AMP-Activated Protein Kinase Restricts Zika Virus Replication in Endothelial Cells by Potentiating Innate Antiviral Responses and Inhibiting Glycolysis

Abstract

Viruses are known to perturb host cellular metabolism to enable their replication and spread. However, little is known about the interactions between Zika virus (ZIKV) infection and host metabolism. Using primary human retinal vascular endothelial cells and an established human endothelial cell line, we investigated the role of AMP-activated protein kinase (AMPK), a master regulator of energy metabolism, in response to ZIKV challenge. ZIKV infection caused a time-dependent reduction in the active phosphorylated state of AMPK and of its downstream target acetyl-CoA carboxylase. Pharmacological activation of AMPK using 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), metformin, and a specific AMPKα activator (GSK621) attenuated ZIKV replication. This activity was reversed by an AMPK inhibitor (compound C). Lentivirus-mediated knockdown of AMPK and the use of AMPKα^-/- mouse embryonic fibroblasts provided further evidence that AMPK has an antiviral effect on ZIKV replication. Consistent with its antiviral effect, AMPK activation potentiated the expression of genes with antiviral properties (e.g., IFNs, OAS2, ISG15, and MX1) and inhibited inflammatory mediators (e.g., TNF-α and CCL5). Bioenergetic analysis showed that ZIKV infection evokes a glycolytic response, as evidenced by elevated extracellular acidification rate and increased expression of key glycolytic genes (GLUT1, HK2, TPI, and MCT4); activation of AMPK by AICAR treatment reduced this response. Consistent with this, 2-deoxyglucose, an inhibitor of glycolysis, augmented AMPK activity and attenuated ZIKV replication. Thus, our study demonstrates that the anti-ZIKV effect of AMPK signaling in endothelial cells is mediated by reduction of viral-induced glycolysis and enhanced innate antiviral responses.

Figure 1.. ZIKV infection alters AMPK activity in endothelial cells.

(A). (A) HRvEC (Human retinal vascular endothelial cells) and HUVEC (Human umbilical cord vein endothelial cells) cells were infected with ZIKV strain PRVABC59 at MOI 1 for 48 h and immunostained for ZIKV envelope antigen 4G2 and detected with an Alexa 594-conjugated secondary antibody (red), while the nuclei were stained with DAPI (magnification, 200X). (B) HRvEC and HUVEC cells were infected with ZIKV (MOI 1) for indicated time points and viral titers were determined using plaque assay. (C) Mock-infected control (C) and ZIKV infected whole cell lysates of HRvEC and HUVEC at the indicated time points were immunoblotted for p-AMPKα (Thr172), p-ACC (Ser79), total AMPKα, ZIKV NS3, and β-actin. (D) The expression levels of p-AMPK and p-ACC were quantified by densitometry and plotted as a percentage of expression relative to the mock-infected controls (mean ± SD, n=3). Band densities of the proteins were normalized to the respective loading control, β-actin. The p-value was calculated using one way ANOVA with Dunnett’s test. *** P <0.0001.

Figure 2.. Pharmacological activation of AMPK inhibits ZIKV replication in endothelial cells.

(A) HRvEC and HUVEC cells were infected with ZIKV at MOI 1 for 48 h in the presence or absence of AMPK activators [AICAR (1 mM), Metformin (20 mM)] and AMPK inhibitor (Compound C; 10 μM). The cells were immunostained for ZIKV envelope antigen 4G2 (red) and the nuclei were counterstained with DAPI (magnification, 200X). Culture supernatants were collected, and viral titers were estimated by plaque assay. Values were plotted on a logarithmic scale (mean ± SD, n=3) for (B) HRvEC, and (C) HUVEC cells. At least four independent fields were counted and plotted as a percentage of ZIKV antigen-positive cells (mean ± SD, n=4) relative to the total number of cells in the captured field for (D) HRvEC and (E) HUVEC. The p-value was calculated using one-way ANOVA Dunnett’s multiple comparison tests. ** P <0.001, *** P <0.0001, ns: not significant.

