Human cytomegalovirus-induced host protein citrullination is crucial for viral replication

Figures

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  Fig. 1. HCMV infection induces protein citrullination. a Protein lysates from HFFs infected with HCMV strain Merlin (HCMV) (MOI 1 PFU/cell) at 48 and 96 h post infection (hpi) or from uninfected HFFs (mock) were exposed to an Rh–PG citrulline-specific probe (left panel) and subjected to gel electrophoresis to detect citrullinated proteins. Equal loading was assessed by Coomassie blue staining (right panel). One representative gel of three independent experiments is shown. b mRNA expression levels of PADI isoforms by RT-qPCR of HCMV-infected (24 hpi) vs. uninfected (mock) HFFs were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase GAPDH and expressed as mean fold change ± SEM over mock-infected cells (n = 3 independent determinations; PADI2, PADI4, and PADI6 P < 0.001, two-way ANOVA followed by Bonferroni’s post test). c PADI2 and PADI4 mRNA levels in HCMV-infected HFFs at the indicated time points (hpi) were normalized to GAPDH mRNA and expressed as mean fold change ± SEM over mock-infected cells (n = 3 independent determinations; PADI2: mock vs. 8 hpi P < 0.001, mock vs. 16 hpi P < 0.001, mock vs. 24 hpi P < 0.001, mock vs. 32 hpi P < 0.001; PADI4: mock vs. 8…

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  Fig. 2. Cl-A blocks HCMV replication. a HFFs were infected with HCMV (MOI 0.1 PFU/cell) and then treated with increasing concentrations of Cl-A, which were given 1 h prior to virus adsorption and kept throughout the whole experiment. At 144 hpi, viral plaques were microscopically counted and expressed as a percentage relative to untreated controls. The number of plaques is plotted as a function of Cl-A concentration. The concentrations resulting in 50% plaque formation (IC50) reduction are represented by the red line. The number of viable cells was determined for each Cl-A concentration using the MTT method (black line). Values are expressed as means ± SEM (error bars) of three independent experiments (untreated vs. 25 µM Cl-A P < 0.001, untreated vs. 50 µM Cl-A P < 0.001, untreated vs. 75 µM Cl-A P < 0.001, untreated vs. 100 µM Cl-A P < 0.001, untreated vs. 200 µM Cl-A P < 0.001, one-way ANOVA followed by Bonferroni’s post test). b To determine the number of viral DNA genomes in HCMV-infected HFFs, viral DNA was isolated at 144 hpi and analyzed by qPCR using primers amplifying a segment of the IE1 gene. GAPDH was used to normalize HCMV genome counts. Values are expressed as mean ± SEM…

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  Fig. 3. Effect of PAD2 and PAD4 gene knockout on HCMV replication. a Knockout (KO) gene variants in HFFs for PAD2 (PAD2 KO) and PAD4 (PAD4 KO) were generated using CRISPR/Cas9 technology. The efficiency of PAD2 and PAD4 protein depletion at 48 hpi was assessed by immunoblotting using antibodies against PAD2, PAD4, or α-tubulin (α-TUB), for equal loading. An anti-UL44 antibody was used to verify HCMV infection. The western blot and relative densitometric analysis are representative of three independent experiments. Values are expressed as fold change in PAD2 and 4 expression normalized to α-tubulin (α-TUB). b HFFs KO cells were infected with HCMV at an MOI of 0.1 PFU/cell. Viral supernatants were collected at the indicated time points and analyzed by standard plaque assay. Values are expressed as means ± SEM of three independent experiments (WT vs. PAD2 KO P < 0.001, WT vs. PAD4 KO P < 0.001, one-way ANOVA followed by Bonferroni’s post test). c To determine the number of viral DNA genomes in HCMV-infected HFFs KO cells (MOI 0.1), viral DNA was isolated at 144 hpi and analyzed by qPCR with primers amplifying a segment of the IE1 gene. GAPDH was used to normalize HCMV genome counts. Values…

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  Fig. 4. Characterization of the citrullinated proteome (citrullinome) of HCMV-infected cells. a Volcano plot depicting the host (upper panel—circle) and viral (lower panel—triangle) citrullinated proteins of infected cells vs. mock-infected cells at 48 hpi (left panel) and 96 hpi (right panel). Cell lysates from uninfected (mock) or HCMV-infected HFFs (MOI 1) were exposed to a biotin-PG to isolate citrullinated proteins on streptavidin agarose. Bound proteins were then subjected to on-bead tryptic digestion and analyzed by LC-MS/MS—in the graph, every identified citrullinated protein corresponds to a dot. The x axis represents the ratio of citrullination between mock and infected cells at the indicated time points, while the y axis indicates the statistical significance. Both variables were plotted on a logarithmic scale (n = 3). b Volcano plot depicting the host citrullinated proteins in HCMV AD169- (left panel) and AD169ΔIE1- (right panel) vs. mock-infected HFFs at 48 hpi. Cell lysates from uninfected (mock) or HCMV-infected (MOI 1) HFFs were exposed to a biotin-PG to isolate citrullinated proteins on streptavidin agarose. Bound proteins were then subjected to on-bead tryptic digestion…

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  Fig. 5. The antiviral role of IFIT1 and Mx1 against HCMV. a HFFs were silenced for IFIT1 and Mx1 using specific siRNAs (siIFIT1, siMx1, respectively). As negative control cells were also similarly transfected with scrambled siRNA (siCTRL). At 24 hpt, cells were infected with HCMV at an MOI of 0.1 PFU/cell. Viral supernatants were collected at 144 hpi and analyzed by standard plaque assay. Values are expressed as means ± SEM of three independent experiments (siCTRL vs. siIFIT1 P = 0.0111, unpaired two-tailed t test; siCTRL vs. siMx1 P > 0.05, unpaired two-tailed t test). b HFFs were transduced with AdVIFIT1 or AdVLacZ at an MOI of 10 PFU/cell. Subsequently, cells were infected with HCMV at an MOI of 1. The extent of virus replication was measured at 144 hpi. Results are expressed as means ± SEM of three independent experiments (AdVLacZ vs. AdVIFIT1 P > 0.05, unpaired two-tailed t test). c Gel electrophoretic mobility shift assay of IFIT1 binding to PPP-7SKas RNA. 50 nM of in vitro transcribed 7SK 5’-ppp-RNA was incubated with only buffer (buffer), 5 µM of recombinant IFIT1, either untreated (UNT rIFIT1) or treated with PAD2 (PAD2’d rIFIT1) and loaded on a tris-glycine agarose gel. Data…