Cells

HEK-293T cells and NIH-3T3 fibroblasts were cultivated in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal (293T) or newborn (3T3) bovine serum, 2 mM L-alanyl-L-glutamine, 100 U mL⁻¹ penicillin, and 100 µg mL⁻¹ streptomycin. Where indicated, cells were synchronized in G0/G1 phase by 48 h growth factor deprivation (0.05% serum). In preparation for proteomic analysis, cells were SILAC-labeled for at least five passages using lysine and arginine-deprived DMEM, supplemented with 10% dialyzed serum (cut-off: 10 kDa), 200 mg/L L-proline (only cells destined for phosphoproteome analysis), heavy (L-[¹³C₆,¹⁵N₂]-lysine (Lys8), L-[¹³C₆,¹⁵N₄]-arginine (Arg10)), medium (L-[²H₄]-lysine (Lys4), L-[¹³C₆]-arginine (Arg6)) or light (natural lysine (Lys0) and arginine (Arg0)) amino acids. Labeling efficiency and arginine–proline conversion was checked using LC–MS/MS.

Viruses

Viruses were derived from the m129-repaired MCMV strain Smith bacterial artificial chromosome (BAC) pSM3fr-MCK-2fl⁶⁶. Infections were carried out at 37 °C under conditions of centrifugal enhancement. In brief, after a virus adsorption period of 30 min, cell cultures were centrifuged for 30 min at 1000 g. Then, the virus inoculum was replaced by fresh medium. Virus titers were determined by flow cytometry of IE1 fluorescent cells at 6 h post infection or by using the median tissue culture infective dose (TCID₅₀) method. Unless otherwise stated, a multiplicity of infection (MOI) of 5 IE protein forming units (IU) per cell was used for experiments.

Bacmids

MCMV-HA-M97 and MCMV-M97-K290Q have been described recently²³. R45A/L47A, L47A/F49A, and M1STOP mutations were introduced into MCMV-HA-M97 by traceless BAC mutagenesis⁶⁷. The oligonucleotide primers used for BAC mutagenesis are specified in Supplementary Data 5. All mutants were controlled by diagnostic PCR and sequencing (Supplementary Fig. 5). To reconstitute infectious virus, BACs together with pp71 expression plasmid were transfected into 3T3 fibroblasts using an Amaxa nucleofector (Lonza).

Plasmids

PCGN-based expression plasmids for HA-tagged HHV1-UL13, HHV3-ORF47, HHV4-BGLF4, HHV5-UL97, HHV6-U69, HHV7-U69, and HHV8-ORF36 (Addgene plasmids #26687, #26689, #26691, #26693, #26695, #26697, #26698) were gifts from Robert Kalejta¹³. PCIneo-3HA-M97 was used as expression plasmid for HA-tagged M97 (ref. ²³). RXL/Cy mutations were introduced by site-directed inverse PCR-mutagenesis (primers, see Supplementary Data 5). PHM830 (Addgene plasmid #20702) was a gift from Thomas Stamminger³⁶. Fragments encompassing the NLS-RXL/Cy modules of M97 and U69 were PCR-amplified and cloned between NheI and XbaI sites of pHM830 (primers listed in Supplementary Data 5). Plasmids were confirmed by Sanger sequencing and purified by cesium chloride-ethidium bromide equilibrium centrifugation. PEI MAX (Polysciences), transfection grade linear polyethylenimine hydrochloride with a molecular weight of 40 kDa, was used for transfection.

