The Gammaherpesvirus m2 Protein Manipulates the Fyn/ Vav Pathway through a Multidocking Mechanism of Assembly Marta Pires de Miranda1,2., Marta Alenquer1,2., Sofia Marques1,2, Le´ nia Rodrigues1,2, Filipa Lopes1,2, Xose´ R. Bustelo3*, J. Pedro Simas1,2* 1 Instituto de Microbiologia e Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal, 2 Instituto Gulbenkian de Cieˆncia, Oeiras, Portugal, 3 Centro de Investigacio´ n del Ca´ncer and Instituto de Biolog´ıa Molecular y Celular del Ca´ncer, Consejo Superior de Investigaciones Cient´ıficas (CSIC), University of Salamanca, Salamanca, Spain Abstract To establish latent infections in B-cells, gammaherpesviruses express proteins in the infected B-cells of the host that spuriously activate signalling pathways located downstream of the B-cell receptor. One such protein is M2, a murine gammaherpesvirus 68-encoded molecule that activates the Vav1/Rac1 pathway via the formation of trimolecular complexes with Scr family members. Previous reports have shown that the formation of this heteromolecular complex involves interactions between a proline rich region of M2 and the Vav1 and Fyn SH3 domains. Here, we show that the optimal association of these proteins requires a second structural motif encompassing two tyrosine residues (Tyr120 and 129). These residues are inducibly phosphorylated by Fyn in non-hematopoietic cells and constitutively phosphorylated in B-cells. We also demonstrate that the phosphorylation of Tyr120 creates specific docking sites for the SH2 domains of both Vav1 and Fyn, a condition sine qua non for the optimal association of these two signalling proteins in vivo. Interestingly, signaling experiments indicate that the expression of M2 in B-cells promotes the tyrosine phosphorylation of Vav1 and additional signaling proteins, a biological process that requires the integrity of both the M2 phosphotyrosine and proline rich region motifs. By infecting mice with viruses mutated in the m2 locus, we show that the integrity of each of these two M2 docking motifs is essential for the early steps of murine gammaherpesvirus-68 latency. Taken together, these results indicate that the M2 phosphotyrosine motif and the previously described M2 proline rich region work in a concerted manner to manipulate the signaling machinery of the host B-cell. Citation: Pires de Miranda M, Alenquer M, Marques S, Rodrigues L, Lopes F, et al (2008) The Gammaherpesvirus m2 Protein Manipulates the Fyn/Vav Pathway through a Multidocking Mechanism of Assembly. PLoS ONE 3(2): e1654. doi:10.1371/journal.pone.0001654 Editor: Olivier Schwartz, Institut Pasteur, France Received November 5, 2007; Accepted January 28, 2008; Published February 27, 2008 Copyright: 2008 Pires de Miranda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant to J.P.S. from the Portuguese Fundac¸a˜o para a Cieˆncia e Tecnologia (POCI/SAU-IMI/57365/2004). X.R.B.’s work is supported by grants from the US National Cancer Institute/NIH (5R01-CA73735-11), the Spanish Ministry of Education and Science (MES) (SAF2006-01789), the Castilla-Leo´ n Autonomous Government (SA053A05), and the Red Tema´tica de Investigacio´ n Cooperativa en Ca´ncer (RTICC) (RD06/0020/0001, Fondo de Investigaciones Sanitarias (FIS), Carlos III Institute, Spanish Ministry of Health). All Spanish funding is co-sponsored by the European Union FEDER program. M.P.M., M.A., S.M., L.R. and F.L. were recipients of fellowships from the Fundac¸a˜o para a Cieˆncia e Tecnologia. Competing Interests: The authors have declared that no competing interests exist. *E-mail: xbustelo@usal.es (XB); psimas@fm.ul.pt (JS) .These authors contributed equally to this work. Introduction Gammaherpesviruses are amongst the most prevalent of human pathogens owing to their ability to establish lifelong persistent infections within their hosts [1]. Persistence is achieved through the establishment of latent infection in conventional memory B lymphocytes. To gain access to this cell type, gammaherpesviruses have developed molecular mechanisms that promote B cell activation in the absence of cognate antigen recognition [2]. Thus, host colonisation by gammaherpesvirus involves the modulation of signalling pathways triggered upon B cell receptor (BCR) activation. This strategy offers an obvious advantage to the virus since infection is not dependent on rare encounters with antigen specific naive B cells. In addition, virus driven proliferation of germinal centre (GC) B cells facilitates the amplification of viral episomes and the subsequent generation of a large pool of latent genomes in long lived memory B cells [3]. Signalling from the BCR complex is initiated when Src family kinases such as Fyn, Blk and Lyn induce the phosphorylation of immunoreceptor tyrosine- based activation motifs (ITAMs) located in the Iga and Igb cytoplasmic tails. Tyrosine-phosphorylated ITAMs then recruit the cytoplasmic Syk protein tyrosine kinase, leading to its membrane translocation and the subsequent trans-phosphoryla- tion of important B-cell signalling proteins such as Vav1, Vav2, phospholipase C-c2 (PLC-c2) and phosphatidylinositol-3 kinase (PI3K). In turn, these molecules promote the generation of a wide spectrum of intracellular signals and biological responses that are essential for the antigenic responses of B lymphocytes [4]. Several gammaherpesvirus proteins have been shown to modulate signal transduction pathways paralleled to those activated by BCR recognition of cognate antigen [2]. One such protein is latent membrane protein (LMP) 2A encoded by Epstein- Barr virus. This transmembrane protein contains two ITAM-like sequences that, upon phosphorylation, can interact with Lyn [5]. PLoS ONE | www.plosone.org 1 February 2008 | Volume 3 | Issue 2 | e1654