Fig. 1: KSHV infection promotes CAD activity and induces RelA deamidation.

A A diagram depicting CAD in pyrimidine synthesis and in RelA deamidation to drive aerobic glycolysis via upregulating glycolysis-associating enzymes and regulators. B A heatmap of intracellular metabolites with z-score normalization for Human Oral Keratinocytes-16B (HOKs) infected with KSHV (MOI = 30, 48 h). Full names of the metabolites are provided in the methods. C HOKs and Lymphatic Endothelial Cells (LECs) were infected with KSHV (MOI = 30 and 10, respectively). Whole cell lysates (WCLs) were prepared at 24 h and analyzed by two-dimensional gel electrophoresis (2DGE) and immunoblotting. HOKs (D) and Tert-immortalized Microvascular Endothelial (TIME) cells (E) were infected with KSHV (MOI = 30 and 3, respectively). Real-time quantitative PCR (RT-qPCR) analyses of the indicated mRNAs were then performed. HOKs (F) and TIME cells (G) were infected with KSHV (MOI = 30 and 3, respectively) for 72 h. WCLs were processed in parallel and analyzed by immunoblotting with the indicated antibodies. H HOKs and LECs were infected with KSHV (MOI = 30 and 10, respectively) for the indicated hours. The culturing medium was collected to determine lactate concentration. I TIME cells were infected with KSHV (MOI = 3) for 72 h. The culturing medium was collected to determine lactate concentration at 16 h post medium replacement. J HOKs were infected with KSHV (MOI = 30) for 24 h. Cells were then analyzed by Seahorse assay and oxygen consumption rate (OCR) was plotted against the extracellular acidification rate (ECAR). K 293 T cells were transfected with plasmids expressing flag-tagged wild-type (WT) CAD or CAD phospho-mimetic mutant S1859E, and then infected with KSHV (MOI = 5) for 24 h. WCLs were analyzed by 2DGE and immunoblotting. Data are presented as mean ± SD of n = 3 biological replicates (1D, 1E, 1H, and 1I) and mean ± SEM of n = 3 biological replicates (1 J). Blots were representative of at least two independent experiments (1 C, 1 F, 1 G, and 1 K). Significance was calculated using two-tailed, unpaired Student’s t-test. Source data are provided as a source data file. See also Fig. S1.