phosphorylation of B-cell proteins upon expression of the wild type M2 protein. These results suggest that if M2 undergoes different levels of phosphorylation in these two residues during the MHV- 68 infection, this information could be computed by the B-cell to trigger intracellular signals of different intensity and/or to stimulate distinct, signal-dependent biological programs. This possibility would add further plasticity for the regulation of the MHV-68 pathogenic program in the host cell. In this regard, it will be interesting to generate phosphospecific antibodies to M2 in the future to monitor the phosphorylation kinetics of M2 during the different phases of MHV-68 latency in vivo. The importance of these two M2 docking motifs for MHV-68 latency is further strengthened by the data derived from the infection of mice with MHV-68 mutant viruses harbouring different M2 mutants. This genetic strategy has confirmed in vivo the important functional role of both the phosphotyrosine and the PRR motifs for the establishment of MHV-68 latency in B cells. Moreover, the observation that the phenotypes obtained by the M2Y and M2P2 proteins are identical, further strengthens the idea regarding the coordinated action of these two docking motifs during the function of M2 in the host cell. Interestingly, the use of this genetic strategy also revealed that the infection of mice with vM2Y and vM2P2 viruses does not recapitulate the overall biological program of the M2 protein during MHV-68 latency. Thus, in contrast to what is observed with a M2 frame shift mutant virus, we have observed that the infection of animals with those viruses does not result in the induction of persistent uncontrolled proliferation of infected GC B cells. These results indicate that other regions of M2 mediate this latter stage of the MHV-68 pathogenic program. Whether this is due to additional, intrinsic functions of M2 or to indirect causes, i.e. clearance of the virus by the immune system, remains to be determined. Collectively, our present data indicates that M2 functions as a multidocking protein that promotes the spurious, BCR-indepen- dent activation of the Vav1/Rac1 pathway and other intracellular routes. Furthermore, we have demonstrated genetically that the intracellular effects triggered by the concerted action of the phosphotyrosine and PRR M2 motifs are essential for the establishment of MHV-68 latency in GC B-cells but not for the subsequent maintenance of long term latency, suggesting the possibility that M2 could trigger distinct signalling branches that contribute independently to different stages of the MHV-68 pathogenic program. Finally, the observation that M2Y and M2P2 disrupt MHV-68 latency raise the prospect of using phosphopep- tides and/or PRR-containing peptides to block latency in vivo or, alternatively, to use in pharmacological approaches to block the activity of specific signalling pathways in B lymphocyte-based diseases. Further studies linking the biochemical properties of M2 with the pathogenesis of MHV-68 should provide valuable insights into the physiological role of this pathway for gammaherpesvirus host colonization. Materials and Methods Plasmids pCMV-Myc constructs, encoding wild type M2, M2Y and M2P2 mutants, and pcDNA3-Vav1 expression plasmid encoding wild type mouse Vav1, have been described previously [22,39]. pCMV-Myc-Fyn, encoding wild type mouse Fyn was generated by subcloning the Fyn cDNA sequences from the corresponding pCMV-HA expression construct [22]. pCMV-Myc constructs encoding Y120F, Y129F, Y120D, Y129D and Y120,129D (2YD) M2 mutants, and pCDNA3 encoding Vav1 with an inactivating amino acid substitution (R696A) in the SH2 domain [40] or encoding Fyn with an inactivating amino acid substitution M2 Phosphorylation and Latency (R176K) in the SH2 [41,42] or (W119L) in the SH3 domains [43], were generated by site directed mutagenesis using a Stratagene Quickchange kit according to the manufacturer’s instructions. A pCDNA3 encoding Vav1 with an inactivating amino acid substitution (P833L) in the most C-terminal SH3 domain has been described [44]. A pGEX-2T construct encoding the SH2 domain of Vav1 has been described [45]. The sequence encoding the SH2 domain of mouse Fyn (residues 145–247) was amplified by PCR and cloned into pGEX-6P-1 expression vector (GE Healthcare). All constructs were verified by DNA sequencing. Antibodies and fusion proteins A rabbit polyclonal antibody to M2 has been described before [22]. Other antibodies used in this work included phosphospecific antibodies to Vav1 phospho-Y174 [46], a monoclonal antibody against the c-Myc epitope (Invitrogen/Clontech), rabbit polyclonal antibodies to Vav1 and Fyn (Santa Cruz), an anti-phosphotyrosine monoclonal antibody (PY99, Santa Cruz) and a goat polyclonal antibody to glutathione S-transferase (GE Healthcare). GST and GST-SH2 fusion proteins were produced in Escherichia coli by IPTG (isopropyl-B-D-thiogalactopyranoside) induction and puri- fied with glutathione-sepharose beads using standard procedures. Cells and transfections COS1 and NIH3T3 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat inactivat- ed foetal bovine serum, 2 mM glutamine and 100 U/ml penicillin and streptamicin. Baby hamster kidney cells (BHK-21) were cultured in Glasgow’s modified Eagle’s medium supplemented as described above plus 10% tryptose phosphate broth. For immunoprecipitation and pull down assays, COS1 cells were transfected with 2–4 mg of the indicated plasmids using the DEAE- dextran method. A20 B cells were propagated in RPMI 1640 medium supplemented with 10% heat inactivated foetal bovine serum, 2 mM glutamine and 100 U/ml penicillin and streptami- cin. For transfection, 26107 A20 cells were electroporated (270 V, 500 mF) with 15 mg of plasmid DNA using a Bio-Rad gene pulser and incubated for 24 h in supplemented RPMI. Immunoprecipitation and in vitro kinase assays Transfected COS1 or A20 B cells were rinsed twice in ice-cold PBS and disrupted with ice-cold lysis buffer containing 10 mM Tris-HCl pH7.4, 100 mM NaCl, 1mM NaF, 1 mM orthovana- date, 0.5% NP-40 and Cwmplete protease inhibitors. Lysates were clarified by centrifugation and incubated with 1 mg of antibodies to either M2 or Vav1 or Fyn for 2 h at 4uC. A20 B cell lysates were precleared with protein-G conjugated Sepharose beads (GE Healthcare), prior to incubation with anti-M2 serum. Immune- complexes were recovered by incubation with protein G- conjugated Sepharose beads for 45 min at 4uC. After 3 washes with ice-cold lysis buffer, proteins were eluted in reducing Laemmli’s buffer, resolved by SDS-PAGE, transferred to nitro- cellulose and immunoblotted with the indicated antibodies. For in vitro kinase assays, Vav1 immunoprecipitates were obtained as described above, washed twice in lysis buffer, and then subjected to an in vitro kinase assay as previously described [22]. Proteins were eluted in reduced Laemmli’s buffer and resolved by SDS- PAGE. Gels were fixed, dried and subjected to autoradiography. Pull-down experiments N-terminal biotinylated, tyrosine phosphorylated (pY) peptides corresponding to a region of M2 incorporating Y120 (SPEENI- pYETANSE) and Y129 (ANSEPVpYIQPIST) were purchased PLoS ONE | www.plosone.org 10 February 2008 | Volume 3 | Issue 2 | e1654