavage of eIF4G-1. Previously, we developed a cleavage site variant of eIF4G-1, termed eIF4G-1(SM), that was 100-fold more resistant to in vitro cleavage by Coxsackievirus 2A protease (2A(Pro)) than wild-type eIF4G-1 (eIF4G-1(WT)), but it was still digested at high protease concentrations. Here we identified a secondary cleavage site upstream of the primary site. We changed Gly at the P1'-position of the secondary site to Ala to produce eIF4G-1(DM). eIF4G-1(DM) was 1,000-10,000-fold more resistant to cleavage in vitro than eIF4G-1(WT). Full functional activity of eIF4G-1(DM) was demonstrated in vitro by its ability to restore cap-dependent translation to a 2A(Pro)-pretreated rabbit reticulocyte system. An isoform containing the binding site for poly(A)-binding protein, eIF4G-1e(DM), was more active in this assay than an isoform lacking it, eIF4G-1a(DM), but only with polyadenylated mRNA. Functional activity was also demonstrated in vivo with stably transfected HeLa cells expressing eIF4G-1(DM) from a tetracycline-regulated promoter. Cap-dependent green fluorescent protein synthesis was drastically inhibited by 2A(Pro) expression, but synthesis was almost fully restored by induction of either eIF4G-1a(DM) or eIF4G-1e(DM). By contrast, encephalomyocarditis virus internal ribosome entry site-dependent green fluorescent protein synthesis was stimulated by 2A(Pro); stimulation was suppressed by eIF4G-1e(DM) but not eIF4G-1a(DM).
ISSN:0021-9258
DOI:10.1074/jbc.M212393200