Ephrin receptor A2 is a functional entry receptor for Epstein–Barr virus

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Epstein–Barr virus (EBV) is an oncogenic virus that infects more than 90% of the world’s population1. EBV predominantly infects human B cells and epithelial cells, which is initiated by fusion of the viral envelope with a host cellular membrane2. The mechanism of EBV entry into B cells has been well characterized3. However, the mechanism for epithelial cell entry remains elusive. Here, we show that the integrins αvβ5, αvβ6 and αvβ8 do not function as entry and fusion receptors for epithelial cells, whereas Ephrin receptor tyrosine kinase A2 (EphA2) functions well for both. EphA2 overexpression significantly increased EBV infection of HEK293 cells. Using a virus-free cell–cell fusion assay, we found that EphA2 dramatically promoted EBV but not herpes simplex virus (HSV) fusion with HEK293 cells. EphA2 silencing using small hairpin RNA (shRNA) or knockout by CRISPR–Cas9 blocked fusion with epithelial cells. This inhibitory effect was rescued by the expression of EphA2. Antibody against EphA2 blocked epithelial cell infection. Using label-free surface plasmon resonance binding studies, we confirmed that EphA2 but not EphA4 specifically bound to EBV gHgL and this interaction is through the EphA2 extracellular domain (ECD). The discovery of EphA2 as an EBV epithelial cell receptor has important implications for EBV pathogenesis and may uncover new potential targets that can be used for the development of novel intervention strategies.

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  1. 1.

    Longnecker, R., Kieff, E. & Cohen, J. I. Fields Virology 6th edn (Lippincott Williams & Wilkins, Philadelphia, 2013).

  2. 2.

    Connolly, S. A., Jackson, J. O., Jardetzky, T. S. & Longnecker, R. Fusing structure and function: a structural view of the herpesvirus entry machinery. Nat. Rev. Microbiol. 9, 369–381 (2011).

  3. 3.

    Sathiyamoorthy, K. et al. Assembly and architecture of the EBV B cell entry triggering complex. PLoS Pathog. 10, e1004309 (2014).

  4. 4.

    Epstein, M. A., Achong, B. G. & Barr, Y. M. Virus particles in cultured lymphoblasts from Burkitt’s lymphoma. Lancet 1, 702–703 (1964).

  5. 5.

    Fingeroth, J. D. et al. Epstein–Barr virus receptor of human B lymphocytes is the C3d receptor CR2. Proc. Natl Acad. Sci. USA 81, 4510–4514 (1984).

  6. 6.

    Wang, X., Kenyon, W. J., Li, Q., Mullberg, J. & Hutt-Fletcher, L. M. Epstein–Barr virus uses different complexes of glycoproteins gH and gL to infect B lymphocytes and epithelial cells. J. Virol. 72, 5552–5558 (1998).

  7. 7.

    Spriggs, M. K. et al. The extracellular domain of the Epstein–Barr virus BZLF2 protein binds the HLA-DR beta chain and inhibits antigen presentation. J. Virol. 70, 5557–5563 (1996).

  8. 8.

    Li, Q. et al. Epstein–Barr virus uses HLA class II as a cofactor for infection of B lymphocytes. J. Virol. 71, 4657–4662 (1997).

  9. 9.

    Mullen, M. M., Haan, K. M., Longnecker, R. & Jardetzky, T. S. Structure of the Epstein–Barr virus gp42 protein bound to the MHC class II receptor HLA-DR1. Mol. Cell 9, 375–385 (2002).

  10. 10.

    Backovic, M., Longnecker, R. & Jardetzky, T. S. Structure of a trimeric variant of the Epstein–Barr virus glycoprotein B. Proc. Natl Acad. Sci. USA 106, 2880–2885 (2009).

  11. 11.

    Kirschner, A. N., Sorem, J., Longnecker, R. & Jardetzky, T. S. Structure of Epstein–Barr virus glycoprotein 42 suggests a mechanism for triggering receptor-activated virus entry. Structure 17, 223–233 (2009).

  12. 12.

    Matsuura, H., Kirschner, A. N., Longnecker, R. & Jardetzky, T. S. Crystal structure of the Epstein–Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) complex. Proc. Natl Acad. Sci. USA 107, 22641–22646 (2010).

  13. 13.

    Sathiyamoorthy, K. et al. Structural basis for Epstein–Barr virus host cell tropism mediated by gp42 and gHgL entry glycoproteins. Nat. Commun. 7, 13557 (2016).

  14. 14.

    Chesnokova, L. S., Nishimura, S. L. & Hutt-Fletcher, L. M. Fusion of epithelial cells by Epstein–Barr virus proteins is triggered by binding of viral glycoproteins gHgL to integrins αvβ6 or αvβ8. Proc. Natl Acad. Sci. USA 106, 20464–20469 (2009).

  15. 15.

    Chesnokova, L. S. & Hutt-Fletcher, L. M. Fusion of Epstein–Barr virus with epithelial cells can be triggered by αvβ5 in addition to αvβ6 and αvβ8, and integrin binding triggers a conformational change in glycoproteins gHgL. J. Virol. 85, 13214–13223 (2011).

  16. 16.

    Molesworth, S. J., Lake, C. M., Borza, C. M., Turk, S. M. & Hutt-Fletcher, L. M. Epstein–Barr virus gH is essential for penetration of B cells but also plays a role in attachment of virus to epithelial cells. J. Virol. 74, 6324–6332 (2000).

  17. 17.

    Chen, J., Rowe, C. L., Jardetzky, T. S. & Longnecker, R. The KGD motif of Epstein–Barr virus gH/gL is bifunctional, orchestrating infection of B cells and epithelial cells. mBio 3, e00290–11 (2012).

  18. 18.

    Wang, X. & Hutt-Fletcher, L. M. Epstein–Barr virus lacking glycoprotein gp42 can bind to B cells but is not able to infect. J. Virol. 72, 158–163 (1998).

  19. 19.

    Chen, J., Jardetzky, T. S. & Longnecker, R. The large groove found in the gH/gL structure is an important functional domain for Epstein–Barr virus fusion. J. Virol. 87, 3620–3627 (2013).

  20. 20.

    Borza, C. M. & Hutt-Fletcher, L. M. Epstein–Barr virus recombinant lacking expression of glycoprotein gp150 infects B cells normally but is enhanced for infection of epithelial cells. J. Virol. 72, 7577–7582 (1998).

  21. 21.

    Kasahara, Y. & Yachie, A. Cell type specific infection of Epstein–Barr virus (EBV) in EBV-associated hemophagocytic lymphohistiocytosis and chronic active EBV infection. Crit. Rev. Oncol. Hematol. 44, 283–294 (2002).

  22. 22.

    Boshoff, C. Ephrin receptor: a door to KSHV infection. Nat. Med. 18, 861–863 (2012).

  23. 23.

    Hahn, A. S. et al. The ephrin receptor tyrosine kinase A2 is a cellular receptor for Kaposi’s sarcoma-associated herpesvirus. Nat. Med. 18, 961–966 (2012).

  24. 24.

    Kania, A. & Klein, R. Mechanisms of ephrin-Eph signalling in development, physiology and disease. Nat. Rev. Mol. Cell Biol. 17, 240–256 (2016).

  25. 25.

    Shao, Z., Zhang, W. F., Chen, X. M. & Shang, Z. J. Expression of EphA2 and VEGF in squamous cell carcinoma of the tongue: correlation with the angiogenesis and clinical outcome. Oral Oncol. 44, 1110–1117 (2008).

  26. 26.

    Shao, Z. et al. EphA2/ephrinA1 mRNA expression and protein production in adenoid cystic carcinoma of salivary gland. J. Oral Maxil. Surg. 71, 869–878 (2013).

  27. 27.

    Wang, D. H. et al. Geldanamycin mediates the apoptosis of gastric carcinoma cells through inhibition of EphA2 protein expression. Oncol. Rep. 32, 2429–2436 (2014).

  28. 28.

    Nakamura, R. et al. EPHA2/EFNA1 expression in human gastric cancer. Cancer Sci. 96, 42–47 (2005).

  29. 29.

    Speck, P. & Longnecker, R. Epstein–Barr virus (EBV) infection visualized by EGFP expression demonstrates dependence on known mediators of EBV entry. Arch. Virol. 144, 1123–1137 (1999).

