Fig 1.
VACV infection reprograms cellular metabolism profoundly and globally under the glutamine-depletion conditions.
(A) Experimental design of global metabolic profiling. Four biological replicates of HFFs per treatment were either mock-infected or infected with VACV at an MOI of 3 for either 8 or 16 hours in medium with glucose (Glc) or glucose plus asparagine (Glc+N). Metabolites were extracted, and their levels were measured. (B & C) Principal component analysis (PCA) showing a clear separation between VACV-infected and uninfected HFFs in glucose plus asparagine medium (B) and in HFFs in glucose only medium (C). Each small circle indicates one sample. The shaded region indicates the 95% confidence interval. PC1 represents the effect of VACV infection and PC2 represents the effect of time.
Fig 2.
VACV infection elevates the levels of TCA cycle intermediates, including citrate.
(A) A simplified overview of the TCA cycle and citrate metabolism. The pyruvate generated from glycolysis can be converted into Acetyl-CoA that reacts with OAA to form citrate in the mitochondria of a cell. The citrate can then be transported out of the mitochondria where it gets converted to Acetyl-CoA and OAA. The cytosolic Acetyl-CoA can act as a precursor for fatty acid biosynthesis. The fatty acids undergo β-oxidation in the mitochondria to convert into Acetyl-CoA to feed the TCA cycle. Glutamine can also feed in the TCA cycle to increase the citrate level by converting it to α-KG. (B) VACV infection increases the levels of most of the TCA cycle intermediates in the absence of exogenous glutamine. The levels of TCA cycle intermediates at 8 hpi in the metabolic profiling of Fig 1A were shown. (C) VACV infection increases the level of glutamate. The level of glutamate in HFFs in the global metabolic profiling of Fig 1A were shown. (D) VACV infection increases the citrate level in HFFs cultured in medium without exogenous glutamine. HFFs infected with indicated viruses at MOI of 5 in media with glucose only (Glc) or glucose plus asparagine (Glc+N). Citrate level was measured at 8 hpi using a citrate assay kit. (E) VACV infection increases the citrate level in HFFs cultured in medium with glutamine. HFFs infected with WT VACV at an MOI of 5 in medium with glucose plus glutamine and the citrate level was measured at indicated time points using a citrate assay kit. (F) VACV infection increases the levels of OAA. HFFs infected with WT VACV at MOI of 5 in HFFs cultured in medium with glucose plus glutamine and the OAA level was measured at 8 hpi. (G) VACV infection increases the ATP levels in HFFs. HFFs were infected with MOI of 2 of WT-VACV or vΔVGF (VACV with VGF gene deleted) in medium containing glucose and glutamine. The ATP levels were measured at 8 hpi by using an ATP assay kit. (H) TCA Cycle activity is important for VACV replication. HFFs infected with WT VACV at MOI of 2 or 0.1 in media with glucose plus glutamine in the presence or absence of 50 μM Enasidenib. VACV titers measured at 24 and 48 hpi for MOI 2 and 0.1 respectively using a plaque assay. (I) Enasidenib treatment has minimal effect on HFF viability. HFFs were treated with 50 μM Enasidenib in medium with glucose plus glutamine. Cell viability measured by a trypan blue assay at 48 h post treatment. Error bars represent the standard deviation of at least three biological replicates. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001.
Fig 3.
VACV infection alters the TCA cycle-related metabolism.
(A) A decrease in Acetyl-CoA upon VACV infection in HFFs cultured in media without glutamine. The level of Acetyl-CoA at 8 hpi in the metabolic profiling of Fig 1A was shown. (B) VACV infection decreases the level of acetyl CoA in HFFs cultured in medium containing glutamine. HFFs infected with WT VACV at an MOI of 2 in media with glucose plus glutamine and the Acetyl-CoA level was measured at 8 hpi using an Acetyl-CoA assay kit. (C) A simplified overview of carnitine metabolism in β-oxidation. The long-chain fatty acids are acylated and then carnitylated by carnitine palmitoyltransferase system, which is then transported into the mitochondrial matrix for β-oxidation to fuel the TCA cycle. (D) VACV infection increases the levels of carnitine-conjugated fatty acids. The metabolic profiling data of fatty acyl carnitines in VACV-infected HFFs (Supplementary File S2) was uploaded to the MetaboAnalyst tool and then a hierarchically clustered heatmap was generated using Ward’s minimum variance and Euclidean distance measure. Color keys indicate the levels of different metabolites; blue: lowest, red: highest. The number on top of the plots represent the p-values comparing the average levels of indicated metabolites levels in mock- and VACV-infected HFFs (E) The levels of long-chain fatty acids are reduced in VACV-infected HFFs. The metabolic profiling data of long-chain fatty acids in VACV-infected HFFs (Supplementary File S2) was processed as in Fig 3D. (F) VACV infection does not affect the level of lactate. The level of lactate in HFFs infected with MOI-3 of WT-VACV in media with glucose (Glc) or glucose plus asparagine (Glc+N) at 8 hpi was determined by global metabolic profiling in Fig 1A. (G) The glycolysis intermediates are either unaffected or reduced by VACV infection. The levels of glycolysis intermediates in HFFs infected with MOI-3 of WT-VACV in medium with glucose (Glc) or glucose plus asparagine (Glc+N) at 8 hpi as determined by global metabolic profiling in Fig 1A. Error bars represent the standard deviation of at least three biological replicates. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Fig 4.
