Antiviral potential of ERK/MAPK and PI3K/AKT/mTOR signaling modulation for Middle East respiratory syndrome coronavirus infection as identified by temporal kinome analysis

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus, and infections with this virus can result in acute respiratory syndrome with renal failure. Globally, MERS-CoV has been responsible for 877 laboratory-confirmed infections, including 317 deaths, since September 2012. As there is a paucity of information regarding the molecular pathogenesis associated with this virus or the identities of novel antiviral drug targets, we performed temporal kinome analysis on human hepatocytes infected with the Erasmus isolate of MERS-CoV with peptide kinome arrays. bioinformatics analysis of our kinome data, including pathway overrepresentation analysis (ORA) and functional network analysis, suggested that extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K)/serine-threonine kinase (AKT)/mammalian target of rapamycin (mTOR) signaling responses were specifically modulated in response to MERS-CoV infection in vitro throughout the course of infection. The overrepresentation of specific intermediates within these pathways determined by pathway and functional network analysis of our kinome data correlated with similar patterns of phosphorylation determined through Western blot array analysis. In addition, analysis of the effects of specific kinase inhibitors on MERS-CoV infection in tissue culture models confirmed these cellular response observations. Further, we have demonstrated that a subset of licensed kinase inhibitors targeting the ERK/MAPK and PI3K/AKT/mTOR pathways significantly inhibited MERS-CoV replication in vitro whether they were added before or after viral infection. Taken together, our data suggest that ERK/MAPK and PI3K/AKT/mTOR signaling responses play important roles in MERS-CoV infection and may represent novel drug targets for therapeutic intervention strategies.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

Heat maps and hierarchical clustering of host kinome responses to MERS-CoV infection. Peptide phosphorylation was assessed by densitometry. The results were scaled and normalized using GeneSpring (version 6.0) software. For hierarchical clustering, 1 − Pearson correlation coefficient was used as the distance metric and the McQuitty method was used as the linkage method. (A) Hierarchical clustering of the MERS-CoV-infected kinome data sets alongside the mock-infected control data sets. (B) Cluster analysis of the MERS-CoV-infected kinome data sets following background subtraction of the time-matched mock-infected control data sets. Spots demonstrating a significant differential phosphorylation between the MERS-CoV-infected and mock-infected control were compiled into a data set for each time point for comparative analysis. The lines at the top of the heat maps indicate the relative similarity between the conditions listed along the bottom edge of the heat maps. Line length indicates the degree of similarity, with shorter lines equating to stronger similarity. The lines on the left side of the heat maps indicate the relative similarity of the signal between the 300 individual peptide targets on the arrays. Red indicates increased phosphorylation; green indicates decreased phosphorylation. (C) Principal component analysis of the mock- and MERS-CoV-infected kinome data sets. (D) MERS-CoV titers from infected cells at each time point during the experiment.

FIG 2

Functional network analysis of temporal kinome responses to MERS-CoV infection in Huh7 cells. Following PIIKA, kinome data sets comparing MERS-CoV-infected cells to mock-infected cells were uploaded to IPA for functional network analysis to identify kinases of potential pharmacological interest. The top two functional networks from each time point are presented. (A) Results at 1 h postinfection. (i) Network 1 (cell morphology, cellular function and maintenance, and carbohydrate metabolism); (ii) network 2 (embryonic development, organ development, organismal development). (B) Results at 6 h postinfection. (i) Network 1 (gene expression, RNA damage and repair, RNA posttranscriptional modification); (ii) network 2 (cell morphology, cellular function and maintenance, cell cycle). (C) Results at 24 h postinfection. (i) Network 1 (cancer, hematological disease, cell death and survival); (ii) network 2 (posttranslational modification, cell morphology, cellular assembly and organization). Red nodes, upregulation of phosphorylation; green nodes, downregulation of phosphorylation; PARP, procyclic acidic repetitive protein; CAT, chloramphenicol acetyltransferase; Cm-csf, granulocyte-macrophage colony-stimulating factor; LDL, low-density lipoprotein; PDGF BB, platelet-derived growth factor with two B chains; GNRH, gonadotropin-releasing hormone; PLC, phospholipase C; FSH, follicle-stimulating hormone; Vegf, vascular endothelial growth factor; IKK, IκB kinase; MHC, major histocompatibility complex; CDK2, cyclin-dependent kinase 2; TCF, T cell factor; HDL, high-density lipoprotein; IL2R, interleukin-2 receptor; PEPCK, phosphoenolpyruvate carboxykinase; FSTL1, follistatin-like 1; PP1C, phosphatase 1 catalytic subunit; TCR, T cell receptor; Pdgfr, platelet-derived growth factor receptor; NMDA, N-methyl-d-aspartate. Solid lines represent direct interactions between proteins, and dashed lines represent indirect interactions.

FIG 3

Western blot array analysis of select phosphorylation events in MERS-CoV-infected and mock-infected cells. Pixel intensities for selected spots on the array (in arbitrary units) are presented on the y axis. Results are presented as the mean ± SD. (A) Results at 1 h postinfection; (B) results at 6 h postinfection; (C) results at 24 h postinfection. The results represent those from one experiment (mean ± SD, n = 3), and the experiment was repeated twice.

FIG 4

Inhibition of MERS-CoV infection by kinase inhibitors. Kinase inhibitors were added either preinfection (−1 h) or postinfection (+2 h) at the concentrations listed. Results are presented as the mean ± SD. (A) Preinfection addition of kinase inhibitors targeting signaling pathways/kinases identified from bioinformatics analysis of temporal kinome data; (B) postinfection addition of kinase inhibitors selected from panel A. The cytotoxicities for all compounds at the highest concentration tested (10 μM) were n = 3), with two technological repeats being performed in each experiment. EGFR, epidermal growth factor receptor.

FIG 5

Inhibitory activity of FDA-licensed kinase inhibitors targeting ERK/MAPK or PI3K/AKT signaling added before or after MERS-CoV infection. Kinase inhibitors were added before (−1 h) or after (+2 h) MERS-CoV infection at the concentrations listed. Results are presented as the mean ± SD. (A) Preinfection addition of kinase inhibitors targeting signaling pathways/kinases identified from bioinformatics analysis of temporal kinome data. (B) Postinfection addition of kinase inhibitors. The cytotoxicities for all compounds tested were n = 3), with two technological repeats performed in each experiment. For the pretreatment experiments, trametinib was significantly more inhibitory than selumetinib at 0.1 μM (P < 0.05). For the postaddition experiments, trametinib was significantly more inhibitory than selumetinib at 1 μM (P < 0.005). VEGFR, vascular endothelial growth factor receptor; PDGFR, platelet-derived growth factor receptor.