Viral growth factor- and STAT3 signaling-dependent elevation of the TCA cycle intermediate levels during vaccinia virus infection
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.