LMP1 Protein from the Epstein-Barr Virus Is a Structural CD40 Decoy in B Lymphocytes for Binding to TRAF3*

Epstein-Barr virus is a human herpesvirus that causes infectious mononucleosis and lymphoproliferative malignancies. LMP1 (latent membrane protein-1), which is encoded by this virus and which is essential for transformation of B lymphocytes, acts as a constitutively active mimic of the tumor necrosis factor receptor (TNFR) CD40. LMP1 is an integral membrane protein containing six transmembrane segments and a cytoplasmic domain at the C terminus that binds to intracellular TNFR-associated factors (TRAFs). TRAFs are intracellular co-inducers of downstream signaling from CD40 and other TNFRs, and TRAF3 is required for activation of B lymphocytes by LMP1. Cytoplasmic C-terminal activation region 1 of LMP1 bears a motif (PQQAT) that conforms to the TRAF recognition motif PVQET in CD40. In this study, we report the crystal structure of this portion of LMP1 C-terminal activation region-1 (204PQQATDD210) bound in complex with TRAF3. The PQQAT motif is bound in the same binding crevice on TRAF3 where CD40 is bound, providing a molecular mechanism for LMP1 to act as a CD40 decoy for TRAF3. The LMP1 motif is presented in the TRAF3 crevice as a close structural mimic of the PVQET motif in CD40, and the intermolecular contacts are similar. However, the viral protein makes a unique contact: a hydrogen bond network formed between Asp210 in LMP1 and Tyr395 and Arg393 in TRAF3. This intermolecular contact is not made in the CD40-TRAF3 complex. The additional hydrogen bonds may stabilize the complex and strengthen the binding to permit LMP1 to compete with CD40 for binding to the TRAF3 crevice, influencing downstream signaling to B lymphocytes and contributing to dysregulated signaling by LMP1.

[1]  Shao-Cong Sun,et al.  An Atypical Tumor Necrosis Factor (TNF) Receptor-associated Factor-binding Motif of B Cell-activating Factor Belonging to the TNF Family (BAFF) Receptor Mediates Induction of the Noncanonical NF-κB Signaling Pathway* , 2005, Journal of Biological Chemistry.

[2]  C. Ni,et al.  Key Molecular Contacts Promote Recognition of the BAFF Receptor by TNF Receptor-Associated Factor 3: Implications for Intracellular Signaling Regulation1 , 2004, The Journal of Immunology.

[3]  G. Bishop,et al.  Roles of TNF Receptor-Associated Factor 3 in Signaling to B Lymphocytes by Carboxyl-Terminal Activating Regions 1 and 2 of the EBV-Encoded Oncoprotein Latent Membrane Protein 11 , 2004, The Journal of Immunology.

[4]  G. Bishop.  The multifaceted roles of TRAFs in the regulation of B-cell function , 2004, Nature Reviews Immunology.

[5]  G. Bishop,et al.  Requirement for TRAF3 in Signaling by LMP1 But Not CD40 in B Lymphocytes , 2004, The Journal of experimental medicine.

[6]  John C Reed,et al.  Structurally Distinct Recognition Motifs in Lymphotoxin-β Receptor and CD40 for Tumor Necrosis Factor Receptor-associated Factor (TRAF)-mediated Signaling* , 2003, Journal of Biological Chemistry.

[7]  G. Bishop,et al.  A Novel Interaction between Protein Kinase D and TNF Receptor-Associated Factor Molecules Regulates B Cell Receptor-CD40 Synergy1 , 2003, The Journal of Immunology.

[8]  C. Benedict,et al.  Death and survival: viral regulation of TNF signaling pathways. , 2003, Current opinion in immunology.

[9]  C. Benedict,et al.  To kill or be killed: viral evasion of apoptosis , 2002, Nature Immunology.

[10]  K. R. Ely,et al.  Structurally adaptive hot spots at a protein interaction interface on TRAF3 , 2002, Journal of molecular recognition : JMR.

[11]  Marnie L. Havert,et al.  Crystallization and preliminary X-ray analysis of the TRAF domain of TRAF3. , 2002, Acta crystallographica. Section D, Biological crystallography.

[12]  Hao Wu,et al.  Distinct molecular mechanism for initiating TRAF6 signalling , 2002, Nature.

[13]  G. Bishop,et al.  Molecular mechanisms of B-lymphocyte transformation by Epstein-Barr virus. , 2002, Microbes and infection.

[14]  G. Bishop,et al.  Mechanisms of TNF receptor‐associated factor (TRAF) regulation in B lymphocytes , 2002, Journal of leukocyte biology.

[15]  Marnie L. Havert,et al.  Downstream regulator TANK binds to the CD40 recognition site on TRAF3. , 2002, Structure.

[16]  Young Chul Park,et al.  All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction. , 2002, Journal of cell science.

[17]  L. Young,et al.  LMP1 structure and signal transduction. , 2001, Seminars in cancer biology.

[18]  G. Bishop,et al.  Multiple Carboxyl-Terminal Regions of the EBV Oncoprotein, Latent Membrane Protein 1, Cooperatively Regulate Signaling to B Lymphocytes Via TNF Receptor-Associated Factor (TRAF)-Dependent and TRAF-Independent Mechanisms1 , 2001, The Journal of Immunology.

[19]  K. Brown,et al.  Differential Signaling and Tumor Necrosis Factor Receptor–Associated Factor (Traf) Degradation Mediated by Cd40 and the Epstein-Barr Virus Oncoprotein Latent Membrane Protein 1 (Lmp1) , 2001, The Journal of experimental medicine.

