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. 2018 Nov 23;9(1):4967.
doi: 10.1038/s41467-018-07179-w.

Orchestration of protein acetylation as a toggle for cellular defense and virus replication

L A Murray  1 , X Sheng  1 , I M Cristea  2
Affiliations

Orchestration of protein acetylation as a toggle for cellular defense and virus replication

L A Murray et al. Nat Commun. .

Abstract

Emerging evidence highlights protein acetylation, a prevalent lysine posttranslational modification, as a regulatory mechanism and promising therapeutic target in human viral infections. However, how infections dynamically alter global cellular acetylation or whether viral proteins are acetylated remains virtually unexplored. Here, we establish acetylation as a highly-regulated molecular toggle of protein function integral to the herpesvirus human cytomegalovirus (HCMV) replication. We offer temporal resolution of cellular and viral acetylations. By interrogating dynamic protein acetylation with both protein abundance and subcellular localization, we discover finely tuned spatial acetylations across infection time. We determine that lamin acetylation at the nuclear periphery protects against virus production by inhibiting capsid nuclear egress. Further studies within infectious viral particles identify numerous acetylations, including on the viral transcriptional activator pUL26, which we show represses virus production. Altogether, this study provides specific insights into functions of cellular and viral protein acetylations and a valuable resource of dynamic acetylation events.

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Conflict of interest statement

The authors declare no Competing Interests.

