Myc transcriptional activity is upregulated during MuHV-4 latency in GC cells.

<p>(A) MuHV-4 infected cells exhibit augmented expression of Myc-dependent genes. FACS purified infected (CD19⁺CD95^hiGL7^hiYFP⁺) or control uninfected (CD19⁺CD95^hiGL7^hiYFP⁻) GC B-cells from C57BL/6 mice infected with MuHV-4-YFP were analysed at 13 days pi for transcription of genes encoding for cyclins (Cyc) B1, D1, D2, D3, E1, cyclin-dependent kinase (Cdk) 4, IL10, B-ATF, MIF and CD70 by qPCR. Transcription of each gene, normalized to GAPDH, is represented as fold induction relative to control uninfected cells purified form the same pool of 5 spleens. Error bars represent SEM from three independent experiments. (B and C) <i>Cγ1-cre^KI/WT</i>; <i>c-myc^fl/fl</i> (GC Myc KO) mice are unable to mount a GC reaction. GC Myc KO and control mice immunized with NP-CGG were analysed at day 10 post-immunization for frequencies of GC (CD19⁺CD95^hiGL7^hi) and IgG1 (CD19⁺IgD⁻IgM⁻IgG1⁺) B-cells, by flow cytometry. (D, E and F) GC Myc KO mice exhibit normal numbers of IgG2a/2b B-cells. GC Myc KO and control mice immunized with CFA-OVA were analysed 14 days later for frequencies of GC, IgG1 and IgG2a/2b (CD19⁺IgD⁻IgM⁻IgG2a/2b⁺) B-cells by flow cytometry. Each point represents an individual mouse; four mice (n = 4) were analyzed for each experimental condition; grey bars indicate the mean. Representative FACS plots from individual animals are shown (top panels).</p

MuHV-4 lacking the H2-K^d-restricted M2 CD8⁺ T cell epitope is positively selected in a H2^d host.

<p>BALB/c or C57BL/6 mice were intranasally infected with equal amounts of the indicated viruses, 10⁴ PFU in total. At 14, 21 or 50 days post-infection germinal centre (B220⁺PNA^high) B cells were recovered from pools of al least three spleens by flow cytometric sorting. The copy number of each viral genome was then measured by quantitative PCR using primers specific for each viral genome. Each sample was assayed in triplicate. Black bars denote the percentage of M2_F85A or vM2_F85AEPI genomes in the total genome load. Error bars show 95% confidence intervals.</p

M2 expression leads to the upregulation of adhesion and co-stimulatory molecules in B cells.

<p>(A) Fold increase of the mean fluorescence intensities (MFI) of several surface molecules, relative to untransduced A20 B cells. A20 B cells (black bars) and A20 B cells stably expressing M2 (open bars) or M2Y (grey bars) were stained with fluorescently labelled antibodies and the surface expression of the indicated molecules was analyzed on a LSR Fortessa flow cytometer. Bars represent the mean of three independent experiments. Error bars represent standard error of the mean. Statistical significance was evaluated using a one-tailed Students t-test. (B) Representative FACS histogram plots for the indicated surface molecules.</p

mLANA modulatory activity towards Myc is independent of Myc phosphorylation status.

<p>(A) HEK 293T cells were transfected to express wild type Myc protein, or specific T58 or S62 residues mutated to alanines, with or without mLANA, as indicated and subjected to immunoblotting analysis with phospho-specific antibodies directed towards Myc phosphorylated on S62 or T58. (B) In vivo interaction of Myc with mLANA is independent of its phosphorylation on T58 and S62 residues. Cellular extracts from HEK 293T cells transfected with the indicated plasmids were subjected to immunoprecipitation with anti-Myc and analysed by immunoblotting. (C & D) mLANA-mediated poly-ubiquitination and activation of Myc is independent of Myc phosphorylation on T58 and S62 residues. (C) HEK 293T cells were transfected with the indicated combinations of plasmids. Cellular lysates were obtained and subjected to Ni-NTA pull down as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003554#ppat-1003554-g003" target="_blank">Figure 3F</a>. (D) HeLa cells were transfected with a Myc (E-box) luciferase reporter vector combined with plasmids for Myc, Myc phospho-mutants, with (grey bars) or without (open bars) mLANA, as indicated and Myc transcriptional activity assayed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003554#ppat-1003554-g003" target="_blank">Figure 3C</a>. Error bars represent SEM from triplicates from three independent transfection experiments. −, without; +, with; α-, anti; TCL, total cellular lysates.</p

Increased germinal center B cell colonization by MuHV-4 lacking M2_84–92 epitope anchor residues.

<p>BALB/c mice were intranasally infected as shown and spleen sections hybridized with viral tRNA-specific riboprobes. A. Representative spleen sections from each group of animals. Dark staining cells express viral tRNAs. Arrows indicate example positive cells. All sections are magnified ×200 and counter stained with haematoxylin. B. Mean±SEM percentage of splenic follicles positive for viral tRNA expression. Six sections per mouse and three mice per group were counted at each time point. Follicles were scored positive if they contained at least one viral tRNA positive cell. At d50 post-infection vM2_F85A and vM2_L92A showed significantly higher latency loads than vWT (p = 0.002 and p<0.001; using a 1 tailed Student's t-Test), whereas vM2_F85AEPI showed no significant difference (p = 0.45). C. BALB/c mice were intranasally infected with 10⁴ PFU of the indicated virus. At 14 or 50 days post-infection, reciprocal frequencies of viral infection in flow cytometrically purified germinal center (B220⁺PNA^high) B cells were determined by limiting dilution and real time PCR. Spleens were pooled from 5 mice per group. Bars show the frequency of viral DNA positive cells with 95% confidence intervals.</p

IFN-γ production in T_H cells conjugated with M2-expressing B cells requires specific peptide presentation.

<p>A20 B cells stably expressing M2 or M2Y were pulsed overnight, or not, with different concentrations of OVA peptide (OVAp) and incubated with OVAp-specific CD4⁺ T cells. (A) Extracellular IFN-γ after 20h of incubation. After incubation the supernatant was recovered and analyzed by sandwich ELISA to determine IFN-γ concentration. Supernatants of three independent experiments were tested in duplicate. (B) Upper panel: percentage of T cells producing IFN-γ after 5h of conjugation in the presence of brefeldin A (BFA). Cells were fixed and stained for CD4 with anti-CD4-APC, and for IFN-γ with anti-IFN-γ-PE, and analyzed on a FACS Calibur. Lower panel: IFN-γ mean fluorescence intensity (MFI) of responding T cells. Average of three independent experiments is shown. Statistical significance of the difference between groups was evaluated by a one-tailed unpaired Student’s t test. (C) Representative FACS plots of intracellular IFN-γ production. (D) Representative confocal images of IFN-γ polarization to the contact zone. Prior to incubation B and T cells were labelled with CMFDA (green) and CMAC (blue) live dyes, respectively. Cells were incubated for 2.5h, fixed and stained for IFN-γ (red), and for pTyr (yellow). Images are from one representative experiment out of three and were obtained using a Zeiss LSM 510 META microscope. (E) Quantification of conjugates with IFN-γ polarization per image. Conjugates were evaluated by confocal microscopy based on B-T_H cell contact, and IFN-γ polarization (red). 45 to 55 images were acquired per sample from three independent experiments. Only images with a minimum of three T cells were considered for analysis. Statistical significance of the difference between groups was evaluated by a Mann-Whitney U test.</p

M2_84–92 epitope expression and CD8⁺ T cells are linked in setting splenic latent loads.

<p>BALB/c mice were intranasally infected with the indicated viruses (10⁴ PFU) and at 11, 13, 15, 17 and 19 days post-infection anti-CD8 monoclonal antibody (MAb) was intraperitoneally injected. At 21 days post-infection spleens were removed for analysis. Control mice are d21 infected littermates that were not injected with MAb. A. Schematic diagram of the experimental setting. B. Splenocytes were stained for CD8. The data show the percentage of CD8⁺ T cells of total lymphocytes (arithmetic means±STDV) in depleted mice (black bars) and control mice (white bars). C. Splenocytes were titrated for reactivation-competent virus by explant co-culture. Each point shows the titre of one mouse. Black symbols represent data for CD8⁺ T cell depleted mice, white symbols represent data for control mice. Horizontal lines indicate arithmetic means. Data were reproducible over two independent experimental groups.</p

mLANA modulates Myc activity through the assembly of heterotypic poly-ubiqutin chains.

<p>(A) mLANA-mediated poly-ubiquitination of Myc is dependent on ubiquitin lysine residues (K) 33, 48 and 63. HeLa cells were transfected to express Myc, mLANA and wild type (WT) or mutant versions of ubiquitin, in which the indicated lysines had been replaced by arginines (R). TCL were subjected to denaturing immunoprecipitation using anti-Myc and analysed by immunoblotting with anti-ubiquitin. (B) mLANA stimulation of Myc transcriptional activity requires K33, K48, and K63 of ubiquitin. HeLa cells were transfected with a Myc (E-box) luciferase reporter vector, the indicated ubiquitin mutants, combined with (open bars) or without (filled bars) mLANA. Myc transcriptional activity was assayed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003554#ppat-1003554-g003" target="_blank">Figure 3C</a>. Error bars represent SEM from triplicates from three independent transfection experiments. (C) mLANA-expressing cells exhibit increased Myc levels when K33, K48 and K63 residues of ubiquitin are preserved. HeLa cells were transfected to express the indicated combination of proteins (top) and Myc protein levels were analysed by immunoblotting. Right panel shows the densitometry analysis of Myc levels in mLANA transfected cells, expressed as fold induction relative to control transfected cells, normalised to Actin. (D) mLANA requires ubiquitin residues K33, K48 and K63 to mediate the poly-ubiquitination of the N-terminus of Myc. HeLa cells were transfected to express K⁻Myc, mLANA and the indicated ubiquitin mutants. Ubiquitination of K⁻Myc was assessed as described in A. (E) mLANA modulation of K⁻Myc transcriptional activity involves ubiquitin residues K33, K48 and K63. HeLa cells were transfected to express K⁻Myc, the indicated ubiquitin mutants, mLANA (open bars) or control transfected (grey bars). Myc transcriptional activity was assayed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003554#ppat-1003554-g003" target="_blank">Figure 3C</a>. Error bars represent SEM from triplicates from three independent transfection experiments. −, without; +, with; α-, anti; IP, immunoprecipitation; TCL, total cellular lysates.</p

Long-term MuHV-4 latent loads depend on viral M2 expression and host H2 haplotype.

<p>Mice were infected intranasally with wild-type (vWT) or M2⁻ (vM2FS) viruses. At d14 or d50 post-infection spleens were removed and titrated for reactivation-competent virus by explant co-culture with BHK-21 cells. Each point shows the titre of one mouse. The horizontal bars show arithmetic means. The dashed horizontal line represents the limit of assay detection. Pre-formed infectious virus, as measured by the parallel titration of equivalent frozen/thawed samples, was below the limit of assay detection. At d50 post-infection the vM2FS latent load was significantly higher than the vWT latent load in BALB/c (P = 0.011) and DBA/2 (P = 3×10⁻⁵) and close to significance in B6.C.H2^d (P = 0.056), but not in C57BL/6 (P = 0.228) and FVB-N (P = 0.347) by a one-tailed Student's t-Test. This was also confirmed by combining all data from H2^d mouse strains (P<0.0001) but not from non-H2^d mice (P = 0.71) using a 2-way non-parametric ANOVA Friedman's test. Data were reproducible over two independent experimental groups.</p

Myc is essential for the amplification of MuHV-4 infection in GC B-cells.

<p>(A and B) MuHV-4 infection of GC Myc KO mice leads to the development of normal GC reactions. (A) GC B-cell percentages were determined by flow cytometry as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003554#ppat-1003554-g001" target="_blank">Figures 1B and 1D</a> at day 14 pi. (B) Dark zone (CD19⁺CD95^hiGL7^hiCXCR4^hiCD86^lo) and light zone (CD19⁺CD95^hiGL7^hiCXCR4^loCD86^hi) percentage of infected cells at day 14 pi. Each point represents an individual mouse; five mice (n = 5) were analyzed for each experimental condition. (C and D) MuHV-4 infected cells from GC Myc KO mice are genotypically and phenotypically wild type for Myc. (C) Total DNA from FACS purified MuHV-4 infected GC B-cells, from GC Myc KO mice, was subjected to PCR analysis with primers specific for deleted (Δ) or floxed (fl) <i>c-myc</i>, as depicted. DNA from total B-cells, in the same pool of splenocytes, was analysed in parallel as a positive control for Δ <i>c-myc</i>. (D) Transcription of Myc, normalized to GAPDH, in purified GC B-cells from GC Myc KO mice is represented as fold induction relative to MuHV-4 infected GC B-cells purified from wild type mice. Error bars represent SEM from three independent experiments. (E and F) MuHV-4 infection induces a strong IgG2a/2b response. GC Myc KO and control mice were analysed at day 21 pi for frequencies of IgG1 and IgG2a/2b B-cells by flow cytometry. Each point represents an individual mouse; four mice (n = 4) were analyzed for each experimental condition; grey bars indicate the mean. Representative FACS plots from individual animals are shown (top panels). (G) MuHV-4 infection of GC Myc KO mice reveals a deficit in the establishment of latency. GC Myc KO and control mice were analysed at days 14 and 21 pi for frequencies of viral infection in GC B-cells. Data were obtained from pools of 5 spleens. Error-bars represent the frequency of viral DNA-positive cells with 95% confidence intervals.</p