Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection

Figures

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  Figure 1. Unique changes in the metabolism of EV71-infected cells. Vero cells were infected without or with EV71 at MOIs of 0.3125, 0.625, or 1.25 for 24 h, and extracted for metabolomic analysis with UPLC-Q-TOF-MS operated in electrospray positive (ESI+) and negative (ESI-) modes (n = 9). The datasets were analyzed by OPLS-DA, and the score plots for the features obtained in ESI+ and ESI– modes are shown (A). Some of the identified metabolites with VIP scores >1.5 are shown (B). The summary plot for metabolite set enrichment analysis for EV71-infected cells (C).

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  Figure 2. EV71 infection is associated with variations in amino acid pools. Vero cells were infected and extracted for metabolomic analysis, as described in Figure 1. Features corresponding to specific amino acids were extracted. The levels of metabolites are expressed relative to those of mock-infected cells. Data are mean ± SD (n = 9). * p < 0.05, ** p < 0.01, *** p < 0.005, significant difference from mock-infected cells.

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  Figure 3. EV71-induced changes in metabolism of epithelial cells. Vero cells were infected and extracted for metabolomic analysis as described in Figure 1. The features of interest were extracted, and the validated metabolites of interest were mapped onto biochemical pathways. The levels of metabolites are expressed relative to those of mock-infected cells. The arrow indicates the metabolic pathway, and the dashed one indicates the transport of biomolecules. Data are mean ± SD (n = 9). * p < 0.05, ** p < 0.01, *** p < 0.005, significant difference from mock-infected cells.

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  Figure 4. Glutamine deprivation rescues cells from viability loss and cytopathic effect. Vero cells were cultured in medium containing the indicated concentrations of glutamine (Gln) (A,B), or in glutamine-free medium supplemented with the indicated concentrations of DM-αKG (C,D). They were mock- (solid bar) or infected (empty bar) with EV71 at MOI of 1.25 for 24 (A,C) and 48 h (B,D), and the viability was assayed by neutral red assay. The data are expressed as percentage relative to those of mock-infected cells and presented as mean ± SD of six separate experiments. *p < 0.05, significant difference from mock-infected cells at a specified concentration. (E) Cells were cultured in glutamine-free medium (no treatment), medium containing 2 mM Gln, or glutamine-free medium containing 3.5 mM DM-αKG, and were mock- (Con) or infected (EV71) with EV71 for 24 h. They were stained with Hoechst 33342 and examined under a fluorescence microscope. The white arrows indicate cells with CPE. The results shown are representative of six experiments (original magnification: × 200). The cells with CPE are those characterized by chromatin condensation and formation of crescent-shaped nuclei. (F) The…

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  Figure 5. Glutamine and DM-αKG are essential to viral replication. (A) Vero cells were infected with EV71 in the medium containing the indicated concentrations of glutamine for 1 h, and were overlaid with 0.3% agarose in MEM/2% FCS, supplemented with the indicated concentrations of glutamine. The infected cells were processed for crystal violet staining as described in Materials and Methods. Representative plates of three experiments are shown here (upper panel). The number of plaques was counted, and is expressed as percentage relative to that formed in the medium supplemented with 2 mM glutamine. Data are mean ± SD. * p < 0.01, significant difference from that of 2 mM glutamine group. (B,C) Vero cells were cultured in medium containing the 0 (solid bar) and 2 (empty bar) mM glutamine (B), or in glutamine-free medium supplemented without (solid bar) or with 3.5 (empty bar) mM DM-αKG (C). They were infected with EV71 at MOI of 1.25. The total RNA was extracted from cells at 0 and 24 h p.i., and qRT-PCR was performed to determine the level of viral genomic RNA. The result is expressed as fold change relative to that at 0 h p.i. Data are mean ± SD of six experiments. * p < 0.01, significant…

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  Figure 6. Increased expression of GDH and CAD proteins during viral infection. (A) Vero cells were mock- (Mock) or infected with virus (EV71) at MOI of 1.25, and were harvested at the indicated time-points p.i. for western blotting with antibodies to GLS, GDH, CAD and actin. A representative result of three experiments is shown. (B) The expression levels were quantified by densitometry and Image J software and normalized to that of actin, and are expressed as fold change relative to that of mock-infected cells at 0 h p.i. * p < 0.05, significant difference from cells at 0 h; § p < 0.05, significant difference from mock-infected cells at the same time-points.