Figures

Fig. 1
Fig. 1
Acetylation contributes to diverse protein functions in uninfected and infected cells. Host pathways that are regulated by acetylation in uninfected cells are manipulated by HCMV during its temporal replication cycle
Fig. 2
Fig. 2
Dynamic regulation and function of protein acetylation during HCMV infection. a Workflow of anti-acetyl lysine IP and MS from whole-cell lysate and enriched virions. b Investigating the relationship between number of acetylation sites and protein abundance. Average protein abundance in parts per million (ppm) sourced from PaxDB; Proteins in this database are scaled in ppm relative to the whole proteome of the reference data set; in this case, the abundances were drawn from the data set: H. sapiens–whole organism (integrated). c Examples of acetylations on low abundance proteins. d Acetylation motifs in uninfected (mock) and infected (96 hpi) cells, analyzed by IceLogo. Numbers of peptides used for motif analyses are indicated. Analysis of all time points is provided in Supplementary Fig. 1
Fig. 3
Fig. 3
Acetylation increases on proteins in immune response and metabolic pathways and decreases on transcriptional regulators. a Protein abundances in infection were normalized to mock abundances and plotted as log2 values. Four biological processes relevant to HCMV infection that display distinct protein abundance trends are illustrated. b K-means clustering was conducted in C3 (k = 7) to cluster changes in peptide acetylation levels at different time points of infection when compared with uninfected cells (right column). Whole-cell protein abundances (left) and peptide acetylation levels normalized to protein abundance (middle) are illustrated. Increased abundance, yellow; decreased abundance, blue; not detected in the proteome, gray. c–e Detected acetylation sites on proteins from indicated functional clusters representing site-specific regulation of acetylation during infection. Circles, proteins; squares, acetylation sites; red lines, previously unknown acetylation sites; black lines, known acetylation sites; red boxes, sites only detected during infection with abundances displayed on a 0–1 scale as they could not be normalized to mock samples; white bars, acetylated peptide not detected. c interferon response, d TCA cycle, e transcription regulation
Fig. 4
Fig. 4
Acetylation in the context of temporal subcellular localization and translocation during infection. a Heatmaps visualizing temporal changes in peptide acetylation levels (right) and acetylation normalized to protein abundance (left) in the nucleus, cytosol, mitochondria, ER, Golgi, plasma membrane (PM), peroxisome, and lysosome. Representative biological processes overrepresented in clusters of increasing and decreasing acetylations within specific subcellular compartments are shown. b Predicted localizations of the identified acetylated proteins in uninfected (mock) cells (vertical) are compared to the localizations of the proteins at 24, 48, 72, and 96 hpi (horizontal) (from Jean Beltran, et al. 2016). Numbers in the upper, light gray triangles along the diagonals indicate non-translocating proteins annotated from, and those in the lower, dark gray triangles indicate proteins annotated from Uniprot. Boxes displaced from the diagonal designate predicted putative translocating protein localization at given time points during infection. Numbers of translocating proteins are indicated by color code. As an example, of the proteins assigned to the ER/Golgi in uninfected cells, 34 were assigned as translocated to the PM at 24 hpi. c Example of a protein translocating from ER/G to PM at 24 hpi. A change in protein acetylation is also observed at 24 hpi. Peptide acetylation levels normalized to protein abundance are represented in parallel with organelle subcellular localization (assigned from) at each infection time point
Fig. 5
Fig. 5
Functional acetylation at the nuclear periphery during HCMV infection. a Nuclear periphery proteins with increased levels of acetylation during infection, scaled from 0 to 1 (left, protein abundance; middle, peptide acetylation level normalized to protein abundance; right, peptide acetylation level) b Acetylation sites detected on lamin and lamin-associated proteins. c Sequence alignment of identified LMNB1 acetyl-lysines from selected animal species. The identified acetyl-lysine residues are highlighted and the six flanking residues were excerpted for representation. d Schematic of LMNB1 protein domains with detected acetylated sites; upper amino acids demarcate domains; lower, acetylated lysines; yellow, acetylation sites that were pursued in this study. e Representative images of MRC5 cells transfected with all seven mCherry-LMNB1 constructs indicated. All constructs localize to the nuclear periphery and do not alter nuclear morphology. Scale bar = 5 μm. f–h Effect of LMNB1 mutants on extracellular virus produced. Cells expressing either wild-type (WT) or mutant LMNB1 were infected, and the supernatant collected at 120 hpi was then used to infect a reporter plate of fibroblasts. Titer (IU/mL) was measured by staining the reporter plate for viral immediate early protein IE1. Average of three biological replicates ± SD using a two-sided Student’s t test, *p value ≤ 0.05, **p value ≤ 0.01. i Cell-associated virus was collected and used to infect a reporter plate. Titer (IU/mL) measured via IE1 staining, **p value ≤ 0.01. Source data are provided as a Source Data file
Fig. 6
Fig. 6
LMNB1 acetylation impedes lamina disruption and viral capsid nuclear egress. a Live cell confocal fluorescence microscopy of infection time course in cells transfected with WT, K134Q, or K134R mCherry-LMNB1 (red) and infected with HCMV AD169 UL32-GFP (green). One slice through the center of the nucleus is shown. Representative images are shown; scale bar = 5 μm. b, c pUL32-GFP nuclear intensity in WT, K134Q, or K134R at each time point; the number of cells (n) is indicated. A two-sided Student’s t test was used. b Mean pUL32-GFP intensity across the center three slices inside the nucleus + SEM. c The ratio of mean pUL32-GFP intensity inside the nucleus to outside the nucleus across the center three slices + SEM. d The volume of the pUL32-GFP particles was analyzed in a 3D rending of each Z-stack, and the number of analyzed cells (n) is indicated. The mean pUL32-GFP particle volume + SEM is shown. A two-sided Student’s t test was used. *p value ≤ 0.05, **p value ≤ 0.01, ***p value ≤ 0.001, ****p value ≤ 0.0001. e Live cell confocal fluorescence microscopy of nuclei at 120 hpi for cells transfected with WT, K134Q, or K134R mCherry-LMNB1 (red) constructs. Left: one representative slice through the center of the nucleus. Right: zoom-in of the square on the left; scale bar = 5 μm. f Percentage of nuclear periphery disruption. Disruption in nuclear periphery are seen as infoldings, as visualized by mCherry-LMNB1 fluorescence across center slices. Mean percentage disruption + SEM is shown and cell numbers are indicated. A two-sided Student’s t test was used; ***p value ≤ 0.001, ****p value ≤ 0.0001. g Proposed model for the function of LMNB1 K134 acetylation. Acetylation may stabilize LMNB1, which may inhibit nuclear periphery deformation and thereby impede HCMV capsid nuclear egress and infectious virus production. Source data are provided as a Source Data file
Fig. 7
Fig. 7
Prevalent acetylation on HCMV proteins, including pUL26 acetylation that restricts viral replication. a Abundances (scaled 0–1) of acetylated viral peptides and their corresponding proteins detected during infection (left, protein abundance; right, peptide acetylation levels normalized to protein abundance). Acetylated lysines and temporal stages of infection (IE (immediate early), DE (delayed early), and L (late)) are indicated. b Lysine content in the proteomes of different viruses. Viral families were clustered by proteome similarities (of representative strains). The lysine percentages of their proteomes were color coded with green (< 5%), blue (5–6%), and yellow (> 6%). c Identified acetylated viral proteins and their corresponding compartments in the virion. d Site-specific acetylations on capsid, envelope, and tegument virion proteins. Functional domains are indicated, and listed in Supplementary Fig. 5. e Schematic of pUL26 domains and putative nuclear localization signal (NLS). f, g, h Viral titers (IU/mL) determined by IE1 staining for infections at MOI 3 (f), MOI 0.25 (g), or MOI 0.05 (h) for WT and K203 mutant viruses (K203Q, K203R). Average of three biological replicates ± SD. A two-sided Student’s t test was used. *p value ≤ 0.05, **p value ≤ 0.01. Source data are provided as a Source Data file
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