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. 2015 Mar 3;10(8):1375-85.
doi: 10.1016/j.celrep.2015.02.003. Epub 2015 Feb 26.

Fatty acid elongase 7 catalyzes lipidome remodeling essential for human cytomegalovirus replication

John G Purdy  1 , Thomas Shenk  2 , Joshua D Rabinowitz  3
Affiliations

Fatty acid elongase 7 catalyzes lipidome remodeling essential for human cytomegalovirus replication

John G Purdy et al. Cell Rep. .

Abstract

Human cytomegalovirus (HCMV) infection rewires host-cell metabolism, upregulating flux from glucose into acetyl-CoA to feed fatty acid metabolism, with saturated very-long-chain fatty acids (VLFCAs) required for production of infectious virion progeny. The human genome encodes seven elongase enzymes (ELOVL) that extend long-chain fatty acids into VLCFA. Here, we identify ELOVL7 as pivotal for HCMV infection. HCMV induces ELOVL7 by more than 150-fold. This induction is dependent on mTOR and SREBP-1. ELOVL7 knockdown or mTOR inhibition impairs HCMV-induced fatty acid elongation, HCMV particle release, and infectivity per particle. ELOVL7 overexpression enhances HCMV replication. During HCMV infection, mTOR activity is maintained by the viral protein pUL38. Expression of pUL38 is sufficient to induce ELOVL7, and pUL38-deficient virus is partially defective in ELOVL7 induction and fatty acid elongation. Thus, through its ability to modulate mTOR and SREBP-1, HCMV induces ELOVL7 to synthesize the saturated VLCFA required for efficient virus replication.

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Figures

Figure 1
Figure 1
HCMV induces fatty acid elongation to produce VLCFAs that are incorporated into the virion envelope. (A) Schematic of pathway from glucose to fatty acid elongation. The straight line down from glucose indicates glycolysis and the circle indicates the TCA cycle. Fatty acid elongases (ELOVLs) use malonyl-CoA as a substrate to elongate shorter fatty acids. (B) Incorporation of labeled carbons from 13C-glucose into saturated FAs in purified virions. Virions were isolated from cells grown in serum-free DMEM containing all glucose U-13C-labeled. For each FA species the data is reported as the percentage that contains at least one 13C-labeled two-carbon unit. (C) The labeling pattern of C26:0 from virions grown as in (b). All data are represented as mean ± SEM of three independent experiments. See also Figure S1.
Figure 2
Figure 2
Fatty acid elongase 7 is strongly induced by HCMV and required for efficient production of infectious progeny. (A) ELOVL1-7 mRNA levels during the course of HCMV replication. MRC-5 fibroblasts infected at 3 IU/cell were compared to mock infected cells for ELOVL1-6. The (*) denotes that, because the ELOVL7 transcript was not detectable in uninfected cells, the ELOVL7 the data is reported as fold change from 4 hpi. (B) HCMV replication in MRC-5 fibroblasts with shRNA knockdown of ELOVL1-7 (compared to non-targeting (NT) sequence control). Knockdown cells were infected at 0.5 IU/cell and at 96 hpi the medium was assayed for infectious progeny. (* p<0.05, t-test) All data are represented as mean ± SEM of at least three independent experiments. See also Figure S2.
Figure 3
Figure 3
ELOVL7 synthesizes fatty acids required for virion infectivity. (A) ELOVL7 protein level during HCMV replication (3 IU/cell). An immediate-early protein (pUL122) is shown as a marker of viral replication. (B) Kinetics of the HCMV replication cycle in ELOVL7 knockdown cells (compared to NT control cells). Immediate-early protein 1 (pUL123; IE1), early proteins pUL44 and pUL26, leaky-late protein pUL83, and late pUL99 protein were examined. (C) C26:0 labeling pattern in knockdown cells grown in DMEM without fetal bovine serum with all glucose U-13C-labeled. Cells were harvested at 72 hpi following infection at (1 IU/cell, 72 hpi). (D) Saturated FA labeling in cells grown under labeling conditions described in part (C). (E) Concentration of saturated FAs in cells grown in serum-free DMEM (72 hpi, 1 IU/cell). (F) The release of infectious progeny, DNA-containing particles, and particle-to-infectious progeny ratio (1 IU/cell). Left panel: Infectious progeny as measured by TCID50 (left panel). Total (infectious and non-infectious) cell-free viral particles were determined by the number of genomes (viral DNA) per ml of medium collected from the cells (middle panel). The particle to IU ratio was also compared (right panel). All data are represented as mean ± SEM of three independent experiments. (* p<0.05, ** p<0.01, t-test)
Figure 4
Figure 4
HCMV replication is enhanced by overexpression of ELOVL7. (A) ELOVL7 protein levels in uninfected MRC-5 fibroblast cells following ELOVL7 overexpression using pLVX-lentiviral stable expression (GFP-expressing cells were used as a control). (B) Kinetics of the HCMV replication cycle (1 IU/cell). (C) C26:0 labeling pattern (1 IU/cell, 72 hpi). (D) Saturated FA labeling (1 IU/cell, 72 hpi). (E) Concentration of saturated FAs (72 hpi, 1 IU/cell). (F) Production of cell-free infectious virus (1 IU/cell). All data are represented as mean ± SEM of three independent experiments. (* p<0.05, ** p<0.01, t-test)
Figure 5
Figure 5
HCMV-induced mTOR and SREBP1 activity is required for lipid remodeling. In all panels, cells were treated with torin 2 to inhibit mTOR or DMSO as a control. (A) Torin 2 blocked HCMV infectivity (bars) at various concentrations without altering cell survival of uninfected cells (circles). (B) ELOVL7 and pUL123 protein levels. AKT and rpS6 are shown as controls to verify mTOR inhibition. (C) C26:0 labeling pattern under 0.1 µM torin 2 treatment (3 IU/cell, 72 hpi). (D) Saturated FA labeling under 0.1 µM torin 2 treatment (3 IU/cell, 72 hpi). (E) Concentration of saturated FAs under 0.1 µM torin 2 treatment (3 IU/cell, 72 hpi). (F) mRNA levels of ELOVL7 in SREBP1 and SREBP2 knockdown cells. FAS and HMGCR levels were used to monitor the decrease in activity of SREBP1 and 2, respectively. (G) Maturation of SREBPs in infected cells treated with various mTOR inhibitors (0.1 µM torin 2, 1 µM PP242, and 0.5 µM INK128). All data are represented as mean ± SEM of three independent experiments (* p<0.05, ** p<0.01, t-test). See also Figure S3.
Figure 6
Figure 6
Viral pUL38 protein drives ELOVL7 expression. (A) ELOVL7 protein level in uninfected cells expressing viral protein pUL38 (or GFP as a control) using pLVX-lentiviral stable expression. (B) ELOVL7 protein expression in cells infected with WT (AD169) or ADdlUL38, a mutant virus lacking the UL38 gene (3 IU/cell, 48 hpi). (C) C26:0 labeling pattern (3 IU/cell, 48 hpi). (D) Saturated FA labeling (3 IU/cell, 48 hpi). (E) Concentration of saturated FAs (3 IU/cell, 48 hpi). All data are represented as mean ± SEM of three independent experiments (* p<0.05, ** p<0.01, t-test). See also Figure S4.
Figure 7
Figure 7
Schematic of mechanisms by which HCMV induces lipidome remodeling by ELOVL7.
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