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. 2013;9(5):e1003333.
doi: 10.1371/journal.ppat.1003333. Epub 2013 May 16.

Saturated very long chain fatty acids are required for the production of infectious human cytomegalovirus progeny

Emre Koyuncu  1 , John G Purdy, Joshua D Rabinowitz, Thomas Shenk
Affiliations

Saturated very long chain fatty acids are required for the production of infectious human cytomegalovirus progeny

Emre Koyuncu et al. PLoS Pathog. 2013.

Abstract

Human cytomegalovirus hijacks host cell metabolism, increasing the flux of carbon from glucose to malonyl-CoA, the committed precursor to fatty acid synthesis and elongation. Inhibition of acetyl-CoA carboxylase blocks the production of progeny virus. To probe further the role of fatty acid metabolism during infection, we performed an siRNA screen to identify host cell metabolic enzymes needed for the production of infectious cytomegalovirus progeny. The screen predicted that multiple long chain acyl-CoA synthetases and fatty acid elongases are needed during infection, and the levels of RNAs encoding several of these enzymes were upregulated by the virus. Roles for acyl-CoA synthetases and elongases during infection were confirmed by using small molecule antagonists. Consistent with a role for these enzymes, mass spectrometry-based fatty acid analysis with ¹³C-labeling revealed that malonyl-CoA is consumed by elongases to produce very long chain fatty acids, generating an approximately 8-fold increase in C26-C34 fatty acid tails in infected cells. The virion envelope was yet further enriched in C26-C34 saturated fatty acids, and elongase inhibitors caused the production of virions with lower levels of these fatty acids and markedly reduced infectivity. These results reveal a dependence of cytomegalovirus on very long chain fatty acid metabolism.

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Conflict of interest statement

I have read the journal's policy and have the following conflicts: JDR and TS are shareholders and receive consulting fees from Kadmon Corp. This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials.