Phosphopeptide enrichment

SILAC-labeled cells were harvested 24 h post MCMV infection via scraping. Cells were lysed with 6 M urea/2 M thiourea in 0.1 M Tris-HCl, pH 8.0. Samples were reduced with 10 mM dithiothreitol (DTT) and alkylated with 50 mM iodoacetamide for 30 min in the dark. Proteins were digested by lysyl endopeptidase (Wako Pure Chemicals) at an enzyme-to-protein ratio of 1:100 (w/w) for 3 h. Subsequently, samples were diluted with 50 mM ammonium bicarbonate to a final concentration of 2 M urea. Digestion with proteomics-grade modified trypsin (Promega) was performed at an enzyme-to-protein ratio of 1:100 (w/w) under constant agitation for 16 h. Enzyme activity was quenched by acidification with trifluoroacetic acid (TFA). The peptides were desalted with C18 Stage Tips prior to nanoLC-MS/MS analysis (whole cell lysate samples). An aliquot of the whole cell lysates were further processed for phosphopeptide enrichment. The tryptic digests corresponding to 200 μg protein per condition were desalted with big C18 Stage Tips packed with 10 mg of ReproSil‐Pur 120 C18‐AQ 5‐μm resin (Dr. Maisch GmbH). Peptides were eluted with 200 μL loading buffer, consisting of 80% acetonitrile (ACN) and 6% TFA (vol/vol). Phosphopeptides were enriched using a microcolumn tip packed with 0.5 mg of TiO₂ (Titansphere, GL Sciences). The TiO₂ tips were equilibrated with 20 μL of loading buffer via centrifugation at 100 g. 50 μL of each sample were loaded on a TiO₂ tip via centrifugation at 100 g and this step was repeated until all the sample was loaded. The TiO₂ column was washed with 20 μL of the loading buffer, followed by 20 μL of 50% ACN/0.1% TFA (vol/vol)). The bound phosphopeptides were eluted using successive elution with 30 μL of 5% ammonium hydroxide and 30 μL of 5% piperidine. Each fraction was collected into a fresh tube containing 30 μL of 20% formic acid. 3 μL of 100% formic acid was added to further acidify the samples. The phosphopeptides were desalted with C18 Stage Tips prior to nanoLC-MS/MS analysis.

Affinity purification

At 12 and 36 h post infection (M97 interactome) or 1 day post transfection (HHV kinases interactomes), cells were harvested by scraping in PBS. After centrifugation at 300 g, cell pellets were lysed in 25 mM Tris-HCl (pH 7.4), 125 mM NaCl, 1 mM MgCl₂, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 5% glycerol, 1 mM DTT, 2 µg mL⁻¹ aprotinin, 10 µg mL⁻¹ leupeptin, 1 µM pepstatin, 0.1 mM Pefabloc. For HA-AP, a µMACS HA isolation kit (Miltenyi Biotec) was employed according to the manufacturer’s instructions, with the following modifications. Lysates were incubated with the magnetic beads for 1 h and SILAC labels were mixed right before the lysates were applied to the column. Lysis buffer was used for the first washing step, lysis buffer without detergent for the second and 25 mM Tris-HCl (pH 7.4) for the final washing step. Samples were eluted in a total volume of 0.2 mL 8 M guanidine hydrochloride at 95 °C. Proteins were precipitated from the eluates by adding 1.8 mL LiChrosolv ethanol and 1 µL GlycoBlue. After incubation at 4 °C overnight, samples were centrifuged for 1 h at 4 °C and ethanol was decanted before samples were resolved in 6 M urea-2 M thiourea buffer. Finally, samples were reduced, alkylated, digested, and desalted as described above (phosphoproteomics).