Figure 3.. AICAR treatment potentiates ZIKV-induced interferon response and decreases the inflammatory response in HRvEC cells.

(A) HRvEC cells were pretreated with AMPK activator, AICAR (1 mM) for 1 h followed by ZIKV challenge (MOI 1) for 48 h. Cells were collected in Trizol for RNA isolation. mRNA levels of the indicated genes were quantified using qRT-PCR, normalized relative to β-actin. (B) Indicated cytokine levels were assessed from the culture supernatant using ELISA. The values are plotted as mean ± SD with n=3; p-values were calculated using one-way ANOVA-Bonferroni’s multiple comparison tests. * P <0.05, *** P <0.0001, ns: not significant.

Figure 4.. AICAR treatment potentiates ZIKV-induced interferon response and decreases the inflammatory response in HUVEC cells.

HUVEC cells were pretreated with AMPK activator, AICAR (1 mM) for 1 h followed by ZIKV challenge (MOI 1) for 48 h. Cells were collected in Trizol for RNA isolation. mRNA levels of the indicated genes were quantified using qRT-PCR, normalized relative to β-actin. (B) Indicated cytokine levels were assessed from the culture supernatant using ELISA. The values are plotted as mean ± SD with n=3; p-values were calculated using one-way ANOVA-Bonferroni’s multiple comparison tests. * P <0.05 **, P <0.001, *** P <0.0001, ns: not significant.

Figure 5:. AICAR treatment inhibits ZIKV-induced glycolysis in endothelial cells.

HRvEC (A, C, F & H) and HUVEC (B, D, G & I) cells were pretreated with AICAR (1 mM) for one hour followed by ZIKV infection for 12 h. (A-D) The extracellular acidification rate (ECAR), an indicator of glycolytic activity, was examined using a Seahorse XFe96 analyzer. The data are presented as mean ± SD. (E) A schematic representation of the glycolysis pathway showing metabolic intermediates and their respective enzymes. (F) HRvEC and (G) HUVEC cells were challenged with ZIKV for 12 h in the presence or absence of AICAR, as described above, and total RNA was extracted and subjected to qRT-PCR analysis of key metabolic genes (HK2, GLUT1, TPI, and MCT4) regulating glycolysis. The gene expression was normalized with ribosomal L-27 as a housekeeping gene. The whole cell lysate of mock-infected, AICAR-treated, ZIKV-infected, and AICAR treated / ZIKV infected HRvEC (H) and HUVEC (I) cells were immunoblotted for the glycolytic enzyme proteins Hexokinase 2 (HK2), GLUT1, and β-actin. The results represent the mean ± SD from three independent experiments, and the statistical analysis was performed using two-way (C & D) and one-way (F & G) ANOVA with Bonferroni’s multiple comparison tests. * P <0.05 **, P <0.001, *** P <0.0001, ns: not significant.

Figure 6.. Inhibition of glycolytic response attenuates ZIKV replication by restoring AMPK activity.

(A) HRvEC cells were treated with a glycolytic inhibitor, 2-deoxyglucose (2-DG), for 1 h followed by infection with ZIKV at MOI 1 or mock infection for 48 h. The cells were fixed and immunostained for ZIKV envelope antigen (red) and nuclei were counterstained using DAPI (magnification 200X). (B) The percentage of ZIKV antigen-positive cells were determined from at least four fields (mean ± SD). (C) HRvEC cells were treated with 2-DG and infected with ZIKV at MOI 1 and the culture supernatant was collected at 48 hpi for virus titration. (D) HRvEC cell lysates from mock-infected, 2-DG treated, ZIKV infected and ZIKV + 2-DG treated cells were immunoblotted for p-AMPK and total AMPK, ZIKV NS3, and β-actin. (E) Glucose uptake was measured in HRvEC cells infected with ZIKV for 48 h in the presence or absence of 1 mM AICAR. 2DG was used as a positive control for inhibition of glucose uptake. The values were plotted as Relative Luminescence units (RLU). (F) ATP levels were measured in HRvEC cells infected with ZIKV in the presence or absence of AICAR (1 mM). The values represent mean ±SD from three independent experiments. The statistical analysis was performed using one-way ANOVA- Bonferroni’s multiple comparison tests. ** P <0.001, *** P <0.0001.

