To confirm that Vav1 binds to both phosphorylation sites and the PRR of M2, we next compared the association of M2 with wild type and mutant Vav1 proteins with inactivated SH2 and the C-terminal SH3 domains either in the absence or presence of Fyn. For these experiments, Vav1 N-terminal SH3 mutants were not analyzed since we have previously shown that M2 only binds to the C-terminal SH3 domain of Vav1 or Vav2 [22]. In the absence of Fyn, the inactivation of the C-terminal Vav1 SH3 domain markedly reduced the M2/Vav1 interaction (Fig. 4C, upper panel, interaction observed only after longer exposure times, data not shown), confirming our previous observations [22]. In contrast, the inactivation of the Vav1 SH2 domain affected only marginally the association of Vav1 with M2 under these conditions (Fig. 4C, upper panel). This is consistent with the low phosphorylation levels of M2 in the absence of Fyn (see above, Fig. 1C, lower panel). As expected [22], co-expression of Fyn enhanced the interaction between M2 and Vav1 (Fig. 4C, upper panel). Under these conditions, a significant level of coimmunoprecipitation was observed between M2 and SH2 and C-terminal SH3 mutants of Vav1 (Fig. 4C, upper panel). Therefore, under conditions of optimal M2 tyrosine phosphorylation, these two domains of Vav1 could efficiently establish physical contacts with the viral protein. Consistent with previous results [22], we found that Fyn formed part of the M2/Vav1 complexes (Fig. 4C, second panel from top). This association could be detected regardless of the Vav1 protein used (Fig. 4C, second panel from top), indicating that the association between Vav1 and Fyn is made indirectly via the scaffolding action of M2. As expected, Fyn also associated with the phosphorylation sites and the PRR of M2, however, in this case binding through the SH2 domain had a stronger influence (Fig.4D, upper panel). Control immunoblots showed equivalent expression levels of M2, Fyn and Vav1 proteins in the appropriate samples (Fig. 4C,D). Taken together, these results indicate that Fyn and Vav1 use two independent docking sites to associate stably with M2. The phosphosites and PRR of M2 contribute to the establishment of latency in GC B cells but they are not responsible for the full complement of M2 functions In order to determine the functions of the identified phospho- sites and the PRR of M2 in physiological context of latency in vivo, we generated MHV-68 recombinant viruses in which the m2 gene was modified to contain the mutations in either the phosphorylation motif or the PRR (designated vM2P2, with proline residues at positions 158, 160, 163 and 167 mutated to alanine). Given that the Y120F M2 mutant still displayed some basal phosphorylation in some cell contexts (i.e., COS1 cells), we decided to use in these experiments a virus encoding the M2Y mutant (designated vM2Y, with tyrosine residues at positions 120 and 129 mutated to phenylalanine), a version of M2 that shows no detectable phosphorylation even under conditions of Fyn overex- pression (see above, Fig. 1A). Moreover, this mutant totally disrupts the interaction with Fyn and the phosphorylation of B-cell proteins (see above, Figs. 1A and 2), thus ensuring the total blockage of the signals emanating from this M2 region. The DNA structures of mutant viruses were verified by PCR, DNA sequencing and examination of restriction enzyme digestion profiles of Escherichia coli-derived BAC DNA and PCR of reconstituted virus DNA. The stability of the introduced mutations was further checked in viruses recovered from latently infected spleens confirming the retention of the engineered point mutations. Spliced M2 transcripts could be readily detected in RNA extracted from fibroblasts infected with each of the viruses, confirming that the mutations did not affect transcription of M2 M2 Phosphorylation and Latency (data not shown). These viruses were used to infect mice and analysed for the previously shown functions of M2 in latency. In addition, we used in this study a previously described M2 frame shift mutant (vM2FS) that does not express this protein [17]. By using this additional virus, we wanted to distinguish whether the phosphosites and the PRR mutants were responsible for either the totality or just a subset of the functions previously ascribed to M2 during B-cell infection [17,30–32]. To analyse the behaviour of these viral mutants during latency, we infected Balb/c mice with each of these viruses and monitored both the establishment and the long-term latency of MHV-68 in B cells. To this end, we subjected these animals to three independent, although comple- mentary biological assays: ex vivo reactivation assays to measure latent infection in total splenocytes, flow cytometry coupled to limiting dilution and real time PCR to quantify the frequency of viral DNA positive GC B cells in spleen, and in situ hybridization analysis to identify virally infected cells within the spleen. Using the former assay, we observed in the case of the wild type virus the expected peak of infection at day 14 post-inoculation, with latent infection subsiding thereafter to become undetectable at day 50 post-inoculation (Fig. 5) [33]. In contrast, the vM2Y and vM2P2 viruses showed a vM2FS-like pattern of infection during the establishment of latency. This was characterised by an approxi- mately 100-fold deficit of latent infection at day 14 post- inoculation and a subsequent increase in infectious centres by day 21 post-inoculation when compared with infections made with wild type viruses (Fig. 5). No preformed infectious virus could be detected by suspension assay of freeze-thawed spleen homogenates at any time point and for any virus analyzed, indicating that splenic infection was only latent. However, unlike the case of the vM2FS mutant virus, the vM2Y and vM2P2 viruses became undetectable during long-term latency (Fig. 5). A revertant virus (vM2Y-R) in which the M2Y locus was restored to wild type status did not show any defects in the course of infection, indicating that phenotypic changes observed with the M2Y mutation were intrinsic to this locus and not the consequence of mutations elsewhere in the viral genome (Fig. 5). These results indicate that the PRR and the phosphosites of M2 are responsible for engaging the cellular responses important for the establishment, but not maintenance, of latency. To assess whether the deficit in the establishment of latency exhibited by the M2 mutant viruses reflected a phenotype in GC B cells, we determined the frequency of GC B-cells harbouring viral genomes. In mice infected with vM2Y or vM2P2, the frequency of viral genome-positive GC B cells only peaked at day 21 post- infection (Table 1). This was in contrast with the frequencies of viral genome-positive GC B cells in mice infected with wild type virus and vM2Y-R, which reached maximal levels at day 14 post- infection (Table 1). After longer post-infection periods (day 50), only mice infected with vM2FS exhibited high frequencies of viral genome-positive B cells (Table 1). To verify that this infection profile was linked to the residency of the virus in GC B cells, we monitored the presence of each of those viruses by detecting transcripts corresponding to the MHV-68- derived microRNAs using in situ hybridization, an assay that allows the analysis of the expansion and cessation kinetics of latent infection within GCs [13,18,34]. Mice infected with either wild type or vM2Y-R viruses showed the expected pattern of infection (Fig. 6A,B) [18]. This pattern was characterized by the detection of large clusters of infected cells within GCs at day 14 post-infection that reflect cellular proliferation and, thereby, expansion of the latently infected cell pool (Fig. 6A, panels a,e). At day 21 post- infection, we observed sharp declines in the total number of infected GCs and in the number of GCs that were associated with PLoS ONE | www.plosone.org 6 February 2008 | Volume 3 | Issue 2 | e1654