Fig. 4. The RXL-NLS module controls M97 localization and substrate phosphorylation.

a Serum-starved 3T3 fibroblasts were infected with the indicated MCMV-HA-M97 variants or left uninfected. At 8, 24, and 48 h post infection, cells were examined by confocal immunofluorescence microscopy for subcellular localization of HA-M97 (green). Nuclei were counterstained with DAPI. Scale bars: 10 μm. Representative images are shown. b The indicated number of cells were categorized based on the subcellular localization of HA-M97 relative to the DAPI stain. c SILAC-labeled 3T3 cells were serum starved and infected with NLS-RXL/Cy-deficient M97^R45A/L47A or RXL/Cy-deficient M97^L47A/F49A viruses (Supplementary Fig. 6). At 24 h post infection cells were harvested and subjected to a phosphoproteomic workflow. Boxplots for phosphosites that belong to proteins with nuclear, cytoplasmic, or uncategorized subcellular GO annotation are depicted with their SILAC log2 fold-change between M97^R45A/L47A and M97^L47A/F49A infection. pSP, pSXXK, LXpSP, or all other sites were assessed individually. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; outliers (data points outside the whiskers) were removed for visibility. P-values are based on an one-sided Wilcoxon rank-sum test comparing nuclear and cytosolic subsets. Data represent the mean of n = 2 replicates.