Saturated Very Long Chain Fatty Acids Are Required for the Production of Infectious Human Cytomegalovirus Progeny
Figure 5
Analysis of the fatty acid content of cells and HCMV virions.
(A) HCMV infection alters cellular (left side) and virion fatty acids (right side). Fibroblasts were infected with BADwt (3 IU/cell) in serum-free medium. At 72 hpi, total lipids from cells or purified virions were extracted and saponified to release fatty acid tails, which were analyzed by LC-MS. Each fatty acid is identified by its chain length and degree of unsaturation, y-axis. The percent of total signal detected for each fatty acid is represented by bars, x-axis. Bar colors represent the log2-fold change over mock-infected cells (black = no change; red = increase; blue = decrease). (B) Treatment with elongase inhibitor blocks virus-induced increase in saturated and monounsaturated VLCFAs (≥C26). Fibroblasts were mock infected or infected with BADwt (3 IU/cell) and treated (50 µM) with Endo-1k, Exo-1w, or DMSO (vehicle). Fresh medium containing the inhibitors was replaced at 48 hpi. Fatty acids were analyzed by LC-MS at 72 hpi. Results report three independent experiments, and error bars represent ±1 SD of the mean. * p<0.05 (t-test, compared to control condition). (C) Rescue from drug block with exogenously added fatty acids. BADinUL99GFP-infected fibroblasts (1 IU/cell) were treated with Endo-1k (50 µM) or DMSO (vehicle) at 2 hpi. Hexacosanoic acid (C26:0), stearic acid (C18:0), oleic acid (C18:1) or ethanol (ETOH, vehicle) were added (50 µM) with the drug. Cells were harvested at 96 hpi, and the yield of infectious virus was assayed. Results report three independent experiments, and error bars represent ±1 SD of the mean. *p<0.05, **p<0.005, non-significant (n.s.) p>0.05 (t-test, ratio of DMSO/Endo-1k in fatty acid treated cells were compared to that of ETOH treated cells). (D) The effect of hexacosanoic acid on viral protein accumulation in infected fibroblasts (BADinUL99GFP, 1 IU/cell) treated with Endo-1k. Cultures received hexacosanoic acid (C26:0) (50 µM) or ethanol (vehicle for C26:0), simultaneously with drug or DMSO (vehicle for the drug) at 2 hpi. Cells were harvested at indicated times after infection and processed for western blot analysis using antibodies specific for IE1, pUL26, and pUL99. β-actin served as a loading control.