Both Glycolysis and β-oxidation contribute towards the citrate level enhancement during VACV infection.
(A) Inhibition of glycolysis and fatty acid oxidation reduces the increase of citrate levels during VACV infection. HFFs were mock-infected or infected with WT-VACV at an MOI of 5 in medium with glucose plus glutamine in the presence or absence of 50 μΜ bromopyruvate, 50 μM PFK-15, 100 μΜ of CPI-613, and 50 μΜ etomoxir. Citrate levels measured at 4 hpi using a citrate assay kit. (B) HFFs treated with indicated chemicals at a concentration as listed in Fig 4A in medium with glucose plus glutamine. Cell viability measured by a trypan blue assay at 48 h post treatment. (C) Glycolysis inhibition suppresses VACV replication. HFFs infected with WT VACV at an MOI of 2 (for 24 h) or MOI of 0.1 (for 48 h) in medium with glucose plus glutamine with or without 50 μΜ bromopyruvate, 50 μM PFK-15. Virus titers measured by a plaque assay. (D) Inhibition of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase reduces VACV titers. HFFs infected with WT VACV at an MOI of 2 (for 24 h) or MOI of 0.1 (for 48 h) in medium with glucose plus glutamine in the presence or absence of 100 μΜ CPI-613. Virus titers were measured by a plaque assay. Error bars represent the standard deviation of at least three biological replicates. ns, P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Fig 5.
VACV growth factor (VGF) deletion abolishes the elevation of citrate level during viral infection.
(A) Inhibition of DNA synthesis does not inhibit the increased citrate level upon VACV infection. HFFs were infected with VACV at an MOI of 5 in medium with glucose plus glutamine in the presence or absence of 40 μg/mL AraC. Citrate level was measured at 8 hpi. (B) Inhibition of protein synthesis reduces citrate level in VACV-infected HFFs. HFFs were infected with VACV at an MOI of 5 in medium with glucose plus glutamine in the presence or absence of 100 μg/mL Cycloheximide. Citrate level was measured at 2 hpi. (C-E) VGF is required for the elevation of citrate level during VACV infection. (C) HFFs were infected with either WT-VACV or vΔVGF or a VGF revertant vΔVGF_Rev at an MOI of 5 in medium with glucose plus glutamine. Citrate level was measured at 4 hpi. (D) HFFs were infected with indicated viruses at an MOI of 5 in medium with glucose only (Glc). Citrate level was measured at 4 hpi. (E) HFFs were infected with indicated viruses at an MOI of 5 in with glucose + asparagine (Glc+N), and citrate level was measured at 4 hpi. (F) VGF mRNA expression in WT-VACV, vΔVGF, and vΔVGF_Rev. RNA was extracted from HFFs infected with indicated viruses at an MOI of 5 for 1 h in medium with glucose plus glutamine, and reverse transcription-quantitative PCR (qRT-PCR) analysis was performed. (G) VGF deletion does not affect the levels of other VACV early proteins. HFFs infected with indicated viruses at an MOI of 5. Western blotting analysis was performed at indicated time post infection to measure the levels of VACV E3 and L2 proteins. Error bars represent the standard deviation of at least three biological replicates. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Fig 6.
Inhibition of the STAT3 pathway and its upstream signaling decreases citrate levels during VACV infection.
(A) Inhibition of the EGFR pathway decreases the citrate level in VACV-infected HFFs. HFFs were infected with WT VACV at an MOI of 5 in the presence or absence of 3 μM afatinib. The citrate level was measured at 4 hpi. (B) Inhibition of the MAPK pathway decreases the citrate level during VACV infection. HFFs were infected with WT VACV at an MOI of 5 in the presence or absence of 20 μM PD0325901. The citrate level was measured at 2 hpi. (C) Inhibition of the STAT3 pathway decreases the citrate level in VACV-infected cells. HFFs were infected with VACV at an MOI of 5 in the presence or absence of 3 μM stattic. The citrate level was measured at 4 hpi. (F) siRNA-mediated knockdown of STAT3. HFFs were transfected with a negative control siRNA or two specific siRNA targeting STAT3 for 48 h. Western blotting analysis was performed to measure the level of STAT3. (G) siRNA-mediated knockdown of STAT3 decreases citrate level during VACV infection. HFFs were transfected with indicated siRNAs for 48 h and then infected with an MOI of 5 of VACV for 4 h, and the citrate level was measured. All the infections were performed in media with glucose plus glutamine. Error bars represent the standard deviation of at least three biological replicates. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
Fig 7.
