AMP-activated protein kinase is required for the macropinocytic internalization of ebolavirus

Abstract

Identification of host factors that are needed for Zaire Ebolavirus (EBOV) entry provides insights into the mechanism(s) of filovirus uptake, and these factors may serve as potential antiviral targets. In order to identify novel host genes and pathways involved in EBOV entry, gene array findings in the National Cancer Institute's NCI-60 panel of human tumor cell lines were correlated with permissivity for EBOV glycoprotein (GP)-mediated entry. We found that the gene encoding the γ2 subunit of AMP-activated protein kinase (AMPK) strongly correlated with EBOV transduction in the tumor panel. The AMPK inhibitor compound C inhibited infectious EBOV replication in Vero cells and diminished EBOV GP-dependent, but not Lassa fever virus GPC-dependent, entry into a variety of cell lines in a dose-dependent manner. Compound C also prevented EBOV GP-mediated infection of primary human macrophages, a major target of filoviral replication in vivo. Consistent with a role for AMPK in filovirus entry, time-of-addition studies demonstrated that compound C abrogated infection when it was added at early time points but became progressively less effective when added later. Compound C prevented EBOV pseudovirion internalization at 37°C as cell-bound particles remained susceptible to trypsin digestion in the presence of the inhibitor but not in its absence. Mouse embryonic fibroblasts lacking the AMPKα1 and AMPKα2 catalytic subunits were significantly less permissive to EBOV GP-mediated infection than their wild-type counterparts, likely due to decreased macropinocytic uptake. In total, these findings implicate AMPK in macropinocytic events needed for EBOV GP-dependent entry and identify a novel cellular target for new filoviral antivirals.

Fig 1

Findings from our comparative genome analysis screen. EBOV GP-mediated transduction and AMPK mRNA expression in the human NCI-60 tumor panel of cell lines. (A) Relative levels of AMPK mRNA in 54 of the 59 cell lines that compose the NCI-60 human tumor cell panel. mRNA levels were obtained from gene array studies that are available to the public at the NIH NCI DTP website (http://dtp.nci.nih.gov). (B) VSVΔG-EBOV transduction efficiency of the same NCI-60 cell lines. NSCLC, non-small-cell lung cancer. Leuk, leukemia; P, prostate; CNS, central nervous system.

Fig 2

Compound C inhibits EBOV transduction of cell lines. (A) Ability of compound C to block EBOV GP transduction in a number of cell lines. The indicated lines were treated with compound C (10 μM) or the DMSO control for 1 h at 37°C. Either EBOV GP or LFV GPC pseudovirions (MOI of 0.05) were added to the cultures. Virus was allowed to internalize for 4 h before virus and inhibitor were removed, and cells were refreshed with medium without inhibitor for an additional 24 h when transduction levels were determined by assessing EGFP expression in transduced cells by flow cytometry. Findings are shown as a percentage of EGFP expression in cells treated with compound C divided by EGFP expression in cells treated with DMSO. (B) Effect of compound C on EBOV GP pseudovirion transduction, LFV GPC pseudovirion transduction, and cellular cytotoxicity (viability) in Vero cells when cells were incubated with compound C or DMSO for 1 h at 37°C for the initial 4 h of the transduction. A range of MOIs from 0.1 to 1 was used for these studies with similar findings (n = 5). After this incubation, cells were refreshed with medium without inhibitor, and cells were assessed for EGFP expression at 24 h following transduction. (C) Effect of compound C on EBOV GP pseudovirion transduction, LFV GPC pseudovirion transduction, and cellular cytotoxicity (viability) in Vero cells incubated with compound C or DMSO for the duration of the 24-h experiment. (D) Time-of-addition experiments with compound C. Vero cells were precooled to 4°C and incubated with EBOV GP pseudovirions. Unbound virus was removed, and the cells were shifted to 37°C. Compound C (50 μM) or DMSO control was added at the indicated time following the temperature shift. At 6 h following initiation of transduction, virus and inhibitor were removed, and cells were refreshed with medium. Viral transduction was assayed 24 h later by flow cytometry. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 3

Compound C inhibits EBOV infection. Vero E6 cells were pretreated with compound C for 1 h, and replication-competent Zaire EBOV that expresses EGFP was added at an MOI of 0.05. After 24 h, cells were imaged and infected cells in micrographs were counted. Data were normalized to untreated cells and studies were performed in triplicate. (A) Bright-field and fluorescent images (EGFP) of a representative micrograph of EBOV infection in the presence of DMSO or 25 μM compound C. (B) Compound C dose-response curve for inhibition of EBOV infection. Shown are mean and standard error of the relative number of EBOV-infected cells normalized to untreated cells after treatment with compound C.

