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. 2011 Feb;85(4):1573-80.
doi: 10.1128/JVI.01967-10. Epub 2010 Dec 8.

Human cytomegalovirus activates glucose transporter 4 expression to increase glucose uptake during infection

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

Human cytomegalovirus activates glucose transporter 4 expression to increase glucose uptake during infection

Yongjun Yu et al. J Virol. 2011 Feb.

Abstract

Glucose transport into mammalian cells is mediated by a group of glucose transporters (GLUTs) on the plasma membrane. Human cytomegalovirus (HCMV)-infected human fibroblasts (HFs) demonstrate significantly increased glucose consumption compared to mock-infected cells, suggesting a possible alteration in glucose transport during infection. Inhibition of GLUTs by using cytochalasin B indicated that infected cells utilize GLUT4, whereas normal HFs use GLUT1. Quantitative reverse transcription-PCR and Western analysis confirmed that GLUT4 levels are greatly increased in infected cells. In contrast, GLUT1 was eliminated by a mechanism involving the HCMV major immediate-early protein IE72. The HCMV-mediated induction of GLUT4 circumvents characterized controls of GLUT4 expression that involve serum stimulation, glucose concentration, and nuclear functions of ATP-citrate lyase (ACL). In infected cells the well-characterized Akt-mediated translocation of GLUT4 to the cell surface is also circumvented; GLUT4 localized on the surface of infected cells that were serum starved and had Akt activity inhibited. The significance of GLUT4 induction for the success of HCMV infection was indicated using indinavir, a drug that specifically inhibits glucose uptake by GLUT4. The addition of the drug inhibited glucose uptake in infected cells as well as viral production. Our data show that HCMV-specific mechanisms are used to replace GLUT1, the normal HF GLUT, with GLUT4, the major glucose transporter in adipose tissue, which has a 3-fold-higher glucose transport capacity.

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Figures

FIG. 1.
FIG. 1.
(A) Glucose (Gluc) consumption is increased in HCMV-infected cells. Serum-starved HFs were either mock treated or HCMV infected (MOI of 3) in serum-free DMEM. Culture medium was collected at the indicated time points, and the glucose concentration in the medium was assayed as described in Materials and Methods. (B) Cytochalasin B inhibition of glucose transport in mock- and HCMV-infected cells. HFs were infected as described for panel A; at 48 hpi, glucose uptake was assayed in the presence of increasing concentrations of cytochalasin B, as described in Materials and Methods.
FIG. 2.
FIG. 2.
HCMV-infected cells have increased GLUT4 expression and decreased GLUT1 expression. Total RNA was isolated at 0, 24, 48, and 72 hpi. GLUT4 (A) and GLUT1 (B) mRNA levels were measured by quantitative RT-PCR and normalized to PPIA mRNA levels. (C) Whole-cell extracts were prepared from mock-infected (M) and HCMV-infected (V) infected HFs at 24, 48, 72, and 96 hpi. GLUT1, GLUT4, MIEPs and actin protein levels were determined by Western analysis.
FIG. 3.
FIG. 3.
GLUT1 expression is inhibited by IE72. (A) GLUT1 mRNA levels decreased in HFs transiently transfected with HCMV IE72. HFs were electroporated with pRSV72, pRSV86, pCD-MIE, or a control vector (see Materials and Methods). At 48 h after electroporation, the cells were refed with serum-free DMEM. Total RNA was isolated at 72 h postelectroporation. GLUT1 and GLUT4 mRNA levels were measured by quantitative RT-PCR and normalized to PPIA mRNA levels. (B) Cultures prepared in parallel with those reported in panel A were harvested for protein level and analyzed by Western blotting for MIE proteins and actin. (C and D) Both the GLUT1 mRNA level and protein levels were decreased in ihfie1.3 cells, which stably express HCMV IE72.
FIG. 4.
FIG. 4.
(A) GLUT4 expression is resistant to glucose concentration in HCMV-infected cells. HFs were mock treated or HCMV infected as described in the text. At 2 hpi the cells were refed with serum-free medium containing 1, 5, or 25 mM glucose. At 48 hpi, total RNA was isolated, and GLUT4 mRNA levels were determined by quantitative RT-PCR. (B) ACL is activated in HCMV infection. Western analysis was performed to determine the levels of total and phosphorylated (S454) ACL in mock-infected (M) or HCMV-infected (V) cell extracts harvested at 48 hpi; MIEPs and actin were also analyzed. (C) ACL is not required for the activation of GLUT4 expression in HCMV infection. Confluent HFs were mock infected or infected with lentiviral vectors expressing a control shRNA (shCTRL) or an shRNA specific for ACL (shACL). Two days after lentiviral infection, the cells were refed with serum-free DMEM. One day later the cells were either mock treated or HCMV infected; at 48 hpi total RNA was isolated and GLUT4 mRNA levels were determined by quantitative RT-PCR. (D) Time course of GLUT4 mRNA levels during HCMV infection. Total RNA was prepared from HCMV-infected HFs at 0, 4, 8, 12, 24, 48, 72, and 96 hpi. GLUT4 mRNA levels were determined by RT-PCR as described in the text.
FIG. 5.
FIG. 5.
Translocation of GLUT4 can bypass Akt signaling in HCMV-infected cells. (A) Akt signaling is not involved in the activation of GLUT4 expression in HCMV-infected cells. Serum-starved HFs were pretreated with 1 μM AKTi for 1 h. Then, the cells were either mock treated or infected with HCMV in serum-free medium in the presence or absence of 1 μM AKTi. Whole-cell extracts were prepared at 48 hpi, and GLUT4, phospho-Akt S473, MIEPs, and actin protein levels were determined by Western analysis. (B) Translocation of GLUT4 can bypass Akt signaling in HCMV-infected cells. Results of fluorescence microscopy analysis of the cellular localization of HA-GLUT4-GFP in HFs are shown. At 2 hpi, mock- or HCMV-infected HFs were electroporated with the HA-GLUT4-GFP reporter plasmid. At 48 hpi the cells were serum starved for 4 h and then either left untreated or treated with insulin and AKTi as described in detail in Materials and Methods. GFP fluorescence (green) is a measure of total GLUT4 expression. Immunofluorescence staining for the HA tag (red) is a measure of GLUT4 on the cell surface. The nuclei were stained with DAPI (blue).
FIG. 6.
FIG. 6.
Inhibition of GLUT4 by indinavir results in reduced HCMV growth. (A) Indinavir inhibits production of infectious HCMV virions. HCMV virus growth curves were generated in the presence of indinavir or water (drug solvent, Control). HFs were infected with HCMV as described in the text. The black line with solid squares indicates the control growth curve in which water was added at 2 hpi. The black line with open squares indicates the indinavir-treated cultures where 200 μM indinavir was added at 2 hpi and staged in the infection until harvesting. The dashed line with open triangles indicates that indinavir was added for 24 h prior to harvest, i.e., 24 to 48, 48 to 72, 72 to 96, or 96 to 120 hpi. (B) Glucose uptake was inhibited by indinavir in HCMV-infected cells. Glucose uptake was measured in HCMV-infected cells in the presence or absence of 200 μM indinavir as described in Materials and Methods. ND, no drug. (C) Virus protein levels in control and indinavir-treated cultures. Whole-cell extracts were prepared from HCMV-infected cultures treated with indinavir as described for panel A and evaluated by Western analysis.
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