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 alte rs several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, N S1 protein enhances the translational rate of viral, but not cellular, mRNA s. To characterize this effect, we looked for targets of NS1 influenza viru s protein among cellular translation factors. We found that NS1 coimmunopre cipitates 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 usi ng a purified His-NS1 protein-containing matrix shelved that the fusion pro tein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI trans lated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in v itro binding experiments with eIF4GI deletion mutants indicated that the NS 1-binding domain of eIF4GI is located between residues 157 and 550, in a re gion where no other component of the translational machinery is known to in teract. Moreover, using overlay assays and pull-down experiments, we shelve d that NS1 and eIF4GI proteins interact directly, in an RNA-independent man ner. 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 previou sly shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data sugges t a model where NS1 recruits eIF4GI specifically to the 5' untranslated reg ion (5' UTR) of the viral mRNA, allowing for the preferential translation o f the influenza virus messengers.