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