M2 Phosphorylation and Latency Figure 6. Modulation of Vav activity is required for normal kinetics of latency in GC B cells. Balb/c mice were intranasally infected with viruses of the indicated viral genotypes. Spleen sections were processed for in situ hybridization with miRNA riboprobes. (A) Representative spleen sections from each group of animals. Dark staining indicates cells positive for viral encoded miRNAs. All sections are magnified at 6200 and counter stained with haematoxylin. (B) Mean percentage 6 SEM of miRNA+ follicles (filled bars) and positive follicles with large clusters of miRNA-positive cells (open bars). Six sections per mouse and at least four mice per group were counted at each time point. Follicles were scored positive if they contained at least one positive miRNA cell and positive for expansion if they contained more than ten miRNA positive cells. doi:10.1371/journal.pone.0001654.g006 to wild type virus (Fig. 6B). In this case, maximal levels of GC infection were only observed at day 21 post-infection (Fig. 6A, panels h,i). These levels declined thereafter and reached values equivalent to those observed in the case of wild type and the vM2Y-R viruses (Fig. 6A, panels m,n). Taken together, these results indicate that the PRR and the phosphotyrosine sites of M2 work coordinately during infection in vivo, where they play essential roles in the M2 functions linked to the establishment of latency within GC B-cells. Furthermore, they suggest that the functions of M2 related to latency maintenance are probably due to other motifs of this molecule, raising the possibility that separate regions of the M2 molecule regulate different branches of the pathogenicity program of MHV-68. Discussion M2 is a MHV-68 encoded protein that modulates signalling pathways linked to the establishment of viral latency in B cells. M2 contributes to the latency program through the manipulation of key signal transduction pathways [22,35]. In order to do so, M2 PLoS ONE | www.plosone.org 8 February 2008 | Volume 3 | Issue 2 | e1654