NanoLC-MS/MS analysis

Phosphopeptides and peptides from whole cell lysates were separated on a MonoCap C18 High Resolution 2000 column (GL Sciences) at a flow rate of 300 nL/min. 6 and 4 h gradients were performed for whole cell peptides and phosphopeptides, respectively. Peptides from HA-AP samples were separated on 45 min, 2 or 4 h gradients with a 250 nL/min flow rate on a 15 cm column (inner diameter: 75 μm), packed in-house with ReproSil-Pur C18-AQ material (Dr. Maisch GmbH). A Q Exactive Plus instrument (Thermo Fisher) was operated in the data-dependent mode with a full scan in the Orbitrap followed by 10 MS/MS scans, using higher-energy collision dissociation. For whole proteome analyses, the full scans were performed with a resolution of 70,000, a target value of 3 × 10⁶ ions, a maximum injection time of 20 ms and a 2 m/z isolation window. The MS/MS scans were performed with a 17,500 resolution, a 1 × 10⁶ target value and a 60 ms maximum injection time. For phosphoproteome analysis, the full scans were performed with a resolution of 70,000, a target value of 3 × 10⁶ ions and a maximum injection time of 120 ms. The MS/MS scans were performed with a 35,000 resolution, a 5 × 10⁵ target value, 160 ms maximum injection time and a 2 m/z isolation window. For AP–MS of M97 interactomes, full scans were performed at a resolution of 70,000, a target value of 1 × 10⁶ and maximum injection time of 120 ms. MS/MS scans were performed with a resolution of 17,500, a target value of 1 × 10⁵ and a maximum injection time of 60 ms. Isolation window was set to 4.0 m/z. A Q-Exactive HF-X instrument (Thermo Fisher) or Q-Exactive Plus instrument was used for AP–MS samples of HHV kinases. The Q-Exactive HF-X instrument was run in Top20 data-dependent mode. Full scans were performed at a resolution of 60,000, a target value of 3 × 10⁶ and maximum injection time of 10 ms. MS/MS scans were performed with a resolution of 15,000, a target value of 1 × 10⁵ and a maximum injection time of 22 ms. The isolation window was set to 1.3 m/z. The Q-Exactive Plus instrument was run in data-dependent top10 mode. Full scans were performed at a resolution of 70,000 a target value of 3 × 10⁶ and maximum injection time of 120 ms. MS/MS scans were performed with a resolution of 35,000, a target value of 5 × 10⁵ and a maximum injection time of 120 ms. The isolation window was set to 4.0 m/z. In all cases normalized collision energy was 26.

Data analysis

Raw data were analyzed and processed using MaxQuant 1.5.2.8 (M97 interactomes) or 1.6.0.1 (phosphoproteomics, whole cell lysates, and interactomes of HHV-CHPKs) software⁶⁸. Search parameters included two missed cleavage sites, fixed cysteine carbamidomethyl modification, and variable modifications including methionine oxidation, N-terminal protein acetylation, asparagine–glutamine deamidation. In addition, serine, threonine, and tyrosine phosphorylations were searched as variable modifications for phosphoproteome analysis. Arg10 and Lys8 and Arg6 and Lys4 were set as labels where appropriate. The peptide mass tolerance was 6 ppm for MS scans and 20 ppm for MS/MS scans. The “match between runs” option was disabled and “re-quantify”, “iBAQ” (intensity-based absolute quantification) and “second peptide” options were enabled. Database search was performed using Andromeda, the integrated MaxQuant search engine, against a protein database of MCMV strain Smith and a Uniprot database of mus musculus proteins (downloaded July 2015) with common contaminants. Raw data from AP–MS samples of HEK-293T cells were searched against a Uniprot database of human proteins (downloaded August 2018 or October 2016) and the sequences of transgenic HHV kinases including common contaminants. FDR was estimated based on target-decoy competition and set to 1% at PSM, protein and modification site level.

Bioinformatics of phosphoproteomic profiles

Phosphosite data and whole proteome data were filtered to exclude contaminants, reverse hits and proteins only identified by site (that is, only identified by a modified peptide). Phosphorylation sites were ranked according to their phosphorylation localization probabilities (P) as class I (P > 0.75), class II (0.75 > P > 0.5) and class III sites (P < 0.5). Class I sites (in at least one of the replicates) were used with a multiplicity of one (that is, only one phosphorylation site on a peptide). MaxQuant normalized site ratios (from Phospho(STY)Sites.txt file) were used and corrected by the ratio of the corresponding protein (from ProteinGroups.txt file) for the respective replicate. SILAC ratios of replicates were log2 transformed, averaged and sites were considered that were quantified in at least one of the replicates. Sites were then categorized as belonging to nuclear or cytosolic proteins, based on the GO annotation of the source protein. Source proteins and corresponding sites with no clear nuclear or cytosolic annotation were classified as “no category”. To assess differences in subcellular phosphoproteomic profiles we used the average SILAC ratio of cells infected with R45A/L47A and L47A/F49A mutant viruses and compared phosphosites that belong to nuclear or cytosolic proteins (see above). One-sided Wilcoxon rank-sum test was performed comparing these two subsets of phosphosites with any possible amino acid in the region +4 to −4 (0 refers to the phosphorylated amino acid). Comparisons were considered when there were at least 19 phosphosites from both cytosolic and nuclear proteins for an amino acid at a given position quantified.