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  Figure 7. GLS, GDH, and CAD are essential to viral replication. (A) Vero cells were transfected with NC siRNA, siGDH, siGLS and siCAD, and 48 h after transfection, the cells were analyzed for expression of GDH, GLS and CAD by western blotting. An equal amount of the lysate of each transfectant was analyzed for expression of actin by western blotting. A representative result of three experiments is shown. (B–D) Vero cells were transfected with NC siRNA, siGDH, siGLS or siCAD for 48 h, and the transfected cells were mock- (solid bar) or infected (empty bar) with virus at MOI of 1.25. After 48 h, the infected cells were analyzed for cell viability (B) and the percentage of CPE (C). The cell viability data are expressed as percentage relative to those of mock-infected cells. Data are mean ± SD of six experiments. (D) Total RNA was extracted from mock- (Mock) or infected (EV71) cells for determination of viral genomic RNA. The result is expressed as fold change relative to that at 0 h p.i. Data are mean ± SD of six experiments. § p < 0.05, significant difference from mock-infected cells; * p < 0.05, significant difference from NC siRNA-transfected cells.

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  Figure 8. Interaction between VP1 and CAD. (A) Vero cells were transfected with the plasmid encoding GFP-VP1-Myc and that coding for N-FLAG-CAD, C-FLAG-CAD, or C34V. Forty-eight hours later, the cell lysate was prepared for immunoprecipitation with anti-c-Myc antibody (αMyc; c-Myc monoclonal antibody 9E10), and the subsequent western blotting with HRP-conjugated anti-FLAG antibody (αFLAG-HRP; MA1-91878) for detection of the co-immunoprecipitated N- or C-FLAG-CAD, or with HRP-conjugated anti-c-Myc antibody (αMyc-HRP; R951-25) for detection of GFP-VP1-Myc. (B) Reciprocal immunoprecipitation with anti-FLAG antibody (αFLAG; FLAG M2 monoclonal antibody) was followed by western blotting with αMyc-HRP for detection of the co-immunoprecipitated GFP-VP1-Myc, or with αFLAG for detection of N-FLAG-CAD. The cell lysate was immunoblotted with αFLAG-HRP, αMyc-HRP or anti-actin (αActin) antibodies. A representative result of three experiments is shown. (C,D) Vero cells were transfected with expression vector of GFP-VP1-Myc (C), GFP-VP1(FY)-Myc (D) or GFP-Myc (negative control) for 48 h, and the cell lysate was subjected to immunoprecipitation with αMyc and western blotting with anti-CAD antibody (αCAD)…

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  Figure 9. VP1 expression promotes pyrimidine biosynthesis that is essential to viral replication. (A) A simplified diagram shows the de novo pyrimidine biosynthesis pathway. It is noted that BRQ selectively inhibits DHODH. (B) (Upper panel) Vero cells were transfected with 0, 1, or 2 μg of VP1 expression vector (plus empty expression vector to maintain the total amount of transfected DNA at 2 μg). Twenty-four hours later, cells were harvested for western blotting with anti-VP1 antibody (αVP1) or anti-actin (αActin) antibodies. A representative result of three experiments is shown. (Lower panel) Vero cells were transfected in a similar manner. Twenty hours later, cells were labeled in NaH13CO3-containing medium and treated with BRQ as described in Materials and Methods. The cell extracts were subjected to mass spectrometric analysis. The abundance of [13C]-dihydroorotate (m/z 158.0283) was quantified from the corresponding extracted ion chromatogram and divided by the labeling time, and the flux is expressed as fold change relative to that of mock-transfected cells. Data are mean ± SD of six replicates. #p < 0.05, significant difference from the mock-transfected cells. (C) Vero cells were…