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

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to express Myc, mLANA and wild type (WT) or mutant versions of ubiquitin, in which the indicated lysines had been replaced by arginines (R).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

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

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

mLANA mediates kTR episome persistence.

<p>Gardella gel after transfection of A20 or A20/mLANA cells with pRepCK vector or k8TR DNA. Lanes contain 2-3x10⁶ cells. Gel was performed at 24 days of puromycin selection. Blot was probed with ³²P-pk8TR DNA. O, gel origin; E, S11 episomes; L, S11 linear genomes due to lytic replication; ccc plasmid DNA is indicated.</p

Episome maintenance of mTR or kTR DNA in mLANA or kLANA expressing cells.

<p>Episome maintenance of mTR or kTR DNA in mLANA or kLANA expressing cells.</p

kLANA mediates mTR episome persistence.

<p>(A) Schematic of kLANA and mLANA. Homologous regions are indicated in grey shading. White and black regions share no homology. Amino acid residue numbers are indicated. P, proline-rich. Gardella gels after transfection of m4TR (B) or m8TR (C) DNA. Blots in B and C were probed with ³²P-pRepCK DNA. (D) Gardella gel after 87 days of indicated cell lines from panels B and C. Blot was probed with ³²P-m8TR DNA. Days of G418 selection are below each panel. O, gel origin; ccc plasmid DNA is indicated. Lanes contain 1.5-2x10⁶ cells. Vertical lines at right (panels B, C, E) indicate positions of episomal bands. Asterisks indicate faint episomal bands. (E) Immune fluorescence for kLANA. m8TR cells are from cell line d (panels C, D). Brightness and contrast were uniformly adjusted in panels from the same field and red signal was uniformly enhanced for k8TR panels using Adobe Photoshop. Magnification, 630x.</p

Generation and lytic growth of MHV68 chimeric viruses.

<p>(A) Schematic diagram. The kLANA cassette was inserted between the M11 stop codon and the mORF72 exon in place of MHV68 103,935–104,709, which includes most of the mLANA ORF. p1, p2, p3, are mLANA promoters[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006555#ppat.1006555.ref038" target="_blank">38</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006555#ppat.1006555.ref039" target="_blank">39</a>]. The mORF72 noncoding exon (black) is located within the mLANA coding region. The E2 splice acceptor site (nt 104,871) and the mORF72 exon splice donor site (nt 104,715) were left intact to ensure expression of kLANA and mORF72. The mLANA start codon and three downstream ATGs were mutated to ATT to prevent initiation of translation (indicated by black dots). The BamHI-G fragment (genomic nt 101,653–106,902) is indicated. mLANA ORF, nt 104,868–103,927. (B) Confocal immunofluorescence detection of mLANA (top panels) or kLANA (lower panels) from yfp viruses. Magnification 630x. (C) Immunoblot of viral proteins. (D) Growth curves of virus in BHK-21 cells after infection with 0.01 PFU/cell. There was no significant difference between infection groups (p>0.05 using one-way non-parametric ANOVA Kruskal-Wallis). (E) Lung virus titers 7 days after infection with 10⁴ PFU of the indicated viruses. Circles represent titers of individual mice (n = 19). Bars indicate the mean. v-Δ1007-21.yfp had significantly lower titers than v-WT.yfp (**p<0.01, using one-way non-parametric ANOVA Kruskal-Wallis followed by Dunn´s multiple comparison test). There were no other statistically significant differences between groups.</p

mLANA and kLANA expression in vivo.

<p>Spleen sections of mice infected with 10⁴ PFU of v-WT or v-kLANA for 14 days. (A) In situ hybridization (brown) with probes for viral miRNAs 1–6. Sections were counter stained with Mayer´s Haemalum. (B, C) Detection of kLANA (B) and mLANA (C) by immunohistochemistry in sections adjacent to those shown in panel A. Arrows in panel B indicate the same kLANA positive cell. Arrow in panel C indicates a mLANA positive cell. Sections were counterstained with haematoxylin. No kLANA signal was detected in sections stained only with secondary antibody. (D, E) mLANA and kLANA nuclear dots detected by indirect immunofluorescence. Images are maximum intensity projections of Z-stacks acquired over the thickness of the spleen sections. No dots were observed in unstained sections or with secondary antibody alone. Magnification 630x. (F) Quantification of mLANA (n = 69 nuclei from 3 mice) or kLANA (n = 67 nuclei from 3 mice) dots per 100 μm³ nuclear volume. Bars indicate means. The number of dots per volume was not significantly different between v-WT and v-kLANA mice (Mann-Whitney test, p>0.05). (G) Viral genomes in FACS sorted YFP⁺ and YFP^- GC B cells from spleens of v-WT.yfp (n = 7) and v-kLANA.yfp (n = 6) infected mice. Circles represent individual mice. Bars indicate means. There was no significant difference between the two infection groups (Mann-Whitney test, p>0.05).</p

v-kLANA latent infection.

<p>Viral latency in spleens of C57 BL/6 mice 14 days after i.n. infection with 10⁴ PFU of the indicated viruses. (A) Latent titers determined by co-culture reactivation assay (closed circles) and titers of pre-formed infectious virus by plaque assay (open circles). Circles are titers of individual mice. Bars indicate mean and dashed line shows the limit of assay detection. v-kLANA titers were significantly lower than v-WT (Mann-Whitney test). *p<0.05. (B) Quantification of viral DNA-positive cells in total splenocytes and in sorted GC B cells (CD19⁺CD95⁺GL7⁺). Data are from pools of five spleens per group. Bars are frequency of viral DNA-positive cells. Error bars indicate 95% confidence intervals. (C-E) Flow cytometry analyses. Representative FACS plots from individual mice are shown in left panels. Quantification graphs in which each point represents an individual mouse are shown at the right. Bars are mean values. Data were combined from 2 independent experiments with 5 mice in each group. (C) Total number of GC B cells (CD19⁺CD95⁺GL7⁺). NS, not significant; *p<0.05 using the Mann-Whitney test. (D) Percentage of GC B cells that were YFP positive. (E) Percentage of YFP positive cells that were GC B cells. ***p<0.001 in (D) and (E) using the Mann-Whitney test.</p