title: Protection of cap-dependent protein synthesis in vivo and in vitro with an eIF4G-1 variant highly resistant to cleavage by Coxsackievirus 2A protease.
abstract: 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).
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932, USA.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Zhao
authors.forename: Barry J
authors.initials: BJ
authors.lastname: Lamphear
authors.forename: Dingding
authors.initials: D
authors.lastname: Xiong
authors.forename: Kirk
authors.initials: K
authors.lastname: Knowlton
authors.forename: Robert E
authors.initials: RE
authors.lastname: Rhoads
mesh_terms: D003384:Coxsackievirus Infections / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism / Q000821:virology; D014162:Transfection; D014764:Viral Proteins
chemical_list: C113920:EIF4G1 protein, human; D039603:Eukaryotic Initiation Factor-4G; D008164:Luminescent Proteins; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D011506:Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus