The ephrin receptor tyrosine kinase A2 is a cellular receptor for Kaposi's sarcoma–associated herpesvirus

Abstract

Kaposi's sarcoma–associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma1, a highly vascularized tumor originating from lymphatic endothelial cells, and of at least two different B cell malignancies2,3. A dimeric complex formed by the envelope glycoproteins H and L (gH-gL) is required for entry of herpesviruses into host cells4. We show that the ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH-gL. EphA2 co-precipitated with both gH-gL and KSHV virions. Infection of human epithelial cells with a GFP-expressing recombinant KSHV strain, as measured by FACS analysis, was increased upon overexpression of EphA2. Antibodies against EphA2 and siRNAs directed against EphA2 inhibited infection of endothelial cells. Pretreatment of KSHV with soluble EphA2 resulted in inhibition of KSHV infection by up to 90%. This marked reduction of KSHV infection was seen with all the different epithelial and endothelial cells used in this study. Similarly, pretreating epithelial or endothelial cells with the soluble EphA2 ligand ephrinA4 impaired KSHV infection. Deletion of the gene encoding EphA2 essentially abolished KSHV infection of mouse endothelial cells. Binding of gH-gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry5,6. Quantitative RT-PCR and in situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from human Kaposi's sarcoma lesions or unaffected human lymphatic endothelium, and in situ in Kaposi's sarcoma specimens, respectively. Taken together, our results identify EphA2, a tyrosine kinase with known functions in neovascularization and oncogenesis, as an entry receptor for KSHV.

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Figure 1: Interaction of EphA2 with KSHV gH-gL.
Figure 2: Analysis of KSHV susceptibility and EphA2 expression in cell culture.
Figure 3: Phosphorylation and endocytosis of EphA2 in response to KSHV and gH-gL.
Figure 4: EphA2 and LANA-1 expression in KS.

References

  1. 1

    Chang, Y. et al. Identification of herpesvirus-like DNA sequences in AIDS- associated Kaposi's sarcoma. Science 266, 1865–1869 (1994).

    CAS  Article  Google Scholar 

  2. 2

    Cesarman, E., Chang, Y., Moore, P.S., Said, J.W. & Knowles, D.M. Kaposi's sarcoma–associated herpesvirus-like DNA sequences in AIDS-related body-cavity–based lymphomas. N. Engl. J. Med. 332, 1186–1191 (1995).

    CAS  Article  PubMed  Google Scholar 

  3. 3

    Soulier, J. et al. Kaposi's sarcoma–associated herpesvirus-like DNA sequences in multicentric Castleman's disease. Blood 86, 1276–1280 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Campadelli-Fiume, G. et al. The multipartite system that mediates entry of herpes simplex virus into the cell. Rev. Med. Virol. 17, 313–326 (2007).

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Akula, S.M. et al. Kaposi's sarcoma–associated herpesvirus (human herpesvirus 8) infection of human fibroblast cells occurs through endocytosis. J. Virol. 77, 7978–7990 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6

    Raghu, H., Sharma-Walia, N., Veettil, M.V., Sadagopan, S. & Chandran, B. Kaposi's sarcoma-associated herpesvirus utilizes an actin polymerization–dependent macropinocytic pathway to enter human dermal microvascular endothelial and human umbilical vein endothelial cells. J. Virol. 83, 4895–4911 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7

    Chandran, B. Early events in Kaposi's sarcoma-associated herpesvirus infection of target cells. J. Virol. 84, 2188–2199 (2010).

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Birkmann, A. et al. Cell surface heparan sulfate is a receptor for human herpesvirus 8 and interacts with envelope glycoprotein K8.1. J. Virol. 75, 11583–11593 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9

    Wang, F.Z., Akula, S.M., Pramod, N.P., Zeng, L. & Chandran, B. Human herpesvirus 8 envelope glycoprotein K8.1A interaction with the target cells involves heparan sulfate. J. Virol. 75, 7517–7527 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10

    Akula, S.M., Pramod, N.P., Wang, F.Z. & Chandran, B. Human herpesvirus 8 envelope-associated glycoprotein B interacts with heparan sulfate–like moieties. Virology 284, 235–249 (2001).

    CAS  Article  PubMed  Google Scholar 

  11. 11

    Hahn, A. et al. Kaposi's sarcoma-associated herpesvirus gH/gL: Glycoprotein export and interaction with cellular receptors. J. Virol. 83, 396–407 (2009).

    CAS  Article  PubMed  Google Scholar 

  12. 12

    Rappocciolo, G. et al. DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages. J. Immunol. 176, 1741–1749 (2006).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Sharma-Walia, N., Naranatt, P.P., Krishnan, H.H., Zeng, L. & Chandran, B. Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 envelope glycoprotein gB induces the integrin-dependent focal adhesion kinase-Src-phosphatidylinositol 3-kinase-Rho GTPase signal pathways and cytoskeletal rearrangements. J. Virol. 78, 4207–4223 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14

    Naranatt, P.P., Akula, S.M., Zien, C.A., Krishnan, H.H. & Chandran, B. Kaposi's sarcoma–associated herpesvirus induces the phosphatidylinositol 3-kinase–PKC-ζ–MEK-ERK signaling pathway in target cells early during infection: implications for infectivity. J. Virol. 77, 1524–1539 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15

    Kaleeba, J.A.R. & Berger, E.A. Kaposi's sarcoma-associated herpesvirus fusion-entry receptor: cystine transporter xCT. Science 311, 1921–1924 (2006).

    CAS  Article  PubMed  Google Scholar 

  16. 16

    Pertel, P.E. Human herpesvirus 8 glycoprotein B (gB), gH, and gL can mediate cell fusion. J. Virol. 76, 4390–4400 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17

    Banfield, B.W., Leduc, Y., Esford, L., Schubert, K. & Tufaro, F. Sequential isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective agent: evidence for a proteoglycan-independent virus entry pathway. J. Virol. 69, 3290–3298 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Zhu, L., Puri, V. & Chandran, B. Characterization of human herpesvirus-8 K8.1A/B glycoproteins by monoclonal antibodies. Virology 262, 237–249 (1999).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Brantley-Sieders, D.M. et al. EphA2 receptor tyrosine kinase regulates endothelial cell migration and vascular assembly through phosphoinositide 3-kinase-mediated Rac1 GTPase activation. J. Cell Sci. 117, 2037–2049 (2004).

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Vieira, J. & O'Hearn, P.M. Use of the red fluorescent protein as a marker of Kaposi's sarcoma–associated herpesvirus lytic gene expression. Virology 325, 225–240 (2004).

    CAS  Article  Google Scholar 

  21. 21

    Stürzl, M. et al. Identification of interleukin-1 and platelet-derived growth factor-B as major mitogens for the spindle cells of Kaposi's sarcoma: a combined in vitro and in vivo analysis. Oncogene 10, 2007–2016 (1995).

    PubMed  Google Scholar 

  22. 22

    Albini, A. et al. The beta-core fragment of human chorionic gonadotrophin inhibits growth of Kaposi's sarcoma–derived cells and a new immortalized Kaposi's sarcoma cell line. AIDS 11, 713–721 (1997).

    CAS  Article  Google Scholar 

  23. 23

    Herndier, B.G. et al. Characterization of a human Kaposi's sarcoma cell line that induces angiogenic tumors in animals. AIDS 8, 575–581 (1994).

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Kaleeba, J.A.R. & Berger, E.A. Broad target cell selectivity of Kaposi's sarcoma–associated herpesvirus glycoprotein-mediated cell fusion and virion entry. Virology 354, 7–14 (2006).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Lupberger, J. et al. EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Nat. Med. 17, 589–595 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26

    Vleck, S.E. et al. Structure−function analysis of varicella-zoster virus glycoprotein H identifies domain-specific roles for fusion and skin tropism. Proc. Natl. Acad. Sci. USA 108, 18412–18417 (2011).

    CAS  Article  PubMed  Google Scholar 

  27. 27

    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).

    CAS  Article  PubMed  Google Scholar 

  28. 28

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29

    Vanarsdall, A.L., Chase, M.C. & Johnson, D.C. Human cytomegalovirus glycoprotein gO complexes with gH-gL, promoting interference with viral entry into human fibroblasts but not entry into epithelial cells. J. Virol. 85, 11638–11645 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30

    Ryckman, B.J., Chase, M.C. & Johnson, D.C. HCMV gH/gL/UL128–131 interferes with virus entry into epithelial cells: evidence for cell type-specific receptors. Proc. Natl. Acad. Sci. USA 105, 14118–14123 (2008).

    CAS  Article  PubMed  Google Scholar 

  31. 31

    Chowdary, T.K. et al. Crystal structure of the conserved herpesvirus fusion regulator complex gH-gL. Nat. Struct. Mol. Biol. 17, 882–888 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32

    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).

    CAS  Article  PubMed  Google Scholar 

  33. 33

    Walker-Daniels, J., Riese, D.J. & Kinch, M.S. c-Cbl–dependent EphA2 protein degradation is induced by ligand binding. Mol. Cancer Res. 1, 79–87 (2002).

    CAS  PubMed  Google Scholar 

  34. 34

    Zhuang, G., Hunter, S., Hwang, Y. & Chen, J. Regulation of EphA2 receptor endocytosis by SHIP2 lipid phosphatase via phosphatidylinositol 3-kinase-dependent Rac1 activation. J. Biol. Chem. 282, 2683–2694 (2007).

    CAS  Article  PubMed  Google Scholar 

  35. 35

    Wang, H.W. et al. Kaposi sarcoma herpesvirus–induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma. Nat. Genet. 36, 687–693 (2004).

    CAS  Article  PubMed  Google Scholar 

  36. 36

    Ogawa, K. et al. The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization. Oncogene 19, 6043–6052 (2000).

    CAS  Article  Google Scholar 

  37. 37

    Scehnet, J.S. et al. The role of Ephs, ephrins and growth factors in Kaposi sarcoma and implications of ephrinB2 blockade. Blood 113, 254–263 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38

    Cheng, N. et al. Blockade of EphA receptor tyrosine kinase activation inhibits vascular endothelial cell growth factor–induced angiogenesis. Mol. Cancer Res. 1, 2–11 (2002).

    CAS  Article  PubMed  Google Scholar 

  39. 39

    Brantley-Sieders, D.M. et al. EphA2 receptor tyrosine kinase regulates endothelial cell migration and vascular assembly through phosphoinositide 3-kinase-mediated Rac1 GTPase activation. J. Cell Sci. 117, 2037–2049 (2004).

    CAS  Article  PubMed  Google Scholar 

  40. 40

    Herndier, B.G. et al. Characterization of a human Kaposi's sarcoma cell line that induces angiogenic tumors in animals. AIDS 8, 575–581 (1994).

    CAS  Article  PubMed  Google Scholar 

  41. 41

    McAllister, R.M., Melnyk, J., Finkelstein, J.Z., Adams, E.C. Jr. & Gardner, M.B. Cultivation in vitro of cells derived from a human rhabdomyosarcoma. Cancer 24, 520–526 (1969).

    CAS  Article  PubMed  Google Scholar 

  42. 42

    Knowles, B.B., Howe, C.C. & Aden, D.P. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209, 497–499 (1980).

    CAS  Article  PubMed  Google Scholar 

  43. 43

    Stürzl, M. et al. Identification of interleukin-1 and platelet-derived growth factor-B as major mitogens for the spindle cells of Kaposi's sarcoma: a combined in vitro and in vivo analysis. Oncogene 10, 2007–2016 (1995).

    PubMed  Google Scholar 

  44. 44

    Vieira, J. & O'Hearn, P.M. Use of the red fluorescent protein as a marker of Kaposi's sarcoma–associated herpesvirus lytic gene expression. Virology 325, 225–240 (2004).

    CAS  Article  Google Scholar 

  45. 45

    Myoung, J. & Ganem, D. Generation of a doxycycline-inducible KSHV producer cell line of endothelial origin: Maintenance of tight latency with efficient reactivation upon induction. J. Virol. Methods 174, 12–21 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  46. 46

    Wickramasinghe, S.R., Kalbfuss, B., Zimmermann, A., Thom, V. & Reichl, U. Tangential flow microfiltration and ultrafiltration for human influenza A virus concentration and purification. Biotechnol. Bioeng. 92, 199–208 (2005).

    CAS  Article  PubMed  Google Scholar 

  47. 47

    Garrigues, H.J., Rubinchikova, Y.E., DiPersio, C.M. & Rose, T.M. Integrin αV3 Binds to the RGD motif of glycoprotein B of Kaposi's sarcoma–associated herpesvirus and functions as an RGD-dependent entry receptor. J. Virol. 82, 1570–1580 (2008).

    CAS  Article  PubMed  Google Scholar 

  48. 48

    Hahn, A. et al. Kaposi's sarcoma-associated herpesvirus gH-gL: glycoprotein export and interaction with cellular receptors. J. Virol. 83, 396–407 (2009).

    CAS  Article  PubMed  Google Scholar 

  49. 49

    Ensser, A., Pfinder, A., Muller-Fleckenstein, I. & Fleckenstein, B. The URNA genes of herpesvirus saimiri (strain C488) are dispensable for transformation of human T cells in vitro. J. Virol. 73, 10551–10555 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Glykofrydes, D. et al. Herpesvirus saimiri vFLIP provides an antiapoptotic function but is not essential for viral replication, transformation, or pathogenicity. J. Virol. 74, 11919–11927 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. 51

    Birkmann, A. et al. Cell surface heparan sulfate is a receptor for human herpesvirus 8 and interacts with envelope glycoprotein K8.1. J. Virol. 75, 11583–11593 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. 52

    König, S. et al. Sodium dodecyl sulfate versus acid-labile surfactant gel electrophoresis: comparative proteomic studies on rat retina and mouse brain. Electrophoresis 24, 751–756 (2003).

    Article  PubMed  Google Scholar 

  53. 53

    Schmidt, K., Wies, E. & Neipel, F. Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor 3 inhibits gamma interferon and major histocompatibility complex class II expression. J. Virol. 85, 4530–4537 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54

    Naschberger, E. et al. Angiostatic immune reaction in colorectal carcinoma: impact on survival and perspectives for antiangiogenic therapy. Int. J. Cancer 123, 2120–2129 (2008).

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the Akademie der Wissenschaften und der Literatur (Mainz), the European Community research project TargetHerpes, Deutsche Forschungsgemeinschaft center grant 643 and graduate school grant 1071 “viruses of the immune system.” M. Stürzl, E.N. and F.N. were supported by the interdisciplinary center for clinical research at the University Hospital of the University of Erlangen. M. Stürzl and E.N. were supported by the German Cancer Aid. We thank L. Wall for experimental assistance and R.C. Desrosiers for helpful discussions and critical reading of the manuscript. KSImm cells were kindly provided by A. Albini (Science and Technology Pole, IRCCS MultiMedica). 4G10 mouse hybridoma supernatant was kindly provided by B. Biesinger (Virologisches Institut, Universitätsklinikum Erlangen).

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A.S.H. and F.N. designed the study. A.S.H., J.K.K. and E.W. performed the key experiments. J.P.-I., K.S., M. Schmidt and A.H. performed real-time PCR experiments, cell culture and infection assays. S.K. performed mass spectrometry and analyzed the data. J.C., A.E., J.M. and N.H.B. contributed key reagents. E.N. and M. Stürzl helped with endothelial cell cultures and performed immunohistochemistry experiments. A.S.H. and F.N. wrote the manuscript. B.F. contributed expertise and helped write the paper.

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Correspondence to Frank Neipel.

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A. Hahn filed a patent to the United States Patent and Trademark Office involving the inhibition of EphA2 and KSHV infection with a small molecule.

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Hahn, A., Kaufmann, J., Wies, E. et al. The ephrin receptor tyrosine kinase A2 is a cellular receptor for Kaposi's sarcoma–associated herpesvirus. Nat Med 18, 961–966 (2012). https://doi.org/10.1038/nm.2805

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