Fig 4

AMPK inhibition significantly reduces EBOV GP-mediated infection of primary human MDMs. (A) MDMs were incubated with DMSO or compound C (50 μM) for 1 h. Treated cells were infected with EBOV GP/rVSV for an additional 4 h. Unbound virus and drug were removed, and viral infection was visualized for EGFP expression at 24 h postinfection by fluorescence microscopy. Experiments were performed in triplicate with two donors. Shown is a representative experiment. (B) Cytotoxicity of compound C in MDMs. Experiments were performed in quadruplicate with two donors, and the mean and standard deviation of the mean are shown.

Fig 5

AMPK^−/− MEFs support lower levels of EBOV infection than WT MEFs. Equal numbers of WT or AMPK^−/− MEFs were infected with VSV, VV, or EBOV GP/rVSV (MOI of 1). Viral infection was assayed by EGFP expression at 24 h postinfection via flow cytometry. ***, P < 0.001.

Fig 6

Actin polymerization is stimulated by EBOV GP pseudovirions in an AMPK-dependent manner, stimulating macropinocytosis. (A) Time course of actin polymerization following EBOV GP pseudovirion incubation in the presence or absence of 10 μM compound C. Vero cells were preincubated with and without drug at 37°C for 2 h. Cells were then incubated with EBOV pseudovirions (MOI of 200) in the presence or absence of drug for 30 m at 4°C. Virus-containing medium was removed, and fresh medium with and without compound C was added before the temperature was shifted to 37°C for 30 m. Cells were fixed in 2% paraformaldehyde and permeabilized, and polymerized actin was imaged by confocal microscopy using Alexa Fluor 647-phalloidin. To quantify the polymerization activity, the total phalloidin staining of 6 to 12 images composed of 6 to 19 cells/image was quantified using the Imaris software program and averaged for the number of voxels/cell. (B) Compound C reduces dextran uptake into Vero cells. FITC-conjugated 70-kDa dextran was incubated with Vero cells treated with either 10 or 50 μM compound C or 5 or 25 μM EIPA, a well-established macropinocytosis inhibitor. One hour later, cells were lifted and assessed for fluorescent dextran uptake by flow cytometry. (C) AMPK^−/− MEFs have reduced dextran (Dex) uptake compared to WT MEFs. FITC-conjugated 70-kDa dextran was incubated with equivalent numbers of WT or AMPK^−/− MEFs for 1 h at 37°C. Cells were washed thoroughly and assessed for dextran uptake by flow cytometry. *, P < 0.05; **, P < 0.001.

Fig 7

AMPK is required for EBOV internalization from the cell surface. (A) Vero cells were incubated with a macropinocytosis inhibitor (EIPA; 25 μM), Cat B inhibitor (CA-074; 25 μM), AMPK inhibitor (compound C; 50 μM), or a DMSO control for 1 h. Treated cells were transduced with EBOV GP pseudovirions (MOI of 0.05) for an additional 4 h. Virus and inhibitor were then replaced with fresh DMEM. Viral transduction was determined 24 h later by EGFP expression via fluorescence-activated cell sorting. (B) Vero cells were incubated with the indicated inhibitor for 1 h before cells were chilled to 4°C. EBOV GP pseudovirions were then allowed to bind to the cells for an additional hour at 4°C. Surface-bound virus was either removed by trypsin treatment prior to shifting the temperature to 37°C (control, 0 min) or allowed to transduce for 1 h at 37°C in the presence of inhibitor or the DMSO control prior to trypsin treatment. After 1 h, cells were refreshed with medium not containing inhibitor. Viral transduction was determined 24 h later by EGFP expression via flow cytometry. **, P < 0.01; ***, P < 0.001.