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. 2014 Feb 4;111(5):1951-6.
doi: 10.1073/pnas.1310779111. Epub 2014 Jan 21.

ChREBP, a glucose-responsive transcriptional factor, enhances glucose metabolism to support biosynthesis in human cytomegalovirus-infected cells

Yongjun Yu  1 , Tobi G Maguire, James C Alwine
Affiliations

ChREBP, a glucose-responsive transcriptional factor, enhances glucose metabolism to support biosynthesis in human cytomegalovirus-infected cells

Yongjun Yu et al. Proc Natl Acad Sci U S A. .

Abstract

Carbohydrate-response element binding protein (ChREBP) plays a key role in regulating glucose metabolism and de novo lipogenesis in metabolic tissues and cancer cells. Here we report that ChREBP is also a critical regulator of the metabolic alterations induced during human cytomegalovirus (HCMV) infection. The expression of both ChREBP-α and ChREBP-β is robustly induced in HCMV-infected human fibroblasts; this induction is required for efficient HCMV infection. Depletion of ChREBP in HCMV-infected cells results in reduction of HCMV-induced glucose transporter 4 and glucose transporter 2 expression, leading to inhibition of glucose uptake, lactate production, nucleotide biosynthesis, and NADPH generation. We previously reported that HCMV infection induces lipogenesis through the activation of sterol regulatory element binding protein 1, which is mediated by the induction of PKR-like endoplasmic reticulum kinase. Data from the present study show that HCMV-induced lipogenesis is also controlled by the induction of ChREBP, in a second mechanism involved in the regulation of HCMV-induced de novo lipogenesis. These results suggest that ChREBP plays a key role in reprogramming glucose and lipid metabolism in HCMV infection.

Keywords: glycolysis; lipid synthesis; viral pathogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
ChREBP expression in HCMV-infected HF cells. (A) ChREBP RNA levels during a HCMV infection time course. Total RNA was isolated at 0, 24, 48, and 72 hpi. Total ChREBP RNA levels were measured by qRT-PCR and normalized to actin RNA levels. (B) ChREBP-α protein levels in the time course of HCMV infection. ChREBP-α and actin protein levels were determined by Western blot analysis. M, mock infection. (C) Cellular localization of HA-tagged ChREBP-α in mock-infected and HCMV-infected cells. At 2 hpi, mock- and HCMV-infected primary HF cells were electroporated with a plasmid expressing an HA-tagged human ChREBP-α. At 1 d after electroporation, cells were serum-starved for overnight, fixed at 48 hpi and stained for nuclear DNA (DAPI, blue), HA (green), and the HCMV MIEPs (red). The DAPI, HA, and MIEP images are overlaid into a composite image (COMP).
Fig. 2.
Fig. 2.
Expression of ChREBP isoforms in HCMV-infected cells. (A) RNA levels of ChREBP-α, ChREBP-β, and total ChREBP in HCMV-infected cells. Total RNA was isolated from mock- and HCMV-infected cells. RNA levels of ChREBP-α, ChREBP-β, and total ChREBP were quantified by qPCR using exon-specific primers. (B) RNA levels of total ChREBP, ChREBP-α, and ChREBP-β in HCMV-infected cells in response to high glucose level. After a 2-h incubation, mock- and HCMV-infected cells were cultured in either low-glucose (2.5 mM) or high-glucose (25 mM) medium. At 48 hpi, total RNA was isolated, and RNA levels of ChREBP-α, ChREBP-β, and total ChREBP were measured by qPCR. Total, total ChREBP; alpha, ChREBP-α; beta, ChREBP-β.
Fig. 3.
Fig. 3.
Induction of ChREBP is critical for HCMV infection. (A) HCMV viral protein expression in ChREBP-depleted HF cells. At 72 hpi, whole-cell extracts were made from mock- and HCMV-infected HF cells pretreated with shGFP or shChREBP1, and levels of cellular and viral proteins were evaluated by Western blot analysis. M, mock infection; V, HCMV infection. (B) HCMV viral titers at 72 and 96 hpi in HF cells treated with shGFP or shChREBP1.
Fig. 4.
Fig. 4.
Metabolic effects of ChREBP depletion on HCMV-infected cells. HF cells were infected with lentiviral vector expressing shGFP or shChREBP1 for at least 4 d. The cells were then serum-starved for 1 d, followed by HCMV infection (MOI of 3). Metabolic gene expression levels and metabolic assays were performed at 48 hpi. (A) Total lipid synthesis in HF cells. Total lipid synthesis was assayed by measuring the incorporation of 14C-acetate into lipids. (B) RNA levels of key lipogenic genes—ACC1, ACL, ELOVL6, and FAS—quantified by qPCR. All RNA data were normalized to actin RNA levels. (C) NADP+/NADPH levels in mock- and HCMV-infected cells pretreated with shGFP or shChREBP1. (D) Glucose uptake. Glc, glucose. (E) Lactate production. Levels of glucose and lactate in the cultured medium were measured using the YSI 7100 Multiparameter Bioanalytical System. (F) Reduction of GLUT4 RNA levels in HCMV-infected cells from depletion of ChREBP. GLUT4 RNA levels were measured as described in B. (G) Protein levels of GLUT2 and GLUT4 in mock- and HCMV-infected cells pretreated with shGFP or shChREBP1. Whole-cell extracts were made at 48 hpi, and GLUT2, GLUT4, and actin protein levels were analyzed by Western blot analysis. (H) Reduction of nucleotide biosynthesis in infected cells from depletion of ChREBP. Total nucleotide synthesis was assayed by measuring the incorporation of d-[U-14C]-glucose into RNA at 48 hpi. M, mock infection; V, HCMV infection; *P < 0.01; **P < 0.005; ***P < 0.002.
Fig. 5.
Fig. 5.
Induction of ChREBP and PERK-mediated SREBP1 activation are independent pathways in HCMV-infected cells. (A) Depletion of ChREBP had little effect on the induction of PERK and the mature forms of SREBP1 during HCMV infection. Whole-cell extracts were made at 72 hpi, and protein levels were measured by Western blot analysis. The M-SREBP1 blot was spliced to match the order of other blots. (B and C) Depletion of PERK had little effect on the induction of ChREBP and GLUT4 during HCMV infection. (B) RNA levels of total ChREBP and GLUT4 in shLuc- and shPERK-treated cells. (C) Protein levels of ChREBP-α, GLUT2, and GLUT4 in PERK-depleted cells. M, mock infection; V, HCMV infection; M-SREBP1, mature forms of SREBP1.
Fig. 6.
Fig. 6.
Diagram of glucose metabolism and lipogenesis in HCMV infection. Ac-CoA, acetyl-CoA; Asp, aspartate; Cit, citrate; Glc, glucose; Lac, lactate; Mito, mitochondria; OAA, oxaloacetate; Pyr, pyruvate.
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