VACV infection induces non-canonical STAT3 phosphorylation at S727 in a VGF-dependent manner.
(A) VACV VGF is indispensable to activate STAT3 (S727) phosphorylation. HFFs infected with indicated viruses at an MOI of 5 for the indicated time. Western blotting analysis was performed to measure the levels of various forms of STAT3. (B) Upregulation of STAT3 S727 phosphorylation starts early during VACV infection. HFFs infected with indicated viruses at an MOI of 5. The samples were collected at 10 min post-infection and 8 hpi, respectively, followed by Western blotting analysis. (C) VACV activates STAT3 (S727) phosphorylation in the absence of glutamine in the medium. HFFs were infected with indicated viruses at an MOI of 5 in medium with glucose only (Glc) or with glucose+asparagine (Glc+N). Protein levels were detected by performing a Western blotting analysis at 4 hpi. (D) Inhibition of the EGFR pathway decreases STAT3 S727 phosphorylation in VACV infected cells. HFFs were infected with MOI of 5 of WT-VACV with or without 3 μM afatinib treatment. Western blotting analysis was performed at 4 hpi to test the levels of different forms of STAT3 protein. (E) Inhibition of the MAPK pathway decreases both Y705 and S727 phosphorylation. HFFs were infected with MOI of 5 of VACV in medium with or without 20 μM PD0325901 treatment. Western blotting analysis was performed at 2 hpi to detect the levels of different forms of STAT3 protein. (F) Stattic treatment inhibits S727 phosphorylation. HFFs were infected with MOI of 5 of WT VACV with or without 3 μM stattic. At 4 hpi, Western blotting analysis was performed to detect the levels of different forms of STAT3 protein. (G) STAT3 S727 phosphorylation is independent of the JAK1/2 pathway. HFFs were infected with an MOI of 5 of VACV in medium with or without 5 μM ruxolitinib treatment. Western blotting analysis was performed at 4 hpi to measure different protein levels. (H) Ruxolitinib treatment decreases the induction of citrate level upon VACV infection. HFFs were infected with WT VACV at an MOI of 5 in the presence or absence of ruxolitinib treatment. The citrate level was measured at 4 hpi. (I) Effects of inhibition of STAT3 and its upstream signaling pathways on VACV early protein expression. HFFs infected with WT VACV at an MOI of 2 in the presence or absence of 3 μM afatinib, 20 μM PD0325901, 3 μM stattic, 5 μM ruxolitinib, 40 μg/mL AraC, or 100 μg/mL cycloheximide. The levels of VACV E3 protein was measured at 2 hpi by a Western blotting analysis. (J) Effects of inhibition of STAT3 and its upstream signaling pathways on VACV early protein levels. HFFs infected at an MOI of 2 with a recombinant VACV expressing Gaussia luciferase under virus early VGF promoter in the presence or absence of 3 μM afatinib, 20 μM PD0325901, 3 μM stattic, 5 μM ruxolitinib, 40 μg/mL AraC, or 100 μg/mL cycloheximide. Early gene expression was measured by a Gaussia luciferase activity assay kit at 2 hpi. All experiments were performed in media with glucose plus glutamine unless otherwise stated. Error bars represent the standard deviation of at least three biological replicates. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. The blots were from different lanes on the same gel and the dashed lines indicate that some non-relevant lanes were removed.
Fig 8.
Proposed model by which VACV infection promotes the TCA cycle.
VACV infection enhances the levels of TCA cycle intermediates and related products. Upon VACV infection, the levels of Acetyl-CoA decrease, while the levels of fatty acyl carnitines (key metabolites for β-oxidation of fatty acids) increase. The increase in the level of citrate can be attributed to the VACV VGF mediated upregulation of non-canonical STAT3 phosphorylation at S727 via EGFR and MAPK pathways. Although not upregulated by VACV, the Y705 phosphorylation of STAT3 is also important for enhancing citrate level. It is unclear if additional viral factors are also required to elevate the TCA cycle and if VGF alone can exert the function in uninfected cells. Red upward arrows indicate increase and black downward arrows indicate decrease of indicated intermediates.