Bioinformatics of HHV–CHPK interactomes

AP–MS data were filtered as described above, ratios were log2 transformed and replicates were averaged (mean) when they were quantified in all three replicates. Two-sided one sample t-tests (null hypothesis: µ₀ = 0) were performed on the experimental data and a set of simulated data where enrichment ratios were permuted for the individual replicates (999 permutations). The t-test p-values were then adjusted according to the permuted data. The p-values in Supplementary Data 1, Fig.1 and Supplementary Fig. 1 were adjusted in this way. Candidate interactors were selected based on a combination of adjusted p-value and means of the three replicates. To harmonize the data obtained from the different CHPK-IPs, we discriminated between candidate interactors and background binders based on volcano plots. For all APs, we used a fixed p-value cut-off of 0.05 and a flexible SILAC fold-change cut-off according to an FDR estimation. For this, we used the simulated data as a false positive set and accepted candidate interactors above a SILAC fold-change that recalled maximum 1% false positives.

FDR calculations were based on the simulated data as false positives, as previously suggested⁶⁹. This yielded a set of high-confidence candidate interactors for APs with individual HHV kinases. To compare individual prey proteins across the APs with different kinases we imputed missing values with random values from a normal distribution (with mean 0 and standard deviation 0.25). Enrichment profiles were clustered using Euclidean distance and assembled into a heatmap using R. GO enrichment of the prey proteins in selected sets of clusters were performed using Metascape⁷⁰.

Bioinformatics of M97 interactomes

AP–MS data were filtered as described above, ratios were log2 transformed and replicates were averaged (mean) when they were quantified in at least two of the replicates. Two-sided one sample t-tests (null hypothesis: µ₀ = 0) were performed on the experimental data and proteins were considered as interactors when they were below a t-test p-value of 0.05 and above a log2 SILAC fold-change of 1.5. Additionally, the molar amount of bait and prey proteins in MS samples was estimated by iBAQ values. Therefore, for samples where M97 was purified, the iBAQ values were summed up, sorted and log10 transformed.

Immunoblot analysis

Whole cells were harvested and subsequently lysed by sonication in 50 mM Tris-Cl (pH 6.8), 2% SDS, 10% glycerol, 1 mM DTT, 2 µg mL⁻¹ aprotinin, 10 µg mL⁻¹ leupeptin, 1 µM pepstatin, 0.1 mM Pefabloc. The lysates were clarified by centrifugation and protein concentration was measured using the Bio-Rad DC protein assay. Lysates were adjusted to equal protein concentration, supplemented with 100 mM dithiothreitol and bromophenol blue, and boiled at 95 °C for 3 min. For subcellular fractionation into nuclei and cytoplasmic extracts, cells were lysed by Dounce homogenization in hypotonic buffer, consisting of 10 mM Hepes (pH 8.0), 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 0.1 mM DTT and protease inhibitors. Nuclei were collected by low-speed centrifugation (4 min, 1300 g, 4 °C). The supernatant was clarified by high-speed centrifugation (15 min, 20,000 g, 4 °C). The nuclei were washed once in the hypotonic extraction buffer and lysed then as described above for the preparation of whole cell lysates. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and blotted to polyvinylidene fluoride membranes. To prevent nonspecific binding, blots were incubated in Tris-buffered saline-0.1% Tween-20 (TTBS) supplemented with 5% skim milk. Afterwards blots were incubated with the following primary antibodies: Cyclin A, C19 (Santa Cruz); cyclin E, M20 (Santa Cruz); CDK2, M2 (Santa Cruz); HA, clone 3F10 (Roche); M99, mouse antiserum (generously provided by Khanh Le-Trilling, University Hospital Essen); IE1, clone Croma 101; M45, clone M45.01; M57, clone M57.02 (all obtained from Center for Proteomics, Rijeka). The blots were developed using horseradish peroxidase-conjugated secondary antibodies in conjunction with the Super Signal West Dura chemiluminescence detection system (Thermo Fisher). All antibodies were diluted to 1 µg per mL in TTBS, 5% skim milk. Uncropped scans of the immunoblots are provided in the Source Data file.

