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An endocytic YXXΦ (YRRF) cargo sorting motif in the cytoplasmic tail of murine cytomegalovirus AP2 ‘adapter adapter’ protein m04/gp34 antagonizes virus evasion of natural killer cells

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Abstract

Viruses have evolved proteins that bind immunologically relevant cargo molecules at the cell surface for their downmodulation by internalization. Via a tyrosine-based sorting motif YXXΦ in their cytoplasmic tails, they link the bound cargo to the cellular adapter protein-2 (AP2), thereby sorting it into clathrin-triskelion-coated pits for accelerated endocytosis. Downmodulation of CD4 molecules by lentiviral protein NEF represents the most prominent example. Based on connecting cargo to cellular adapter molecules, such specialized viral proteins have been referred to as ‘connectors’ or ‘adapter adapters.’ Murine cytomegalovirus glycoprotein m04/gp34 binds stably to MHC class-I (MHC-I) molecules and suspiciously carries a canonical YXXΦ endocytosis motif YRRF in its cytoplasmic tail. Disconnection from AP2 by motif mutation ARRF should retain m04-MHC-I complexes at the cell surface and result in an enhanced silencing of natural killer (NK) cells, which recognize them via inhibitory receptors. We have tested this prediction with a recombinant virus in which the AP2 motif is selectively destroyed by point mutation Y248A, and compared this with the deletion of the complete protein in a Δm04 mutant. Phenotypes were antithetical in that loss of AP2-binding enhanced NK cell silencing, whereas absence of m04-MHC-I released them from silencing. We thus conclude that AP2-binding antagonizes NK cell silencing by enhancing endocytosis of the inhibitory ligand m04-MHC-I. Based on a screen for tyrosine-based endocytic motifs in cytoplasmic tail sequences, we propose here the new hypothesis that most proteins of the m02–m16 gene family serve as ‘adapter adapters,’ each selecting its specific cell surface cargo for clathrin-assisted internalization.

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Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft, SFB490, individual project E4 ‘antigen presentation under the influence of murine cytomegalovirus immune evasion proteins.’ NAWL received intramural funding from the young investigator program MAIFOR of the University Medical Center Mainz.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standard

All procedures performed in studies involving animals were approved by the ethics committee of the Landesuntersuchungsamt Rheinland-Pfalz, Permission No. 177-07/G09-1-004 in accordance with German Federal Law §8 Abs. 1 TierSchG (animal protection law). BALB/c mice were bred and housed under specified pathogen-free conditions at the Central Laboratory Animal Facility (CLAF) at the University Medical Center of the Johannes Gutenberg-University, Mainz, Germany.

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  1. Marina Babic Cac

    Present address: German Rheumatism Research Centre, Innate Immunity Group, Berlin, Germany

  2. Stefan Ebert

    Present address: Section for Experimental Tumor Immunology, Department for Obstetrics and Gynaecology, University of Wuerzburg Medical School, Würzburg, Germany

Authors and Affiliations

  1. Institute for Virology, University Medical Center of the Johannes Gutenberg-University Mainz and Research Center for Immunotherapy (FZI), Obere Zahlbacher Strasse 67, Hochhaus am Augustusplatz, 55131, Mainz, Germany

    Annette Fink, Franziska Blaum, Stefan Ebert, Niels A. W. Lemmermann & Matthias J. Reddehase

  2. Department of Histology and Embryology, School of Medicine, University of Rijeka, Rijeka, Croatia

    Marina Babic Cac

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  1. Annette Fink
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Correspondence to Annette Fink.

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This article is part of the Special Issue on Cytomegalovirus.

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Fink, A., Blaum, F., Babic Cac, M. et al. An endocytic YXXΦ (YRRF) cargo sorting motif in the cytoplasmic tail of murine cytomegalovirus AP2 ‘adapter adapter’ protein m04/gp34 antagonizes virus evasion of natural killer cells. Med Microbiol Immunol 204, 383–394 (2015). https://doi.org/10.1007/s00430-015-0414-1

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