Figure 7.. Specific modulation of AMPK activity alters ZIKV replication and antiviral responses in endothelial cells.

(A) HRvEC cells were infected with ZIKV at MOI 1 for 48 h in the presence or absence of 10 μM GSK621. Infected and treated cells were immunostained for ZIKV envelope antigen 4G2 and images were captured at 200X magnification. (B) The culture supernatant from 48 h infected HRvEC cells were used for viral titer determination by plaque assay, and (C) cell lysate were subjected to western blot for p-AMPK, p-ACC, total AMPK, ZIKV NS3, and β-actin. (D & E) Cells transfected with control or AMPK shRNA lentivirus were challenged with ZIKV at MOI 1 for 48 h. ZIKV replication was assessed by immunostaining of ZIKV 4G2 antigen, and (F) plaque assay. (G) Under similar experimental conditions, the whole cell lysate was used for western blot to assess the expression of p-AMPK, p-ACC, total AMPK, ZIKV NS3, and β-actin. (H) The quantitative PCR analysis was performed to quantify the relative expression of inflammatory mediators (TNFα, CCL5, and CXCL10), IFNs (IFNα2, IFNβ1, IFNγ), and IFN-inducible genes (OAS2, ISG15, and MX1) normalized to β-actin. The values represent mean ± SD from three independent experiments, and the statistical analysis was performed using one way ANOVA with Bonferroni’s multiple comparison tests. ** P <0.001, *** P <0.0001.

Figure 8.. AMPK deficient mouse embryonic fibroblasts (MEFs) are susceptible to ZIKV infection.

Wild-type (WT) and AMPK α1/α2 knockout (KO) MEFs were infected or mock-infected with ZIKV at MOI 1 for 48 h. (A) Viral infectivity was assessed by immunostaining for ZIKV envelop antigen 4G2 (red) and the nuclei were counterstained using DAPI. Images were captured at 200X magnification. (B) ZIKV antigen-positive cells were counted relative to the total number of cells and plotted as a percentage of ZIKV antigen-positive cells. (C) Culture supernatants from ZIKV infected WT and AMPK KO MEFs were collected at 48 hpi and used for viral titer determination by plaque assay. (D) qRT-PCR was performed on ZIKV-infected (48 h) WT and AMPK KO MEFs to quantify the relative expression of indicated genes. A house-keeping gene, β-actin, was used for normalization. Values represent mean ± SD from three independent experiments. The statistical analysis was performed using one-way ANOVA with Bonferroni’s correction for multiple comparison tests. * P <0.05 **, P <0.001, *** P <0.0001.

Figure 9.. AMPK deficient MEFs elicit diminished antiviral response upon poly I:C challenge.

Wild-type (WT) and AMPK_α1/α2 knockout (KO) MEFs were challenged with poly I:C (100 ng/ml) for indicated time points (3 to 48h). Cells were collected in Trizol for RNA isolation and cDNA preparation. qRT-PCR was performed to quantify the relative expression of PRRs, antiviral genes (Rig-I, Ifnβ1, and Ifnγ), and IFN-inducible genes (Oas2, Isg15, and Mx1), with normalization to GAPDH mRNA. The values represent mean ± SD from three independent experiments, and the statistical analysis has been performed using one-way ANOVA with Bonferroni’s correction for multiple comparison tests. *** P < 0.0001.