Immunoprecipitation

Cells were lysed by freezing-thawing in IP buffer (IPB): 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 10 mM NaF, 0.5 mM Na₃VO₄, 0.5% Nonidet P-40, 10% glycerol, 1 mM DTT, 2 µg mL⁻¹ aprotinin, 1 mM leupeptin, 1 mM Pefabloc. Cell extracts were clarified by centrifugation at 20,000 g. Cyclin A IPs were performed by incubating the IPB extracts with Cyclin A, H432, conjugated agarose beads (Santa Cruz)⁴⁶. For HA IPs, a µMACS HA isolation kit (Miltenyi Biotec) was employed according to the manufacturer’s instructions, except that IPB was used as both lysis and washing buffer.

Kinase assay

First, HA-M97 was immunoprecipitated from infected cells. To this end, IPB extracts were prepared and incubated with HA antibody clone 3F10 (1 µg per mL) and Protein G-conjugated agarose beads. The precipitates were washed several times with IPB and twice with 20 mM Tris-Cl (pH 7.4), 10 mM MgCl₂, 1 mM DTT. Then, they were incubated under constant agitation for 60 min at 30 °C in kinase reaction buffer: 20 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 1 mM DTT, 10 mM β-glycerophosphate, 50 μM ATP, 5 μCi [γ-³²P]ATP. Kinase reactions were analyzed by 8% SDS-PAGE followed by autoradiography.

Immunofluorescence microscopy

3T3 fibroblasts were grown and infected on glass coverslips. Where indicated, cells were incubated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 30 min. To analyze for M97 localization and sites of EdU incorporation, coverslips were washed with PBS and incubated for 10 min in PBS-4% paraformaldehyde fixation solution, followed by additional washing and incubation in PBS-T permeabilization solution (PBS, 0.1% Triton X-100, 0.05% Tween 20) and 2% bovine serum albumin (BSA) fraction V/ PBS-T blocking solution. Afterwards, incorporated EdU was conjugated to Alexa Fluor 488 using the Click-iT EdU labeling kit (Thermo Fisher). Then samples were incubated overnight at 4 °C with the following antibodies: anti-HA clone 3F10 or anti-M57 clone M57.02 (Center for Proteomics, Rijeka), both diluted to 1 μg mL⁻¹ in PBS-T containing 2% BSA. After washing in PBS, cells were incubated for 1 h at 25 °C with Alexa Fluor 488 or 647-coupled anti-IgG antibodies (Thermo Fisher). Coverslips were mounted on glass slides in 4′,6-diamidino-2-phenylindole (DAPI) containing Fluoromount-G medium (Thermo Fisher). Images were acquired by an Eclipse A1 laser-scanning microscope, using NIS-Elements software (Nikon Instruments). Equal microscope settings and exposure times were used to allow direct comparison between samples. For quantification, the microscope slides were randomly scanned and all cells in the randomly acquired frames were analyzed by ImageJ.

Flow cytometry

Cells were fixed and permeabilized in ice-cold PBS-80% ethanol for at least 16 h. After washing, cells were incubated on ice for at least 16 h with one of the following primary antibodies: anti-IE1 (clone Croma 101) or anti M57 (clone m57.02), both diluted to 1 µg mL⁻¹ in PBS-1% BSA. After washing, cells were incubated in Alexa Fluor 647 conjugated anti-mouse IgG for 1 h at 25 °C. Cells were washed again and incubated then for 15 min at 25 °C in PBS supplemented with 0.1 mg mL⁻¹ RNAse A and 25 μg mL⁻¹ propidium iodide. All washing steps and antibody dilution were performed using PBS-1% BSA. Flow cytometry was performed using a FACSCanto II instrument equipped with FACSDiva and CellQuest Pro software (Becton Dickinson).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.