Eukaryotic Translation Initiation Factor 4GI Is a Cellular Target for NS1 Protein, a Translational Activator of Influenza Virus

Abstract

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.

Large-scale analysis of influenza A virus sequences reveals potential drug target sites of non-structural proteins

“…Some of the highly conserved residues of the ED, which are buried inside, most probably fulfil a role in stabilizing the protein structure, such as Ala132, Leu144, Ala149, Ile160, Ala177 and Leu181. Residues 81-113 have been reported to interact with a translation initiation factor eIF4F (Aragó n et al, 2000). Based on the present analysis, we postulate that the conserved residues Tyr89, Ser99, Met104, Leu105, Pro107, Gln109 and Lys110 are involved in interaction with eIF4F, while other highly conserved residues in this region, such as Met93 and Trp102, stabilize the structure, since they are not as exposed to the surface as the previous residues.…”

“…The major role of NS1 is to antagonize the antiviral response of the host by preventing the activation of NF-kB and induction of alpha/betainterferon (IFN-a/b) (Wang et al, 2000). However, NS1 is a multifunctional protein that is additionally involved in several processes: (i) inhibiting the pre-mRNA 39-end processing (Fortes et al, 1994) by binding to two 39-end processing factors, namely cleavage and polyadenylation specificity factor and poly(A)-binding protein II (Chen et al, 1999;Nemeroff et al, 1998); (ii) blocking the post-transcriptional processing and nuclear export of cellular mRNA (Fortes et al, 1994); (iii) stimulating the translation of matrix (M1) proteins (Enami et al, 1994;Marió n et al, 1997); (iv) inhibiting the activation of a protein kinase that phosphorylates the elf-2 translation initiation factor by binding to doublestranded (ds)RNA (Lu et al, 1995;Aragó n et al, 2000); (v) induction of the phosphatidylinositol-3-kinase (PI3K)/Akt signalling pathway in order to support virus replication (Ehrhardt et al, 2006(Ehrhardt et al, , 2007. The NS1 protein is 230-237 aa, depending on the strain, and has a molecular mass of approximately 26 kDa (reviewed by Hale et al, 2008b).…”

Conducting the initiation of protein synthesis: the role of eIF4G

“…This interaction specifically favors translation of influenza virus transcripts by allowing selective recruitment and positioning of eIF4F on the 5' end of viral mRNAs (Aragon et al, 2000).…”

EIF3G is associated with narcolepsy across ethnicities

“…Interestingly, EIF3B:G:I also interacts directly with certain viral mRNAs to promote the translation of viral proteins. 18 EIF4G, which interacts with EIF3, 19 contains a binding domain for the non-structural influenza protein, NS1, 20 and is recruited to the viral mRNA to initiate translation of influenza virus. This is particularly interesting as recent findings have implicated a role of the H1N1 pandemic virus of 2009 as a trigger for narcolepsy development.…”