Glycolytic shift during West Nile virus infection provides new therapeutic opportunities

Abstract

Background: Viral rewiring of host bioenergetics and immunometabolism may provide novel targets for therapeutic interventions against viral infections. Here, we have explored the effect on bioenergetics during the infection with the mosquito-borne flavivirus West Nile virus (WNV), a medically relevant neurotropic pathogen causing outbreaks of meningitis and encephalitis worldwide.

Results: A systematic literature search and meta-analysis pointed to a misbalance of glucose homeostasis in the central nervous system of WNV patients. Real-time bioenergetic analyses confirmed upregulation of aerobic glycolysis and a reduction of mitochondrial oxidative phosphorylation during viral replication in cultured cells. Transcriptomics analyses in neural tissues from experimentally infected mice unveiled a glycolytic shift including the upregulation of hexokinases 2 and 3 (Hk2 and Hk3) and pyruvate dehydrogenase kinase 4 (Pdk4). Treatment of infected mice with the Hk inhibitor, 2-deoxy-D-glucose, or the Pdk4 inhibitor, dichloroacetate, alleviated WNV-induced neuroinflammation.

Conclusions: These results highlight the importance of host energetic metabolism and specifically glycolysis in WNV infection in vivo. This study provides proof of concept for the druggability of the glycolytic pathway for the future development of therapies to combat WNV pathology.

Keywords: Glycolysis; Immunometabolism; Neuroinflammation; West Nile virus.

Fig. 1

Literature search and meta-analysis for glucose levels in human patients infected with WNV. A Flowchart for the systematic literature search and meta-analysis of glucose levels in patients infected with WNV according to PRISMA guidelines. B–D Results from individualized patients identified in the literature search for B CSF glucose (n = 231); C blood glucose (n = 33); D CSF/blood glucose ratio (n = 60). Dashed lines denote normal values in healthy individuals (see text for details on reference values)

Fig. 2

Bioenergetics of WNV infection in cultured cells. A Changes in mitochondrial respiration profile induced by WNV replication. Real-time monitorization of oxygen consumption rate (OCR) in Vero cells infected or not with WNV RVPs at 1 or 48 hpi (n = 5). Arrows indicate the time of injection of oligomycin, FCCP and rotenone/antimycin A. Respiratory parameters determined are indicated by colored areas. B Comparison of respiratory parameters determined by OCR measurement between control (uninfected) and infected cells with RVPs at 48 hpi. Fold change over uninfected at 1 hpi is represented. ***, P < 0.001 for two-way ANOVA and Sidak’s multiple comparisons test (n = 9). C Changes in glycolytic rate induced by WNV replication. Real-time monitorization of extracellular acidification rate (ECAR) was measured in Vero cells infected or not with WNV RVPs at 1 or 48 h pi (n = 6). Arrows indicate the time of injection of glucose, oligomycin and 2-DG. Glycolytic parameters determined are indicated by colored areas. D Comparison of glycolytic parameters determined by ECAR measurement between control (uninfected) and cell infected with SRIPs at 48 hpi. Fold change over uninfected samples at 1 hpi is represented. *P < 0.05; **P < 0.01; ****P < 0.0001 for two-way ANOVA and Sidak’s multiple comparisons test (n = 10). E–H Effect of glucose metabolism manipulation on WNV infection. Vero cells were infected with WNV (MOI of 1 PFU/cell) and subjected to glucose depletion (E), treatment with 10 µM AL-429 (F), 10 mM 2-DG (G) or 50 mM oxamate (H) and virus yield was determined at 24 hpi by plaque assay. Cell viability was evaluated in uninfected cells treated in parallel by quantification of cellular ATP. **P < 0.01, ***P < 0.001; ****P < 0.0001 for unpaired t-test applying Welch’s correction when differences between variances were found (n = 4–6)

Fig. 3

WNV infection reprograms host bioenergetics towards increased glycolysis in mouse neural tissues. A Experimental design and sample collection. Mice were intraperitoneally infected with 10⁴ PFU of WNV and humanly euthanized at 7 days after infection (n = 6). Brain and cerebellum were harvested for transcriptomic analyses. Figure was created with BioRender. B WNV alters the transcriptomic profile in the CNS. Virus load was quantified in the brain and cerebellum hemisphere by RT-qPCR at 3, 7 and 10 days after infection. The changes in the number of differentially expressed genes (DEGs) with |FC|> = 2 and FDR corrected P < 0.05 are indicated. Upregulated denotes FC > 2 and downregulated FC < 2 over uninfected animals. C Assessment of immune cell activation and infiltration in the brain and the cerebellum of WNV-infected mice by using bulk RNA-seq expression data to estimate the abundance of cell types during the progress of WNV neuroinvasion with CellKb. D DEGs related to energy metabolism in the glycolysis and oxidative phosphorylation in the brain and cerebellum of infected animals. The list of DEGs in WNV-infected tissues was filtered for genes available. Reactome pathways of glycolysis, pyruvate metabolism and TCA cycle, pentose phosphate pathway and respiratory electron transport to identify energy-related DEGs. Heat maps indicate the fold change of identified DEGs at each time post-infection

Fig. 4

2-DG and DCA alleviate neuroinflammation in a mouse model of WNV infection. A Experimental design and sample collection. Mice were intraperitoneally infected with 10⁴ PFU of WNV and treated with the glycolysis inhibitors 2-DG (500 mg/kg) or DCA (200 mg/kg) diluted in saline serum by intraperitoneal injection once daily (QD) for 7 days, starting on day 0 after infection. Control mice were treated with saline serum. Animals were humanely euthanized at 7 days after infection (n = 5). Figure was created with BioRender. B WNV viral burden in the brain. Virus load was quantified in the brain by RT-qPCR. C Assessment of immune cell activation and infiltration in the brain of WNV-infected mice by using bulk RNA-seq expression data to estimate the abundance of cell types during the progress of WNV neuroinvasion with CellKb tool (n = 5 for 2-DG and DCA and n = 3 for vehicle). D, E Effect of the treatment with 2-DG or DCA on the DEGs related to the antiviral mechanism by IFN-stimulated genes (D) and signaling by interleukins (E) in the brain of infected animals (n = 5 for 2-DG and DCA and n = 3 for vehicle). F–I Cytokine induction in the brain of infected mice assessed by RT-qPCR: E Ccl2; F Cxcl10; G Cxcl11; H Tnf-α. *p < 0.05; **p < 0.01; for Kruskal–Wallis test and Dunn’s correction for multiple comparisons (F, H, I) or one-way ANOVA and Dunnett’s correction for multiple comparison (G) (n = 5)