Journal: mSystems

Article Title: Sirtuin 2 promotes human cytomegalovirus replication by regulating cell cycle progression

doi: 10.1128/msystems.00510-23

Figure Lengend Snippet: SIRT2 deacetylase activity supports an early stage of the HCMV replication cycle. ( A ) Diagram of the AGK2 treatment and HCMV infection procedure used throughout this study. Unless otherwise indicated, fibroblasts were treated with AGK2 (or DMSO, vehicle control) 12 hours prior to infection with HCMV. Cells were then incubated in either complete growth media (for uninfected mock samples) or in complete growth media containing HCMV inoculum (for infected samples). Infection was allowed to proceed for 1 hour to allow for virus entry, after which time media containing virus inoculum was replaced with dimethylsulfoxide (DMSO)- or AGK2-treated media, and the infection time course was initiated at 0 HPI. ( B ) Quantification of supernatant virus titers collected from cells treated with DMSO (vehicle control), 2.5- or 5.0-µM AGK2 at 120 and 144 HPI with either AD169 HCMV or TB40/E HCMV. ( C ) Confocal analysis of endogenous SIRT2 localization in uninfected cells and at 24, 48, 72, and 96 HPI with AD169 HCMV. Infection was verified by antibody-based detection of viral protein IE1 (24 and 48 HPI) or pUL99 (72 and 96 HPI). Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining. Scale bars, 50 µM. ( D ) Diagram of the 5-day HCMV replication cycle, with numbers corresponding to the replication cycle stages analyzed in panels E–H. ( E ) Assessment of HCMV entry into host cells following DMSO or AGK2 treatment, analyzed using qPCR-based quantification of intracellular viral genomes at 0 HPI with AD169 HCMV. ( F ) Virus genomes produced at 48 or 72 HPI with AD169 HCMV in DMSO- or AGK2-treated cells, quantified by qPCR. ( G ) Titer of cell-associated HCMV collected from DMSO- or AGK2-treated cells at 72 HPI with AD169 HCMV. ( H ) Particle-to-infectious unit ratio of virus released by 120 HPI with AD169 HCMV from DMSO- or AGK2-treated cells. ( I ) Quantification of supernatant virus titers collected at 120 HPI with AD169 HCMV. For this assay, cells were not pre-treated with AGK2 prior to infection but were instead treated with either DMSO or AGK2 at 0, 24, 48, or 72 HPI. N = 3 biological replicates for panels B and E–I. Significance was determined by analysis of variance for panel B and Student’s t -test for panels E–I. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars indicate standard deviation. GO, gene ontology; HPI, hours post-infection; IP, immunoaffinity purification; LC-MS/MS, liquid chromatography tandem mass spectrometry; MS, mass spectrometry; n.s., not significant; qPCR, quantitative PCR.

Article Snippet: AGK2 stock solution was generated by resuspending powdered high-performance liquid chromatography (HPLC)-grade AGK2 (Fisher Scientific) in DMSO.

Techniques: Histone Deacetylase Assay, Activity Assay, Infection, Control, Incubation, Virus, Staining, Produced, Standard Deviation, Immunoaffinity Purification, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Real-time Polymerase Chain Reaction

Journal: mSystems

Article Title: Sirtuin 2 promotes human cytomegalovirus replication by regulating cell cycle progression

doi: 10.1128/msystems.00510-23

Figure Lengend Snippet: Acetylome analysis reveals altered acetylation status of cell cycle proteins following inhibition of SIRT2 with AGK2 treatment. ( A ) Acetylome workflow for investigating the impact of SIRT2 on protein abundance and acetylation status during AD169 HCMV infection. ( B ) Diagram depicting the total number of unique acetylated peptides identified in our data set with the proportion that were increased in abundance following SIRT2 inhibition. Subcellular localization analysis was performed with the parent proteins of the 393 differentially regulated acetylated peptides using GO and UniProt databases. ( C ) Diagrams of known SIRT2 substrates with temporally altered lysine acetylation following SIRT2 inhibition. Protein length, acetylated lysine location, and functional domains (green) are shown. See also Fig. S4D. Bar graphs depict the temporal abundance of each acetylated peptide at 0, 12, and 24 HPI with AGK2 treatment relative to DMSO control. ( D ) The number of acetylation sites per protein that increased in abundance with SIRT2 inhibition. ( E ) Heatmap of acetylated peptides that increased in abundance at least twofold with AGK2 treatment relative to DMSO control. The top heatmap shows acetylated peptide abundances. The bottom heatmap shows the protein-normalized acetylated peptide abundances. ( F ) Gene ontology enrichment analysis of all proteins containing acetylation sites that were increased in abundance with AGK2. The dotted line provides false discovery rate (FDR), and the bars correspond with fold enrichment. ( G ) Proteins that were identified as high-confidence SIRT2 interacting proteins in our IP-MS analysis, which also contain an acetylated lysine that increased in abundance with SIRT2 inhibition. Donut charts depict the min-max normalized temporal abundance of each protein in the IP-MS data set. Rectangular heatmaps depict the temporal fold change in abundance of acetylated peptides belonging to each protein. ( H ) Diagrams of cyclin-dependent kinase proteins with temporally altered lysine acetylation driven by SIRT2 inhibition. Protein length, acetylated lysine location, and functional domains (green) are shown. Bar graphs depict the temporal abundance of each acetylated peptide at 0, 12, and 24 HPI with AGK2 treatment relative to control. N = 2 biological replicates.

