A novel interaction of pokeweed antiviral protein with translation initiation factors 4G and iso4G: a potential indirect mechanism to access viral RNAs.
Pokeweed antiviral protein (PAP) is a ribosome inactivating protein recognized primarily for its ability to depurinate the sarcin/ricin loop of the large rRNA. Studies have demonstrated that PAP also depurinates other RNA templates, such as Human immunodeficiency virus-1 RNA and Brome mosaic virus RNAs. However, the mechanism by which PAP accesses viral RNAs is not known. Considering that PAP was shown recently to bind the m(7)G of the cap structure, we speculated that PAP may interact with other factors involved in translation initiation. By far western analysis, we show that PAP binds specifically to eIF4G and eIFiso4G of wheat germ and analysis with truncation mutants of eIFiso4G indicates that a region of this protein, between amino acids 511 and 624, is required for PAP binding activity. The yeast two-hybrid system supports these results by showing reduced growth and alpha-galactosidase expression with truncation in this region of eIFiso4G. PAP binds m(7)GTP-Sepharose and this interaction does not diminish the binding of PAP to purified eIFiso4G, indicating that a complex can form among the cap structure, PAP and eIFiso4G. We incubated PAP with uncapped and non-polyadenylated transcripts containing a 3' translation enhancer sequence (TE) known to increase translation of the RNA in an eIF4F dependent manner. We show that in the presence of wheat germ lysate, PAP depurinates the uncapped and non-polyadenylated transcripts containing a functional wild-type 3'TE, but does not depurinate messages containing a non-functional mutant 3'TE. These results support our hypothesis that binding of PAP to eIF4G and eIFiso4G can provide a mechanism for PAP to access both uncapped and capped viral RNAs for depurination.
Department of Biology, York University Toronto, Ontario M3J 1P3, Canada.
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Hudak
D001665:Binding Sites; D039603:Eukaryotic Initiation Factor-4G / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008957:Models, Genetic; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D010442:Peptide Chain Initiation, Translational; D010940:Plant Proteins / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011687:Purines / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D054789:Ribosome Inactivating Proteins, Type 1; D017384:Sequence Deletion; D020798:Two-Hybrid System Techniques
D039603:Eukaryotic Initiation Factor-4G; D010940:Plant Proteins; D020033:Protein Isoforms; D011687:Purines; D012315:RNA Caps; D012367:RNA, Viral; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein