Fig. 5: MHV68 latency protein M2 causes p53 induction via Src-family kinase signaling. | Nature Communications

Fig. 5: MHV68 latency protein M2 causes p53 induction via Src-family kinase signaling.

From: Intrinsic p53 activation restricts gammaherpesvirus driven germinal center B cell expansion during latency establishment

Fig. 5

a, b Flow cytometry analysis of p53 induction in primary murine B cells after transduction with retroviruses that encode MHV68 latency proteins. Representative histograms and mean fluorescence intensities of p53 staining in transduced cells are shown. Data in (a) show p53 staining 4 days after transduction of cells with retroviruses that encode v-Cyclin, LANA, M2, M11, or a frame-shift/translation-stop mutant M2 (M2.Stop) as a negative control. Data are biological replicates, n = 2. Data in (b) show p53 staining 2 days after transduction with M2.Stop, M2-WT, or a tyrosine mutant M2-Y120F/Y129F (M2-Tyr). Data represent mean ± SEM. Two-tailed Student’s t test, ns - not significant, *p < 0.05, and ****p < 0.0001. The experiment was performed in technical triplicate. Data in (c) show p53 staining 2 days after transduction with M2.Stop or M2-encoding retroviruses in the presence of DMSO (1:1000), mouse IgG (20 μg/mL), PP2 (10 μM), PP3 (10 μM), CsA (500 ng/mL), or rat anti-IL10R (20 μg/mL). Inhibitors were added 24 h post-transduction. Data represent mean ± SEM. Ordinary one-way ANOVA, ns - not significant and ****p < 0.0001. The experiment was performed in technical triplicate. Gating strategies and additional controls are shown in Supplementary Fig. 11. d, e RNA-seq was performed 4 days after transduction of primary B cells with M2 or M2.Stop retroviruses to evaluate changes in transcription. Data in (e) show gene set enrichment analysis (GSEA) and (d) hallmark gene z-score heatmaps comparing M2 to M2.Stop for the hallmark p53 gene set. Statistical scores are inset into the top right of analysis images. NES normalized enrichment score, q-val FDR-adjusted p-value. The genes presented were derived from GSEA and DEseq2 analysis of all genes with significant expression changes where the expression level increased from M2 to M2.Stop at least 1.5-fold and decrease from M2 to M2.Stop at least 1.5-fold. A table of hallmark gene sets with significant changes caused by M2 is shown in Supplementary Fig. 14.

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