Article Snippet: AGK2 stock solution was generated by resuspending powdered high-performance liquid chromatography (HPLC)-grade AGK2 (Fisher Scientific) in DMSO.

Techniques: Inhibition, Quantitative Proteomics, Infection, Functional Assay, Control, Protein-Protein interactions

Journal: mSystems

Article Title: Sirtuin 2 promotes human cytomegalovirus replication by regulating cell cycle progression

doi: 10.1128/msystems.00510-23

Figure Lengend Snippet: SIRT2 inhibition and CDK2(K6) acetylation drive cell cycle progression from G1 to S phase. ( A ) Illustrative contour maps depicting the gating parameters utilized for flow cytometry-based cell cycle profiling experiments. Single cells were isolated by gating based on FSC, SSC, and DAPI profiles. Mitotic cells were isolated based on the presence of phosphorylated histone H3 S10 (pH3-S10) and non-mitotic cells were further gated into G1, S, or G2 phase populations. Cells in S phase were gated based on EdU staining, as EdU is incorporated into newly synthesized DNA. Cells in G1 and G2 were gated based on DAPI profiles, as cells in G2 possess double the DNA content of cells in G1. ( B ) Representative contour maps for both treatment conditions (DMSO or AGK2) showing the proportion of cells in G1, S, or G2 phase. The flow cytometry-based cell cycle profiling experiment was performed for uninfected cells (mock) and infected cells at 0, 6, 12, and 24 HPI with AD169 HCMV. ( C ) Quantification of the data shown in panel B across three biological replicates. The average percentage of cells in each cell cycle stage is shown for each analyzed infection time point and treatment condition. ( D ) Cell cycle diagram depicting the cell cycle stages (G0, G1, S, G2, and M) and the key cyclin and CDK proteins that regulate progression through and between stages. ( E ) Quantification of supernatant virus titer at 144 HPI following treatment with a CDK2 inhibitor (CDK2-IN-4) and AGK2. Cells were incubated with (+) or without (−) CDK2-IN-4 for 12 hours prior to infection with AD169 HCMV. AGK2 or DMSO (vehicle control) were added to the cells at 0 HPI, following removal of HCMV inoculated cell media. Virus titer was calculated as AGK2-treated samples relative to DMSO-treated samples for each CDK2-IN-4 treatment condition. ( F ) Quantification of the change in the percentage of cells existing in G1, S, or G2, driven by stable expression of acetyl mimic CDK2(K6Q) or lysine charge mimic CDK2(K6R) constructs relative to WT CDK2(K6) expression. N = 3 biological replicates. Significance was determined by Student’s t -test for panels C and E and analysis of variance for panel F. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars indicate standard deviation. FSC, forward scatter; SSC, side scatter.

Article Snippet: AGK2 stock solution was generated by resuspending powdered high-performance liquid chromatography (HPLC)-grade AGK2 (Fisher Scientific) in DMSO.

Techniques: Inhibition, Flow Cytometry, Isolation, Staining, Synthesized, Infection, Virus, Incubation, Control, Expressing, Construct, Standard Deviation

Journal: mSystems

Article Title: Sirtuin 2 promotes human cytomegalovirus replication by regulating cell cycle progression

doi: 10.1128/msystems.00510-23

Figure Lengend Snippet: SIRT2 inhibition delays HCMV replication kinetics. ( A ) Our TRUSTED targeted mass spectrometry assay was utilized for detection and quantification of HCMV protein abundances throughout infection with AD169 HCMV following DMSO or AGK2 treatment. Heatmaps depict the abundance of HCMV proteins throughout infection following treatment with DMSO (top heatmap) or AGK2 (center heatmap). The bottom heatmap depicts the fold change in abundance (AGK2 relative to DMSO treatment) for each viral protein across analyzed infection time points. Proteins are grouped by their temporal class: IE, DE, LL, and L. N = 3 biological replicates. ( B ) Euclidean distance analysis showing the impact of SIRT2 inhibition on the temporality of HCMV protein abundance profiles. ( C ) Proposed model for the function of SIRT2 deacetylase activity in promoting HCMV replication. DE, delayed early; IE, immediate-early; L, late; LL, leaky late;N.D., not detected.

Article Snippet: AGK2 stock solution was generated by resuspending powdered high-performance liquid chromatography (HPLC)-grade AGK2 (Fisher Scientific) in DMSO.

Techniques: Inhibition, Mass Spectrometry, Infection, Quantitative Proteomics, Histone Deacetylase Assay, Activity Assay