Supporting Information Edgar et al. 10.1073/pnas.1601895113 SI Methods (Actimetrics), and recordings were analyzed using LumiCycle Mice. Sample size was determined using the resource equation: Data Analysis software (Actimetrics). E (degrees of freedom in ANOVA) = (total number of exper- – Cell Cycle Analysis of Confluent Cell Monolayers. NIH 3T3, primary imental animals) (number of experimental groups), with −/− sample size adhering to the condition 10 < E < 20. For com- WT, and Bmal1 fibroblasts were sequentially transduced − − parison of MuHV-4 and HSV-1 infection in WT vs. Bmal1 / with lentiviral fluorescent ubiquitin-based cell cycle indicators mice at ZT7 (Fig. 2), the investigator did not know the genotype (FUCCI) mCherry::Cdt1 and amCyan::Geminin reporters (32). of the animals when conducting infections, bioluminescence Dual reporter-positive cells were selected by FACS (Influx Cell imaging, and quantification. For bioluminescence imaging, Sorter; BD Biosciences) and seeded onto 35-mm dishes for mice were injected intraperitoneally with endotoxin-free lucif- subsequent analysis. To confirm that expression of mCherry:: Cdt1 and amCyan::Geminin correspond to G1 (2n DNA con- erin (Promega E6552) using 2 mg total per mouse. Following < ≤ anesthesia with isofluorane, they were scanned with an IVIS tent) and S/G2 (2 n 4 DNA content) cell cycle phases, Lumina (Caliper Life Sciences), 15 min after luciferin admin- respectively, cells were stained with DNA dye DRAQ5 (abcam) and analyzed by flow cytometry (LSR-Fortessa; BD Biosci- istration. Signal intensity was quantified using Living Image ences). To examine dynamics of replicative activity under ex- software (Caliper Life Sciences), obtaining maximum radiance perimental confluent conditions, synchronized FUCCI reporter for designated regions of interest (photons per second per − − − monolayers were observed by time-lapse live cell imaging over square centimeter per Steradian: photons·s 1·cm 2·sr 1), relative 3 d (Nikon Eclipse Ti-E inverted epifluorescent microscope). to a negative control region. At 24 d after infection, mice were At stated circadian times, numbers of mCherry::Cdt1- and culled, and tissue was removed for analysis of latent infection. amCyan::Geminin-positive cells were counted, and their ratio Analysis of Virus Latency. For analysis of MuHV-4 latent infection was determined. in mice, viral genome loads were measured by real-time PCR Viruses. To generate MuHV-4 and HSV-1 stocks, infected BHK21 using DNA extracted from spleen tissue as previously described cells were harvested following the development of cytopathic (15). MuHV-4 M2 gene was amplified using a Rotor Gene 3000 effect. Virus titer was determined by plaque assay as follows. For (Corbett Research), and PCR products were quantified by hy- MuHV-4, BHK21 or NIH 3T3 cell monolayers were incubated bridization with a Taqman probe. Genome copy number was (2 h at 37 °C) with 10-fold dilutions of virus then overlaid with determined by comparison with a standard curve of cloned M2 0.6% carboxymethylcellulose to limit progeny virus spread to ad- plasmid templates. Cellular DNA was quantified in parallel by jacent cells only. After 4 d, cells were fixed in 10% (wt/vol) form- amplifying part of the adenosine phosphoribosyl transferase gene aldehyde and stained with 0.1% toluidine blue for plaque counting. (APRT) and the ratio of MuHV-4 genome copies to APRT For HSV-1, BHK21 cells were incubated (45 min at 37 °C) in determined. Reactivation of latent MuHV-4 was measured by suspension with 10-fold dilutions of virus before supplementation infectious center assay: spleens were disrupted into single-cell with 1% carboxymethylcellulose. After 1–2 d, cells were fixed in suspensions and serial dilutions were cocultured with cell 10% formaldehyde and stained with 0.1% toluidine blue for plaque monolayers. After 6 d, cells were fixed and stained for plaque counting. WT and PB2::GLUC IAV stocks were amplified in 10-d- counting, as above. For analysis of HSV-1 latent infection in old embryonated chicken eggs and titrated by plaque assay. For mice, viral genome loads were measured by real-time PCR IAV titration, Madin Darby canine kidney (MDCK) cell mono- using DNA extracted from dorsal root ganglion tissue. HSV-1 layers were incubated (1 h at 37 °C) with virus in serum-free me- ICP0 gene were amplified using a Rotor Gene 3000, and PCR diumandthenoverlaidwith1.2%Avicel(IMCD)/DMEMplus products were quantified by hybridization with a Taqman 1 μg/mL TPCK-trypsin (Worthington Biochemical), 0.14% BSA probe. Genome copy number was determined by comparison (Sigma), and 1× Glutamax (Life Technologies). After 2 d, cells with a standard curve of cloned ICP0 plasmid templates. Cel- were fixed in 10% formaldehyde and stained with 0.1% toluidine lular DNA was quantified in parallel by amplifying part of the blue for plaque counting. Additionally, single-cycle