  30. 30.

    Hahn, A. S. & Desrosiers, R. C. Binding of the Kaposi’s sarcoma-associated herpesvirus to the ephrin binding surface of the EphA2 receptor and its inhibition by a small molecule. J. Virol. 88, 8724–8734 (2014).

  31. 31.

    Kumar, B. & Chandran, B. KSHV entry and trafficking in target cells-hijacking of cell signal pathways, actin and membrane dynamics. Viruses 8, E305 (2016).

  32. 32.

    Dutta, D. et al. EphrinA2 regulates clathrin mediated KSHV endocytosis in fibroblast cells by coordinating integrin-associated signaling and c-Cbl directed polyubiquitination. PLoS Pathog. 9, e1003510 (2013).

  33. 33.

    Kirschner, A. N., Lowrey, A. S., Longnecker, R. & Jardetzky, T. S. Binding-site interactions between Epstein–Barr virus fusion proteins gp42 and gH/gL reveal a peptide that inhibits both epithelial and B-cell membrane fusion. J. Virol. 81, 9216–9229 (2007).

  34. 34.

    Hahn, A. S. & Desrosiers, R. C. Rhesus monkey rhadinovirus uses eph family receptors for entry into B cells and endothelial cells but not fibroblasts. PLoS Pathog. 9, e1003360 (2013).

  35. 35.

    Tugizov, S. M., Berline, J. W. & Palefsky, J. M. Epstein–Barr virus infection of polarized tongue and nasopharyngeal epithelial cells. Nat. Med. 9, 307–314 (2003).

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The authors acknowledge help and advice from members of the Longnecker and Jardetzky laboratories, especially N. Susmarski. The authors thank M. Lingen and T. Li from the Human Tissue Resource Center (HTRC) at the University of Chicago for performing experiments during the revision, and M. Manzano for providing reagents. The research was supported by AI076183 (to R.L. and T.J.) from the National Institute of Allergy and Infectious Diseases, by CA117794 (to R.L. and T.J.) from the National Cancer Institute, by AI119480 (to R.L., H.Z. and T.J.) from the National Institute of Allergy and Infectious Diseases as well as by the Chicago Biomedical Consortium (Fall 14-0121 and 15-0257, to J.C.).

Author information


  1. Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    • Jia Chen
    • , Samantha Schaller
    •  & Richard Longnecker
  2. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA

    • Karthik Sathiyamoorthy
    •  & Theodore S. Jardetzky
  3. Department of Pharmacology, University of Illinois at Chicago College of Medicine, Chicago, IL, USA

    • Xianming Zhang
  4. Department of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    • Bethany E. Perez White


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J.C. and R.L. designed the overall study, with input from the co-authors. J.C. performed the key experiments. K.S. performed the initial microarray analysis, cloning gH and gL expression plasmids, purified protein preparations and carried out EphA2 binding experiments. X.Z. performed the RNA-seq analysis and helped with the sgRNA constructs and statistical analysis. S.S. helped with cell cultures. B.E.P.W. contributed key reagents. J.C. and R.L. wrote the manuscript. X.Z., K.S. and T.S.J. contributed expertise and helped write the paper. All authors analysed the results, read and approved the manuscript for submission.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Richard Longnecker.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–6, Supplementary Table 5.

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    Candidate receptor genes identified by comparing GeneRNA seq data from HEK293 and B cells (HEK293>5 FPKM, B< 5 FPKM, HEK293/B >10, membrane protein).

  4. Supplementary Table 2

    Candidate receptor genes identified by comparing GeneRNA seq data from HEK293 and B cells (HEK293>5 FPKM, B< 5 FPKM, HEK293/B >10, membrane protein, AGS/HEK293>2).

  5. Supplementary Table 3

    Candidate receptor genes identified by comparing GeneRNA seq data from AGS and B cells (AGS>5 FPKM, B< 5 FPKM, AGS/B >10, membrane protein).

  6. Supplementary Table 4

    Candidate receptor genes identified by comparing RNA seq from AGS and B cells (AGS>5 FPKM, B< 5 FPKM, AGS/B >10, membrane protein, AGS/HEK293>2).