EBV membrane protein LMP2A interactions with ubiquitin ligases and a signaling scaffold

Over 90% of the adult human population carries Epstein-Barr Virus (EBV). EBV stays for life, striking a balance with the immune system. The virus evades immune elimination by severely restricting its own gene expression. The latent infection is established with the resting memory type B-cell as the reservoir. In the majority of infected individuals worldwide the coexistence with EBV is harmless. The dependence on a functioning immune system is evident from the long list of EBV-associated diseases that also feature accompanying immune dysfunction. An RNA message for the Latent Membrane Protein 2A (LMP2A) of EBV is constantly detected both in peripheral B-cells of healthy EBV carriers and in EBV-associated tumors. Elucidation of LMP2A interactions with the host cell will therefore contribute both to a better understanding of cellular signaling pathways, the regulation of EBV latency and to treatment of EBV-associated malignancies. Years of LMP2A research lead to the conclusion that LMP2A serves as a safeguard for the latency of EBV in the resting memory type B cells by interfering with and intercepting B-Cell Receptor (BCR) functions. In the normal resting B cell, BCR provides tonic signals to promote cell survival until the B-cell encounters its cognate antigen, upon which antigen-induced BCR signals will initiate differentiation to antibody-producing plasma cells. LMP2A sends surrogate tonic survival signals but interferes with the antigen-induced signals from the BCR. It does so by activating a key survival molecule in the cell, the serine-threonine kinase Akt and by down-regulating the Src-family tyrosine kinase Lyn, which is essential for antigen driven B-cell activation and differentiation. This knowledge was gained by using in-vitro cultured cells, biochemical and reverse genetic methods, as were the present studies, since the in-vivo cells that harbor the latent EBV infection are so scarce that direct studies can not be performed. Studies of viral perturbation of cellular systems are relevant model systems for learning about the normal functions of the cell and for pointing to vulnerable functions that are targeted also in non-viral disease, as in cancer. In the present thesis I provide evidence that: 1. Activation of LMP2A by constitutive tyrosine phosphorylation requires clustering of LMP2A molecules in the raft compartments of cell membranes (Paper I). 2. Src-family kinases, normally associated with the BCR, are labeled for degradation by E3 ubiquitin ligases of the Nedd4/AIP4 HECT-domain family, which are bound to LMP2A (Paper II); 3. LMP2A interacts with the Shb signaling scaffold, which can mediate Akt activation (Paper III). LIST OF PUBLICATIONS: I. Matskova, L. , Ernberg, I. and Pawson, T. and Winberg, G. 2001. The C-terminal domain of the Epstein-Barr virus LMP2A membrane protein contains a clustering signal. J. Virol. 75:10941-10949. II. Winberg G, Matskova L. , Chen F, Plant P, Rotin D, Gish G, Ingham R, Ernberg I, Pawson T. 2000. Latent membrane protein 2A of Epstein-Barr virus binds WW domain E3 protein-ubiquitin ligases that ubiquitinate B-cell tyrosine kinases. Mol Cell Biol. 20(22):8526-8235. III. Matskova, L.V. , Ingham, R. , Gish, G. , Lindholm, C.K. , Pawson, T. , Ernberg, I and Winberg, G. 2004. The Shb signalling scaffold interacts with Nedd4/AIP4 HECT domain E3 ubiquitin ligases and mediates Akt activation by interaction with the Epstein-Barr virus LMP2A membrane protein. Submitted. ASSOCIATED PUBLICATIONS: I. Ingham, R.J., Raaijmakers, J., Lim, C.S. H. , Mbamalu, G. , Gish, G. , Chen, F. , Matskova, L. , Ernberg, I. , Winberg, G. , and Pawson. T. 2004. The Epstein-Barr virus protein, LMP2A, co-opts tyrosine kinases used by the T cell receptor. Submitted. II. Kis, L.L. , Nishikawa, J., Takahara, M. , Nagy, N. , Matskova, L. , Takada, K. , Elmberger, G. , Ohlsson, A. , Klein, G and Klein, E. 2004. The in vitro EBV-infected subline of KMH2, derived from Hodgkin lymphoma, expresses only EBNA-1; CD40ligand and IL-4 induces LMP1 but not EBNA-2. Oncogene In press. TABLE OF CONTENTS