Functionally, LMP2A has been shown to drive B cell development in vivo [6], thus mimicking the presence of a functional BCR [7]. Another example is provided by the Kaposi’s sarcoma-associated herpesvirus (KSHV) K1 protein. This transmembrane protein also contains a functional ITAM that mediates interactions with a large cohort of signalling proteins, including Syk, Lyn, Vav1, PLC-c2 and PI3K [8–11]. Although many studies have addressed the function of these proteins [12], due to the restricted host range tropism of the human gammaherpesviruses, their role in vivo has not been fully demonstrated. Therefore, we still lack studies that can correlate the biochemical and biological properties of these viral proteins with the different biological aspects involved in the pathogenesis of those viruses in their host cells. A suitable model to circumvent this problem is the use of murine gammaherpesvirus 68 (MHV-68), because this pathogen can infect laboratory mice [13,14]. Despite this difference in tropism, MHV-68 can establish long term latent infection in memory B cells upon amplification of viral episomes in GC B cells [15–19]. During the establishment of latency by MHV-68 a selective number of viral-encoded proteins are expressed that are predicted to orchestrate biological programs in the host B cell [20]. One of these proteins is M2, a 192 amino acid-long polypeptide that bears no sequence similarity to any other known protein [21]. One of the functions of M2 is to induce the phosphorylation and activation of Vav1 and Vav2 in a BCR-independent, but Src family-dependent manner [22]. Vav1 and Vav2 work as phosphorylation-dependent guanosine nucleotide exchange factors (GEFs) for members of the Rho/Rac family [23], a group of GTPases involved in the regulation of cytoskeletal, mitogenic, and antigenic responses [24]. The activation of Vav proteins by M2 requires the formation of a trimeric complex between these two proteins and the tyrosine kinase Fyn [22], a process mediated by the recognition of a M2 proline rich region (PRR) by the SH3 domains of both Vav and Fyn proteins [22]. Interestingly, we have previously shown that the formation of this complex results also in the phosphorylation of M2 on a region that contains two tyrosine residues (Tyr 120 and Tyr 129) [22]. These results suggested that in addition to protein interaction events, the function of M2 could be modulated by phosphorylation-based signals. However, they could not exclude the possibility that this phosphorylation was a bystander defect derived from the close physical proximity of M2 and Fyn in the heteromolecular complex. To investigate the possible implication of phosphorylation- dependent effects in the functional cycle of M2, we performed in this work experiments aimed at identifying the specific residues that were phosphorylated by Fyn on M2. In addition, we investigated the functional capabilities of M2 mutant proteins lacking different combinations of these two putative phosphory- lation sites at the biochemical and signalling level. To obtain additional information about the regulatory properties of M2 in vivo, we also compared the pathogenicity of M2 mutant viruses with disrupted phosphorylation sites, with wild type MHV-68 and with recombinant viruses bearing previously reported inactive M2 mutant proteins, such as an M2 frame shift mutant [17] and a PRR-mutant version that cannot trigger the stimulation of the Fyn/Vav1/Rac1 pathway [22]. Interestingly, these experiments demonstrated that the phosphorylation of M2 on the tyrosine 120 is critical for the optimal assembly of the M2/Fyn/Vav1 complex and, perhaps more importantly, for the activation of the latency program of MHV-68 in infected mice. They also provided evidence suggesting that the action of M2 in that pathogenic program involves the manipulation of additional signalling molecules within B-cells. Taken together, these results reveal additional mechanistic aspects of the cross-talk established among M2 Phosphorylation and Latency M2 and elements of the B-cell signaling machinery that are crucial for the orchestration of specific biological programs linked to gammaherpesvirus latency in the host B-cell. Results Phosphorylation of M2 on Tyr120 is required for Vav1 activation We have previously shown that a M2 mutant protein (M2Y) containing two missense mutations on tyrosine residues 120 and 129 (the amino acid residue positions used in this study for M2 refer to the final spliced form of this protein, [25]) could not be phosphorylated by Fyn in vivo or in vitro [22], indicating that these residues were the actual Fyn targets in M2. To identify the exact acceptor site of the phosphate group, we generated two M2 mutant proteins in which either the Y120 (M2Y120F mutant) or the Y129 (M2Y129F mutant) residues were mutated to phenylalanine. To monitor the phosphorylation of these mutants, these proteins were expressed in COS1 cells in the presence of Myc-tagged Fyn, immunoprecipitated, and subjected to immunoblot analysis with anti-phosphotyrosine (PTyr) antibodies. As a control, we used COS1 expressing either the wild type or the previously described M2Y mutant version of M2 [22]. These experiments revealed a graded effect of the mutations in the total levels of M2 phosphorylation. Thus, the M2Y129F mutant displayed only marginal defects on tyrosine phosphorylation when compared to its wild type counter- part (Fig. 1A, upper panel), indicating that this site is not the main target of Fyn. In contrast, the phosphorylation of M2 was drastically reduced and totally abolished in the case of the M2Y120F and the M2Y mutant versions, respectively (Fig. 1A, upper panel). These results indicate that the main phosphorylation site of M2 is Tyr120. To investigate the effect of the Y to F mutations in the function of M2, we used the above anti-M2 immunoprecipitates to monitor the association of this protein with Fyn. Using anti-Myc immunoblots, we observed that Fyn could co-immunoprecipitate at similar levels with both wild type M2 and the M2Y129F mutant protein. In contrast, its association with M2 was severely reduced in the case of the M2Y120F mutant and totally abolished when co- expressed with the M2Y mutant (Fig. 1A, second panel from top). As previously described [22], Fyn was also tyrosine phosphorylated in this complex as demonstrated by the detection of phospho-Fyn in the anti-M2 immunoprecipitates derived from cells expressing either the wild type or the M2Y129F versions (Fig. 1A, upper panel). These results suggest that Y120 is involved in the physical interaction of M2 with Fyn in vivo. We examined next the role of these two M2 phosphorylation sites in the coassembly of Fyn and Vav1 and the subsequent phosphorylation of Vav1. To this end, we co-transfected COS1 cells with Vav1, Fyn and wild type M2 or M2 tyrosine mutants in the indicated combinations, and examined Vav1 immunoprecip- itates for the presence of Fyn and M2 proteins (Fig. 1B). While wild type M2 and the M2Y129F mutant co-immunoprecipitated equally well with Vav1, the interaction of both M2Y and M2Y120F mutants with this GEF was significantly reduced although, unlike the case of the interaction of Fyn with M2Y, not completely abolished (Fig. 1B, top panel). Likewise, Vav1 associated optimally with Fyn when co-expressed with either wild type M2 or the M2Y129F mutant. Instead, this complex was partially or totally lost when M2 was replaced by the M2Y120F and M2Y mutants in the transfections, respectively (Fig. 1B, second panel from top). In agreement with these results, in vitro kinase assays indicated that the optimal phosphorylation of Vav1 by Fyn could be only triggered by wild type M2 and M2Y129F proteins but not by the rest of Y to F M2 mutants used in the study (Fig. 1C). Taken together, our results indicate that Y120 is the PLoS ONE | www.plosone.org 2 February 2008 | Volume 3 | Issue 2 | e1654