Saturated Very Long Chain Fatty Acids Are Required for the Production of Infectious Human Cytomegalovirus Progeny
Figure 6
VLCFAs are synthesized from pre-existing fatty acids during HCMV infection.
(A) Elongation of pre-existing C18 fatty acids. Fibroblasts were either mock-infected or infected with BADwt (3 IU/cell) and feed U-13C-glucose starting at 0 hpi. Lipids were extracted at 72 hpi and the 13C-labeling of C26:0 fatty acid tails is shown. Inset within the figure is a schematic representation of C26:0 fatty acid. The first 16 carbons are added by fatty acid synthase (FAS, black) and the remaining carbons nearest the carboxylic group are added by elongases (red). (B) Kinetic analysis by carbon labeling of ≥C26:0 VLCFAs during HCMV infection. MRC5 cells were infected with BADwt (3 IU/cell), U-13C-glucose was added at 0 hpi and saponified fatty acids from total cellular lipids were analyzed by LC-MS at the indicated times. Results report three independent experiments, and error bars represent ±1 SD of the mean. (C) The average amount of 13C-label incorporated into C18:0-C30:0 at 48 hpi. As in part A, fibroblasts were infected and fed 13C-glucose at 0 hpi. The unlabeled fraction (C18:0 and shorter) existed prior infection and the labeled forms (i.e. the longer chains) were formed following infection. (D) Analysis of lipid droplets. Fibroblasts were infected with BADinUL99GFP (10 IU/cell). Cells were fixed at 96 hpi and stained with oil red O to visualize lipid droplets (visible in red and phase channels). Infected cells were identified by pUL99-GFP expression (green), and DNA was stained with Hoechst 33258 (blue). The image is representative of >5 different visual fields analyzed in two independent experiments.