Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus.
Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix showed that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we showed that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5' untranslated region (5' UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.
Centro Nacional de Biotecnología (CSIC), Universidad Autonoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain.
T
T
Aragón
S
S
de la Luna
I
I
Novoa
L
L
Carrasco
J
J
Ortín
A
A
Nieto
D020121:5' Untranslated Regions; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D039603:Eukaryotic Initiation Factor-4G; D017353:Gene Deletion; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010957:Plasmids; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D039603:Eukaryotic Initiation Factor-4G; C085820:INS1 protein, influenza virus; D010448:Peptide Initiation Factors; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D012313:RNA