growth curves APRT gene and the ratio of HSV-1 genome copies to APRT were performed as follows: parallel cell cultures were infected with determined. WT IAV (MOI = 5 pfu/cell; 37 °C) and after 1 h any remaining input virus was removed by acid washing (40 mM citric acid, pH 3, Cell Culture and Bioluminescence Assays. All cells were propagated 135 mM NaCl, 10 mM KCl). At stated times after infection, cul- in DMEM containing 4.5 g/L glucose (Sigma D6546), supple- tures were harvested and stored at −80 °C. The amount of in- mented with 10% (vol/vol) FetalClone III serum (Thermo fectious virus produced within the time stated was determined by × Scientific HyClone), 1 Glutamax (Life Technologies), 100 U plaque assay as above. penicillin/mL, and 100 μg/mL streptomycin (Penicillin–Strepto- mycin Solution; Sigma P0781). Transfections were conducted Correlation of Viral Bioluminescence and Infectious Particle Production. using GeneJuice transfection reagent (Millipore). After syn- To correlate M3:luciferase MuHV-4 (M3:luc MuHV-4) and chronization, cells were transferred to “Air Medium,” containing CMV:luciferase HSV-1 (CMV:luc HSV-1) bioluminescence with DMEM (Sigma D5030) supplemented with 5 g/L glucose, production of infectious particles, parallel confluent fibroblast 20 mM Hepes, 100 U penicillin/mL, 100 μg/mL streptomycin, monolayers were incubated with luciferase viruses at a range 0.035% NaHCO3, FetalClone III serum, 1× Glutamax, 2× B-27 of multiplicities of infection (MOI; 2–0.001 pfu/cell for M3:luc Supplement (Life Technologies 17504-044), and 0.3 mM Lucif- MuHV-4; 10–0.001 pfu/cell for CMV:luc HSV-1). Input virus erin (Biosynth L8220). All experiments were initiated >24 h after was then removed by acid washing (40 mM citric acid, pH 3, cells were transferred to constant conditions, so that they would 135 mM NaCl, 10 mM KCl). Bioluminescence was monitored not be subject to any effects of the synchronization treatment. in real time using a Lumicycle-32 (Actimetrics). At stated times Bioluminescence was monitored using a LumiCycle-32 system after infection, parallel cultures were harvested and stored at Edgar et al. www.pnas.org/cgi/content/short/1601895113 1of17 −80 °C. The amount of infectious virus produced was de- WT: CT18 vs. CT30 termined by plaque assay. The total bioluminescence acquired during stated times after infection was determined by the area TMT label Time point under curve (AUC) method. Correlation analysis was per- 126 WT CT30 Rep A formed using linear regression (total bioluminescence vs. log 10 127 WT CT30 Rep B pfu produced by equivalent cultures over the same time frame 128 WT CT30 Rep C during exponential growth). 129 WT CT18 Rep A 130 WT CT18 Rep B ICP0 in Vitro Expression Assay. ICP0 adenoviral vectors were a kind gift from Anna Salvetti and Marie-Claude Geoffroy (INSERM 131 WT CT18 Rep C U649, Nantes, France). This Tet-On system comprises Ad.CMV. rtTA (expressing rtTA from the HCMV immediate early pro- −/− moter), Ad.TRE.ICP0 [expressing ICP0 from a tetracycline- Bmal1 : CT18 vs. CT30 responsive (TRE) promoter], and Ad.TRE.FXE [expressing a nonfunctional RING-finger deletion mutant (FXE) of ICP0 TMT label Time point −/− from a TRE promoter] as previously described (33). NIH 3T3 126 Bmal1 CT18 Rep A cells expressing the Bmal1:luc reporter construct were infected 127 Bmal1−/− CT18 Rep B with the adenovirus vectors alone or in combination (Ad.CMV. 128 Bmal1−/− CT18 Rep C −/− rtTA: MOI = 10 pfu/cell; Ad.TRE.ICP0 or Ad.TRE.FXE: MOI = 129 Bmal1 CT30 Rep A −/− 4 pfu/cell). After 46 h of bioluminescence recording, doxycy- 130 Bmal1 CT30 Rep B cline (Dox) was added (1 μg/mL) and bioluminescence re- 131 Bmal1−/− CT30 Rep C cording resumed. Proteomics: Lysis and Alkylation. Primary fibroblasts from WT +/+ −/− (Bmal1 )andBmal1 mice were grown to confluence in Proteomics: Cleanup and LC-MS/MS Analysis. Peptides dried by 6-well plates (n = 3 per time point) and synchronized with 100 nM vacuum centrifugation were cleaned up in preparation for LC- dexamethasone for 20 min, and their medium was exchanged MS/MS analysis using C18 Stage Tips with a centrifuge-based and then incubated under constant conditions (37 °C in dark- protocol. Peptides were then aliquoted and taken to dryness ness). Cells were then harvested by briefly washing with ice-cold by vacuum centrifugation and may be stored at −80°C until re- PBS and incubating with lysis buffer (250 mM Hepes, 1% SDS, quired for LC-MS/MS analysis. Labeled peptide samples were 1% Nonidet P-40, and 10 mM DTPA) for 20 min. The first time resuspended in 50 μL 0.1% TFA, sonicated for 15 min, and in- point was designated Circadian Time (CT)18 and the other jected (5 μL per injection). Peptide mixtures were separated on a CT30 based on the assessment of cells expressing the Bmal1:luc 50-cm, 75-μm-ID Pepmap column over a 3-h gradient at 40 °C or the Per2:luc reporter that were synchronized in parallel (Figs.