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. 2013;9(8):e1003497.
doi: 10.1371/journal.ppat.1003497. Epub 2013 Aug 1.

Viperin regulates cellular lipid metabolism during human cytomegalovirus infection

Jun-Young Seo  1 , Peter Cresswell
Affiliations

Viperin regulates cellular lipid metabolism during human cytomegalovirus infection

Jun-Young Seo et al. PLoS Pathog. 2013.

Abstract

Human cytomegalovirus (HCMV) has been shown to induce increased lipogenesis in infected cells, and this is believed to be required for proper virion envelopment. We show here that this increase is a consequence of the virus-induced redistribution of the host protein viperin to mitochondria and its capacity to interact with and block the function of the mitochondrial trifunctional protein (TFP), the enzyme that mediates fatty acid-β-oxidation. The resulting decrease in cellular ATP levels activates the enzyme AMP-activated protein kinase (AMPK), which induces expression of the glucose transporter GLUT4, resulting in increased glucose import and translocation to the nucleus of the glucose-regulated transcription factor ChREBP. This induces increased transcription of genes encoding lipogenic enzymes, increased lipid synthesis and lipid droplet accumulation, and generation of the viral envelope. Viperin-dependent lipogenesis is required for optimal production of infectious virus. We show that all of these metabolic outcomes can be replicated by direct targeting of viperin to mitochondria in the absence of HCMV infection, and that the motif responsible for Fe-S cluster binding by viperin is essential. The data indicate that viperin is the major effector underlying the ability of HCMV to regulate cellular lipid metabolism.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Viperin expression is necessary for HCMV-induced metabolic effects.
HFtelo cells stably expressing no shRNA, control luciferase (Luc) shRNA or viperin shRNAs were infected with HCMV at an moi of 2 for the indicated days. (A) AMPK activity. Cells were treated with DMSO or the AMPK inhibitor, Compound C (CompC; 5 µM). At 2 days post infection (dpi), the cells were harvested in lysis buffer and the lysates from 2.5×104 cells were assayed for AMPK activity. Data are presented as mean ± SEM of duplicate samples and are representative of two individual experiments. *, P<0.01. (B and C) mRNA levels of GLUT4 (B), ChREBPα and ChREBPβ (C). Total RNA was isolated, and the mRNA levels were measured by quantitative RT-PCR and normalized to β actin mRNA. Data are presented as means ± SEM of triplicate samples and are representative of three individual experiments. (D) Cells were stained with antibody specific to ChREBP and antibody to the HCMV protein IE-1 to identify infected cells. The filled arrows indicate ChREBP localized to the nuclei, and the open arrows indicate ChREBP localized to cytoplasm. Fifty infected cells were examined for ChREBP nuclear localization. ChREBP was detected at the nuclei in over 50% of HCMV-infected control cells and less than 1% of viperin knockdown cells. Scale bar, 20 µm.
Figure 2
Figure 2. Viperin expression induces HCMV-induced lipogenesis.
HFtelo cells stably expressing no shRNA, control luciferase (Luc) shRNA or viperin shRNAs were infected with HCMV at an moi of 2 for the indicated days. (A) Lipogenic enzyme mRNA levels. Total RNA was isolated, and the mRNA levels were measured by quantitative RT-PCR and normalized to β actin mRNA. Data are presented as means ± SEM of triplicate samples and are representative of three individual experiments. ACL, ATP-citrate lyase; ACC2, acetyl-coenzyme A (CoA) carboxylase 2; FAS, fatty acid synthase; DGAT1 and DGAT2, diacylglycerol acyltransferases 1 and 2. (B) Total lipid synthesis. The infected cells were labeled with [14C]-acetate for 3 hr. Total lipids from 1×105 cells were extracted and [14C] incorporation assessed. Data are presented as mean ± SEM of triplicate samples and are representative of two individual experiments. *, P<0.0001. (C) Cells were stained with antibodies specific to adipose differentiation-related protein (ADRP), a marker for lipid droplets (LDs), and gB, an HCMV glycoprotein. The filled arrows indicate the accumulated LDs, and the open arrows indicate barely detectable LDs in HCMV-infected viperin knockdown cells. Scale bar, 20 µm. (D) Quantification of LDs. LD diameter: mean of 500 LDs ± SEM.; LD number: mean of 50 cells ± SEM *, P<0.0001.
Figure 3
Figure 3. Viperin-dependent lipid synthesis is required for formation of HCMV viral envelope and production of infectious virion.
