from Sigma. A non-phosphorylated version of the Y120 peptide was obtained by incubation with 50 U of YOP tyrosine phosphatase (New England Biolabs), for 30 min at 30uC. The enzyme was inactivated by the addition of 1 mM orthovanadate. For pull-down experiments, 10 mg of each peptide were incubated with clarified cell lysates or purified GST proteins overnight at 4uC. Peptide complexes were recovered with 20 ml of streptavidin-conjugated Sepharose beads (GE Healthcare) for 1 h at 4uC. After 3 washes in lysis buffer, proteins were eluted from the beads in reducing Laemmli’s buffer, resolved by SDS-PAGE, transferred to nitrocellulose and analysed by western blot with the indicated antibodies. Generation of recombinant viruses MHV-68 vM2Y (with tyrosine residues at positions 120 and 129 mutated to phenylalanines) and vM2P2 (with proline residues at positions 158, 160, 163 and 167 mutated to alanines) viruses were generated by mutagenesis of the viral genome cloned as a bacterial artificial chromosome (BAC) [47,48]. The following point mutations were introduced on the M2 gene by overlapping PCR using MHV-68 genomic DNA as a template: T4221, 4228A in vM2Y and G4108, 4120, 4129, 4135C in vM2P2. PCR products were inserted into the HindIII E MHV-68 fragment cloned in the pST76K-SR shuttle plasmid [17], using BlnI (nt 3908) and XhoI (nt 5361) restriction sites. The PCR-derived regions were sequenced to confirm the integrity of the mutations. Recombinant HindIII E shuttle plasmids were transformed into an Escherichia coli strain (DH10B) containing the wild type MHV-68 BAC (pHA3). Following a multi-step selection procedure, recombinant BAC clones were identified by DNA sequencing. To generate a vM2Y revertant virus (vM2Y-R), the wild type HindIII E pST76K-SR shuttle plasmid was transformed into DH10B cells containing the mutant BAC genome. All viruses were reconstituted by transfec- tion of BAC DNA into BHK-21 cells using FuGENE 6 (Roche Molecular Biochemicals). The loxP-flanked BAC cassette was removed by viral passage through NIH Cre 3T3 cells and limiting dilution to obtain GFP-negative viruses. Analysis of recombinant viruses Groups of 6- to 8-week old female BALB/c mice (Instituto Gulbenkian de Cieˆncia, Portugal) were inoculated intranasally with 104 p.f.u. in 20 ml of PBS under halothane anaesthesia. At 14, 21 or 50 days post-infection, spleens were removed and processed for subsequent analysis. Titres of infectious virus were determined by suspension assays of freeze-thawed spleen homogenates using BHK-21 cells. Latent virus load was quantified by using explant References 1. Rickinson AB, Kieff E (2007) Epstein-Barr Virus. In: Knipe DM, Howley P, eds. Fields Virology. Philadelphia: Lippincott-Raven. pp 2655–2670. 2. Damania B (2004) Oncogenic gamma-herpesviruses: comparison of viral proteins involved in tumorigenesis. Nat Rev Microbiol 2: 656–668. 3. Thorley-Lawson DA (2001) Epstein-Barr virus: exploiting the immune system. Nat Rev Immunol 1: 75–82. 4. Jumaa H, Hendriks RW, Reth M (2005) B cell signaling and tumorigenesis. Annu Rev Immunol 23: 415–445. 5. Fruehling S, Longnecker R (1997) The immunoreceptor tyrosine-based activation motif of Epstein-Barr virus LMP2A is essential for blocking BCR- mediated signal transduction. Virology 235: 241–251. 6. Caldwell RG, Wilson JB, Anderson SJ, Longnecker R (1998) Epstein-Barr virus LMP2A drives B cell development and survival in the absence of normal B cell receptor signals. Immunity 9: 405–411. 7. Merchant M, Swart R, Katzman RB, Ikeda M, Ikeda A, et al. (2001) The effects of the Epstein-Barr virus latent membrane protein 2A on B cell function. Int Rev Immunol 20: 805–835. 8. Lagunoff M, Majeti R, Weiss A, Ganem D (1999) Deregulated signal transduction by the K1 gene product of Kaposi’s sarcoma-associated herpesvirus. Proc Natl Acad Sci U S A 96: 5704–5709. M2 Phosphorylation and Latency cocultures of single-cell suspension splenocytes with BHK-21 cells. Plates were incubated for four (suspension assays) or five (coculture assays) days, then fixed with 10% formal saline and counterstained with toluidine blue. Viral plaques were counted with a microscope. The frequency of MHV-68 genome-positive GC B cells was determined by limiting dilution combined with real-time PCR, essentially as previously described [20]. GC B cells (B220+; PNAhi) were obtained from pools of five spleens using a BD FACSAria Flow Cytometer (BD Biosciences). The purity of sorted cells was always .98%. Real-time PCR was performed on a ABI Prism 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer’s instructions, using the fluorescent Taqman methodology. It is important pointing out that the change in the value range in these experiments when compared with previous published work using the Light Cycler apparatus from Roche Molecular Biochemicals, is due to the higher sensitivity of the real- time PCR methodology performed here. Noteworthy, that this change in sensitivity affects the total values but not the fold difference in the frequencies of viral DNA positive cells for wild type MHV-68 when compared to vM2FS. In fact, if we compare the fold changes in the present and previous manuscripts, they are actually very similar (compare current manuscript and [20]). Primer/probe sets used were specific for the MHV-68 M9 gene (59 primer: GCCACGGTGGCCCTCTA; 39 primer: CAGGCCTC- CCTCCCTTTG; probe: 6-FAM-CTTCTGTTGATCTTCC– MGB). Samples were subjected to a melting step of 95uC for 10 min followed by 40 cycles of 15 s at 95uC and 1 min at 60uC. In situ hybridization with a digoxigenin-labelled riboprobe encompassing both MHV-68 vtRNAs and microRNAs 1 to 4 was performed on formalin-fixed, paraffin-embedded spleen sections, as previously described [49]. Probes were generated by T7 transcription of a pEH1.4 using a commercial kit from Roche Molecular Biochemicals, according to the manufacturer’s instructions. Acknowledgments We thank Ma´rio Matos for histology processing and Ana Caetano for technical help with FACS. Author Contributions Conceived and designed the experiments: XB JS MP MA SM. Performed the experiments: MP MA LR. Analyzed the data: XB JS MP MA SM. Contributed reagents/materials/analysis tools: MP MA SM LR FL. Wrote the paper: XB JS. 9. Lee BS, Lee SH, Feng P, Chang H, Cho NH, et al. (2005) Characterization of the Kaposi’s sarcoma-associated herpesvirus K1 signalosome. J Virol 79: 12173–12184. 10. Lee H, Guo J, Li M, Choi JK, DeMaria M, et al. (1998) Identification of an immunoreceptor tyrosine-based activation motif of K1 transforming protein of Kaposi’s sarcoma-associated herpesvirus. Mol Cell Biol 18: 5219–5228. 11. Tomlinson CC, Damania B (2004) The K1 protein of Kaposi’s sarcoma-associated herpesvirus activates the Akt signaling pathway. J Virol 78: 1918–1927. 12. Brinkmann MM, Schulz TF (2006) Regulation of intracellular signalling by the terminal membrane proteins of members of the Gammaherpesvirinae. J Gen Virol 87: 1047–1074. 13. Simas JP, Efstathiou S (1998) Murine gammaherpesvirus 68: a model for the study of gammaherpesvirus pathogenesis. Trends Microbiol 6: 276–282. 14. Stevenson PG, Efstathiou S (2005) Immune mechanisms in murine gamma- herpesvirus-68 infection. Viral Immunol 18: 445–456. 15. Flano E, Kim IJ, Moore J, Woodland DL, Blackman MA (2003) Differential gamma-herpesvirus distribution in distinct anatomical locations and cell subsets during persistent infection in mice. J Immunol 170: 3828–3834. 16. Flano E, Kim IJ, Woodland DL, Blackman MA (2002) Gamma-herpesvirus latency is preferentially maintained in splenic germinal center and memory B cells. J Exp Med 196: 1363–1372. PLoS ONE | www.plosone.org 11 February 2008 | Volume 3 | Issue 2 | e1654