Figures

Figure 1
Figure 1. siRNA screen for cellular metabolic enzymes required for efficient HCMV replication.
Fibroblasts were transfected in 96-well plates with siRNAs and mock-infected or infected with BADinUL99GFP (0.5 IU/cell) 24 h later. HCMV yield in the supernatant was determined at 96 hpi. (A) Effect of siRNA controls used in the screen on HCMV yield. siRNAs targeting the cellular kinase PLK1, non-targeting siRNA (siNT), HCMV IE2 (siIE2) and the cellular kinase VPS34 (siVPS34_1–3) were included on each 96-well plate along with siRNAs to be screened for antiviral activity. The values represent the normalized yields from 18 independent experiments. siNT was assayed in octuplicate (n = 144) and siPLK1 was assayed in quadruplicate in each experiment (n = 72). Error bars represent ±1 standard deviation of the mean. (B) siRNAs targeting genes associated with fatty acid metabolism were identified as hits in the screen. Three siRNAs were tested for each target, and their effect on HCMV yield was quantified, log2-transformed and plotted. The siRNA showing the most robust effect on HCMV yield are identified for several enzymes. siRNAs designated in green type reduce virus yield and those in red type elevate virus production. Hits marked by an asterisk (*) are cases where only one siRNA out of three affected the virus yield. The dashed black horizontal lines delimit the spread of the 72 controls in the assay (1.4 times the mean) and the dashed colored horizontal lines mark the deviation from the control mean (3× SD) required to consider an siRNA a hit. (C) siRNA hits grouped by gene ontology analysis.
Figure 2
Figure 2. Identification of genes involved in fatty acid metabolism that are induced during HCMV infection.
(A) RT-qPCR analysis of cellular RNAs. RNA was analyzed at 48 hpi with BADinUL99GFP (10 IU/cell). Results were normalized to the expression level of 4 housekeeping genes, except in the case of elongases where GAPDH alone was used, and presented as log2-transformed fold change (infected/mock). Fold increases of ELOVL7, ACSL6, ELOVL3 and SLC27A2 are indicated. The results show the average of two independent experiments. (B) RT-qPCR analysis of acyl-CoA synthetase RNAs in HCMV-infected cells. Results are from data in panel A. Error bars represent ±1 SD of the mean. (C) ACSL1 protein accumulates during HCMV infection. Fibroblasts were mock-infected or infected with BADinUL99GFP (3 IU/cell), harvested after indicated time intervals and analyzed by western blot by using antibody to ACSL1. β-actin was monitored as a loading control. Band intensities were quantified using ImageJ software and ratio of ACSL1/β-actin after infection was compared to that of mock infected cells (M). (D) RT-qPCR analysis of elongase RNAs. RNA was analyzed at 48 hpi with BADinUL99GFP (10 IU/cell). Results are from two independent experiments assayed in triplicate. Error bars represent ±1 SD of the mean. (E) ELOVL3 protein accumulates in HCMV-infected cells. Cells were treated as in panel C and analyzed by western blot by using an antibody to ELOVL3. β-actin was monitored as a loading control. Band intensities were quantified as in panel C.
Figure 3
Figure 3. Triacsin C inhibits HCMV replication.
(A) Triacsin C blocks the production of HCMV progeny. Fibroblasts were infected with BADinUL99GFP (1 IU/cell) and maintained in medium containing 10% serum. Cultures were treated at 2 hpi with DMSO (vehicle) or triacsin C at indicated concentrations. At 96 hpi, cell viability and virus yield (intracellular plus extracellular) was assayed. The results are from two biological replicates. Error bars represent ±1 SD of the mean. (B) Triacsin C does not block HCMV cytoplathic effect or pUL99-GFP expression. Fibroblasts were infected with BADinUL99GFP (1 IU/cell) and treated with triacsin C (250 nM) at 2 hpi. At 96 hpi images were captured by phase microscopy and fluorescent microscopy. (C) Triacsin C does not affect the accumulation of immediate early, early and late viral proteins. Fibroblasts were infected with BADinUL99GFP (1 IU/cell) and treated with DMSO or triacsin C (250 nM) 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.
Figure 4
Figure 4. Pharmacological inhibitors of fatty acid elongases reduce HCMV yield.
(A) Elongase inhibitors, but not inactive derivatives, block the production of infectious progeny. At 2 hpi with BADinUL99GFP (1 IU/cell), fibroblasts were treated with DMSO (vehicle) or drugs at indicated concentrations. At 96 hpi, virus yield (intracellular plus extracellular) was assayed, and cell viability was monitored for indicated samples. The results report two biological replicates. Error bars represent ±1 SD of the mean. (B) Elongase inhibitors block cytopathic effect and pUL99-GFP accumulation. Cells were infected with BADinUL99GFP (1 IU/cell) and treated with drugs (50 µM) at 2 hpi. At 96 hpi images were captured by phase microscopy and fluorescent microscopy. (C) Effect of Endo-1k on the accumulation of viral proteins. Fibroblasts were infected with BADinUL99GFP (1 IU/cell) and treated with DMSO or Endo-1k (50 µM) at 2 hpi. Cells were harvested at indicated times after infection and processed for western blot analysis using antibodies specific for IE1, pUL44, pUL26, pUL55, pUL83 and pUL99. β-actin served as a loading control. (D) Elongase inhibitors reduce the infectivity of virions . Cells were treated with drugs (50 µM) at 2 hpi, and received fresh medium and drugs at 48 hpi (Medium replaced) or maintained without re-feeding (Medium not replaced). At 96 hpi, virus particles and infectivity (IU) were quantified. Results report three independent experiments, and error bars represent ±1 SD of the mean. **p<0.005 (t-test, ratio of virus particles/infectivity after drug treatment was compared to that of DMSO treated cells).
Figure 5
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.
Figure 6
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.
Figure 7
Figure 7. Genes linked to VLCFA synthesis identified by the siRNA screen and RT-qPCR analysis.
Genes whose knockdown inhibited HCMV replication are designated red. Blue genes were not assayed in the screen. Red arrows mark genes whose transcript levels are increased after infection and black hyphens mark the genes whose transcript levels do not change more than 3-fold. The expression levels of unmarked genes were not assayed.
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