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

doi:10.1128/MCB.20.17.6259-6268.2000

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Molecular and Cellular Biology, September 2000, p. 6259-6268, Vol. 20, No. 17
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Tomás Aragón,¹ Susana de la Luna,¹^, Isabel Novoa,²^, Luis Carrasco,² Juan Ortín,¹ and Amelia Nieto¹^,^*

Centro Nacional de Biotecnología (CSIC)¹ and Centro de Biología Molecular (CSIC-UAM),² Universidad Autonoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain

Received 31 January 2000/Returned for modification 2 March 2000/Accepted 5 June 2000

Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes,like polyadenylation, splicing, and nucleocytoplasmic transportof cellular mRNAs. In addition, NS1 protein enhances the translationalrate of viral, but not cellular, mRNAs. To characterize this effect,we looked for targets of NS1 influenza virus protein among cellulartranslation factors. We found that NS1 coimmunoprecipitates witheukaryotic initiation factor 4GI (eIF4GI), the large subunit ofthe cap-binding complex eIF4F, either in influenza virus-infectedcells or in cells transfected with NS1 cDNA. Affinity chromatographystudies using a purified His-NS1 protein-containing matrix showedthat the fusion protein pulls down endogenous eIF4GI from COS-1cells and labeled eIF4GI translated in vitro, but not the eIF4Esubunit of the eIF4F factor. Similar in vitro binding experimentswith eIF4GI deletion mutants indicated that the NS1-binding domainof eIF4GI is located between residues 157 and 550, in a regionwhere no other component of the translational machinery is knownto interact. Moreover, using overlay assays and pull-down experiments,we showed that NS1 and eIF4GI proteins interact directly, in anRNA-independent manner. Mapping of the eIF4GI-binding domain inthe NS1 protein indicated that the first 113 N-terminal aminoacids of the protein, but not the first 81, are sufficient tobind eIF4GI. The first of these mutants has been previously shownto act as a translational enhancer, while the second is defectivein this activity. Collectively, these and previously publisheddata suggest a model where NS1 recruits eIF4GI specifically tothe 5' untranslated region (5' UTR) of the viral mRNA, allowingfor the preferential translation of the influenza virusmessengers.

^* Corresponding author. Mailing address: Centro Nacional de Biotecnología (CSIC), Universidad Autonoma de Madrid, Campus deCantoblanco, 28049 Madrid, Spain. Phone: 34 91 585 4533. Fax:34 91 585 4506. E-mail: anmartin{at}cnb.uam.es.

Present address: Centre de Medicina Medica i Molecular-IRO, Hospital Duran i Reynals, 08907 Barcelona,Spain.

Present address: Department of Biochemistry, New York University Medical Center, New York, NY10016.

Molecular and Cellular Biology, September 2000, p. 6259-6268, Vol. 20, No. 17
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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