[20]  J C Reed,et al.  Molecular basis for CD40 signaling mediated by TRAF3. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[21]  G. Mosialos,et al.  Epstein-Barr virus transformation: involvement of latent membrane protein 1-mediated activation of NF-κB , 1999, Oncogene.

[22]  N. Raab-Traub,et al.  Mimicry of CD40 signals by Epstein-Barr virus LMP1 in B lymphocyte responses. , 1999, Science.

[23]  E. Kieff,et al.  The structural basis for the recognition of diverse receptor sequences by TRAF2. , 1999, Molecular cell.

[24]  E. Kieff,et al.  The Epstein-Barr Virus Oncoprotein Latent Membrane Protein 1 Engages the Tumor Necrosis Factor Receptor-Associated Proteins TRADD and Receptor-Interacting Protein (RIP) but Does Not Induce Apoptosis or Require RIP for NF-κB Activation , 1999, Molecular and Cellular Biology.

[25]  J. Holton,et al.  Crystallographic analysis of CD40 recognition and signaling by human TRAF2. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[26]  G. Bishop,et al.  Cutting edge: contrasting roles of TNF receptor-associated factor 2 (TRAF2) and TRAF3 in CD40-activated B lymphocyte differentiation. , 1999, Journal of immunology.

[27]  Hao Wu,et al.  Structural basis for self-association and receptor recognition of human TRAF2 , 1999, Nature.

[28]  R. Finberg,et al.  U937 cells overexpressing bcl-xl are resistant to human immunodeficiency virus-1-induced apoptosis and human immunodeficiency virus-1 replication. , 1999, Virology.

[29]  G. Bishop,et al.  The EBV transforming protein, latent membrane protein 1, mimics and cooperates with CD40 signaling in B lymphocytes. , 1999, Journal of immunology.

[30]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[31]  Lyons Sf,et al.  The roles of human viruses in the pathogenesis of lymphoma. , 1998 .

[32]  W. Hammerschmidt,et al.  Epstein–Barr virus‐mediated B‐cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor , 1998, The EMBO journal.

[33]  E. Kieff,et al.  The Epstein–Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-κB , 1997 .

[34]  R. Noelle,et al.  Assembly and Regulation of the CD40 Receptor Complex in Human B Cells , 1997, The Journal of experimental medicine.

[35]  J. Russo,et al.  Kaposi's sarcoma-associated herpesvirus encodes a functional Bcl-2 homologue , 1997, Nature Medicine.

[36]  E. Cheng,et al.  A Bcl-2 homolog encoded by Kaposi sarcoma-associated virus, human herpesvirus 8, inhibits apoptosis but does not heterodimerize with Bax or Bak. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[37]  E. Kieff,et al.  Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-kappaB activation , 1996, Molecular and cellular biology.

[38]  G. Bishop,et al.  Different CD40-mediated signaling events require distinct CD40 structural features. , 1996, Journal of immunology.

[39]  D. Thorley-Lawson,et al.  Epstein-Barr virus and the B cell: that's all it takes. , 1996, Trends in microbiology.

[40]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[41]  M. Miura,et al.  Tumor necrosis factor-induced apoptosis is mediated by a CrmA-sensitive cell death pathway. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[42]  H. Gendelman,et al.  Tumor necrosis factor alpha-induced apoptosis in human neuronal cells: protection by the antioxidant N-acetylcysteine and the genes bcl-2 and crmA , 1995, Molecular and cellular biology.

[43]  M. Tewari,et al.  Fas- and Tumor Necrosis Factor-induced Apoptosis Is Inhibited by the Poxvirus crmA Gene Product (*) , 1995, The Journal of Biological Chemistry.

[44]  M. Rowe,et al.  The Epstein-Barr virus latent membrane protein-1 (LMP1) mediates activation of NF-kappa B and cell surface phenotype via two effector regions in its carboxy-terminal cytoplasmic domain. , 1995, Oncogene.

[45]  Jeffrey A. Ledbetter,et al.  How B and T cells talk to each other , 1994, Nature.

[46]  E. Kieff,et al.  Epstein-Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[47]  M. Rowe,et al.  Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[48]  D. Rock,et al.  An African swine fever virus gene with similarity to the proto-oncogene bcl-2 and the Epstein-Barr virus gene BHRF1 , 1993, Journal of virology.

[49]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[50]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[51]  E. Kieff,et al.  Orientation and patching of the latent infection membrane protein encoded by Epstein-Barr virus , 1986, Journal of virology.

[52]  Talapady N. Bhat,et al.  OMITMAP: An electron density map suitable for the examination of errors in a macromolecular model , 1984 .

[53]  W. Leiserson,et al.  Establishment of B cell hybridomas with B cell surface antigens. , 1982, Journal of immunology.

[54]  G. Mosialos,et al.  Cellular signaling pathways engaged by the Epstein-Barr virus transforming protein LMP1. , 2002, Frontiers in bioscience : a journal and virtual library.

[55]  G. Bishop,et al.  Signaling by CD40 and its mimics in B cell activation , 2001, Immunologic research.

[56]  J Bajorath,et al.  Immune regulation by CD40 and its ligand GP39. , 1996, Annual review of immunology.

[57]  J. Banchereau,et al.  The CD40 antigen and its ligand. , 1994, Annual review of immunology.