(A) Transmission electron micrographs of HCMV-infected HFtelo cells stably expressing the indicated shRNAs. Monolayers were infected with HCMV at an moi of 2 and processed for EM at 7 dpi. Multiple frames from each sample were imaged and photographed. Particles from a representative cell are shown. White and black arrows indicate non-enveloped particles and enveloped particles, respectively. Nu, nucleus; Cyto, cytoplasm. Scale bar, 1 µm. (B) The numbers of enveloped and non-enveloped particles were counted in each frame and calculated as a ratio of enveloped particles to total particles in the cytoplasm of infected cells. The graphs indicate the mean percent of the enveloped particles per total 20–50 particles in each frame (from >10 cells of each sample) ± SEM *, P<0.0001. (C) HFtelo cells expressing the indicated shRNAs were infected with HCMV at an moi of 0.2, and supernatants and cells were harvested at 6 dpi. Virus yield was quantified by a fluorescence-based virus infectivity assay. Data are presented as means ± SEM of duplicate samples and are representative of two individual experiments. *, P<0.01; **, P<0.005. (D) Viral genome copy numbers in viral particles harvested from supernatants and cells at 6 dpi (initial moi of 2). The viral DNAs were extracted and assayed by real-time PCR. (E) Expression of HCMV viral proteins, MCP, gB, pp65, and pp28 in HFtelo cells expressing the indicated shRNAs at 6 dpi (initial moi of 2). Each protein was detected by immunoblot using specific monoclonal antibodies. Grp94 served as a protein-loading control.
Figure 4
Figure 4. Targeting viperin to mitochondria leads to increased lipogenesis.
The N-terminal 42 residue α-helical region of mouse viperin (WT), a mouse viperin-GFP chimera, or viperin (DCA) was replaced by the mitochondrial localization sequence (MLS) of the HCMV protein vMIA. The MLS was also directly fused to enhanced green fluorescent protein (EGFP) as a negative control (MLS-GFP). (A and B) mRNA levels of GLUT4 and ChREBPs (A) and lipogenic enzymes (B) in viperin knockdown HFtelo cells transiently expressing the indicated chimeric viperin proteins. The mRNA level in the cells expressing a control vector was set at 1. Data are represented as means ± SEM of triplicate samples and are representative of two individual experiments. *, P<0.01; **, P<0.05; ***, P<0.005. (C) LDs were monitored in viperin knockdown HFtelo cells transiently expressing the indicated chimeric viperin proteins. Cells were stained with ADRP and viperin antibodies. The filled arrows indicate the accumulated LDs in the transfected cells. The open arrows indicate the small basal LDs in the non-transfected or transfected cells. Scale bar, 20 µm. (D) Total lipid synthesis. Viperin knockdown HFtelo cells transiently expressing the indicated chimeric viperin proteins were labeled with [14C]-acetate for 3 hr. Total lipids from 5×104 cells were extracted in chloroform-methanol and [14C] incorporation assessed. Data are presented as mean ± SEM of triplicate samples and are representative of two individual experiments. *, P<0.001. (E) The viperin knockdown HFtelo cells were transfected with the indicated constructs. At 1 day after transfection, the cells were transferred to serum-free medium in the presence and absence of glucose and incubated for 24 hr at 37°C. GLUT4, ChREBPs and lipogenic enzyme mRNA levels were measured. Data are represented as means ± SEM of triplicate samples and are representative of two individual experiments. *, P<0.005; **, P<0.05.
Figure 5
Figure 5. Viperin interaction with TFP is responsible for increased lipogenesis.
(A) mRNA levels of GLUT4, ChREBPs and lipogenic enzymes in TFP β subunit deficient (HADHB (−)) cells transiently expressing the indicated chimeric viperin proteins were measured as described above. Data are represented as means ± SEM of triplicate samples and are representative of two individual experiments. *, P<0.05. (B) LDs were monitored in HADHB (−) cells transiently expressing the indicated chimeric viperin proteins as described above. The arrows indicate the small basal LDs in the non-transfected or transfected cells. Scale bar, 20 µm.
Figure 6
Figure 6. Inhibition of fatty acid β-oxidation induces lipogenesis.
(A and B) mRNA levels of GLUT4 and lipogenic enzymes (A) and ChREBPs (B) in HFtelo cells treated with etomoxir (0.2 mM), an inhibitor of mitochondrial long chain fatty acid oxidation, for 3 or 24 hr. Data are represented as means ± SEM of triplicate samples and are representative of two individual experiments. *, P<0.005; **, P<0.05. (C) LDs were monitored in etomoxir-treated HFtelo cells. Cells were stained with ADRP and viperin antibodies. Scale bar, 20 µm.
Figure 7
Figure 7. Specificity of the viperin effects on modulation of lipid metabolism in HCMV infection.
(A–C) HFtelo cells stably expressing the indicated shRNAs were infected with recombinant HCMV.mVIP, in which the loci US7–US16 were replaced by doxycycline (Dox)-inducible mouse viperin-GFP, at an moi of 2 for 1 or 3 days. Dox (2 µg/ml) was added before infection. mRNA levels of GLUT4 and ChREBPs (A) and lipogenic enzymes (B) were measured. Data are presented as means ± SEM of triplicate samples and are representative of two individual experiments. Cells were stained with antibody specific to ADRP (C). The HCMV.mVIP-infected cells were detected by expression of viperin-GFP. The arrows indicate the accumulated LDs. Scale bar, 20 µm. (D) HFtelo cells expressing the indicated shRNAs were infected with HCMV.mVIP at an moi of 0.2, and supernatants and cells were harvested at 6 dpi. Dox (2 µg/ml) was added on day 0 and 3. Virus yield was quantified by a fluorescence-based virus infectivity assay. Data are presented as means ± SEM of duplicate samples and are representative of two individual experiments.
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