The ERK Mitogen-Activated Protein Kinase Pathway Contributes to Ebola Virus Glycoprotein-Induced Cytotoxicity

Vaccine Research Center, NIAID, National Institutes of Health, Room 4502, Bldg. 40, MSC-3005, 40 Convent Drive, Bethesda, MD 20892-3005, USA.
Journal of Virology (Impact Factor: 4.44). 03/2007; 81(3):1230-40. DOI: 10.1128/JVI.01586-06
Source: PubMed


Ebola virus is a highly lethal pathogen that causes hemorrhagic fever in humans and nonhuman primates. Among the seven known viral gene products, the envelope glycoprotein (GP) alone induces cell rounding and detachment that ultimately leads to cell death. Cellular cytoxicity is not seen with comparable levels of expression of a mutant form of GP lacking a mucin-like domain (GPDeltamuc). GP-induced cell death is nonapoptotic and is preceded by downmodulation of cell surface molecules involved in signaling pathways, including certain integrins and epidermal growth factor receptor. To investigate the mechanism of GP-induced cellular toxicity, we analyzed the activation of several signal transduction pathways involved in cell growth and survival. The active form of extracellular signal-regulated kinases types 1 and 2 (ERK1/2), phospho-ERK1/2, was reduced in cells expressing GP compared to those expressing GPDeltamuc as determined by flow cytometry, in contrast to the case for several other signaling proteins. Subsequent analysis of the activation states and kinase activities of related kinases revealed a more pronounced effect on the ERK2 kinase isoform. Disruption of ERK2 activity by a dominant negative ERK or by small interfering RNA-mediated ERK2 knockdown potentiated the decrease in alphaV integrin expression associated with toxicity. Conversely, activation of the pathway through the expression of a constitutively active form of ERK2 significantly protected against this effect. These results indicate that the ERK signaling cascade mediates GP-mediated cytotoxicity and plays a role in pathogenicity induced by this gene product.

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    • "GP has pleiotropic effects on host cells which might contribute to the effects of virus and VLPs on macrophage and DC function. These include the induction of MAPK and other signaling pathways (Martinez et al., 2007; Zampieri et al., 2007). GP also masks surface molecules, including class I MHC and beta integrins, which may influence the ability of infected cells to trigger immune responses (Simmons et al., 2002, Reynard, 2009)Francica et al., 2010) (Takada et al., 2000). "
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    ABSTRACT: The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), are highly lethal zoonotic agents of concern as emerging pathogens and potential bioweapons. Antigen-presenting cells (APCs), particularly macrophages and dendritic cells, are targets of filovirus infection in vivo. Infection of these cell types has been proposed to contribute to the inflammation, activation of coagulation cascades and ineffective immune responses characteristic of filovirus hemorrhagic fever. However, many aspects of filovirus-APC interactions remain to be clarified. Among the unanswered questions: What determines the ability of filoviruses to replicate in different APC subsets? What are the cellular signaling pathways that sense infection and lead to production of copious quantities of cytokines, chemokines and tissue factor? What are the mechanisms by which innate antiviral responses are disabled by these viruses, and how may these mechanisms contribute to inadequate adaptive immunity? A better understanding of these issues will clarify the pathogenesis of filoviral hemorrhagic fever and provide new avenues for development of therapeutics.
    Antiviral research 02/2012; 93(3):416-28. DOI:10.1016/j.antiviral.2012.01.011 · 3.94 Impact Factor
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    • "Constitutive activation of ERK2, but not ERK1, is 391 critical for the acquired resistance to Imatinib Mesylate in chronic myelogenous 392 leukemia management (Aceves-Luquero et al., 2009). In addition to cancers, Ebola 393 virus envelope glycoprotein reduced phosphorylation and kinase activity of ERK2, 394 but not ERK1, correlating with induction of cell death (Zampieri et al., 2007). 395 Vaccinia virus M2L protein blocks ERK2 phosphorylation, inhibiting virus-induced 396 NF-κB activation (Gedey et al., 2006). "
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    ABSTRACT: Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a deubiquitinating enzyme, reportedly blocks poly I : C-induced activation of interferon regulatory factor 3 and nuclear factor kappa B, reducing interferon (IFN) induction. This study investigated type I IFN antagonist mechanism of PLpro in human promonocytes. PLpro antagonized IFN-α-induced responses such as interferon-stimulated response element- and AP-1-driven promoter activation, protein kinase R, 2'-5'-oligoadenylate synthetase (OAS), interleukin (IL)-6 and IL-8 expression, and signal transducers and activators of transcription (STAT) 1 (Tyr701), STAT1 (Ser727) and c-Jun phosphorylation. A proteomics approach demonstrated downregulation of extracellular signal-regulated kinase (ERK) 1 and upregulation of ubiquitin-conjugating enzyme (UBC) E2-25k as inhibitory mechanism of PLpro on IFN-α-induced responses. IFN-α treatment significantly induced mRNA expression of UBC E2-25k, but not ERK1, causing time-dependent decrease of ERK1, but not ERK2, in PLpro-expressing cells. Poly-ubiquitination of ERK1 showed a relationship between ERK1 and ubiquitin proteasome signalling pathways associated with IFN antagonism by PLpro. Combination treatment of IFN-α and the proteasome inhibitor MG-132 showed a time-dependent restoration of ERK1 protein levels and significant increase of ERK1, STAT1 and c-Jun phosphorylation in PLpro-expressing cells. Importantly, PD098059 (an ERK1/2 inhibitor) treatment significantly reduced IFN-α-induced ERK1 and STAT1 phosphorylation, inhibiting IFN-α-induced expression of 2'-5'-OAS in vector control cells and PLpro-expressing cells. Overall results proved downregulation of ERK1 by ubiquitin proteasomes and suppression of interaction between ERK1 and STAT1 as type I IFN antagonist function of SARS-CoV PLpro.
    Journal of General Virology 04/2011; 92(Pt 5):1127-40. DOI:10.1099/vir.0.028936-0 · 3.18 Impact Factor
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    • "Data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR). Clone 36 and similar monoclonal antibodies to p38 MAPK (T180/Y182) are commonly used in flow cytometry and Western blot analysis to measure p-p38 (Rius et al. 2010; Zampieri et al. 2007). In vitro studies were conducted to demonstrate antibody specificity and the impact of IFN-alpha on intracellular p38 protein and p38 phosphorylation in specific lymphocyte subsets (Supplementary Materials). "
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    ABSTRACT: Cytokine-induced stimulation of p38 mitogen activated protein kinase (MAPK) has been shown to influence behaviorally-relevant pathophysiologic pathways including monoamine neurotransmission and neuroendocrine function and thus may contribute to behavioral changes that occur during chronic administration of the innate immune cytokine, interferon (IFN)-alpha. Accordingly, in the current study, phosphorylation (activation) of intracellular p38 MAPK in peripheral blood lymphocytes was analyzed by flow cytometry every 2 h for 12 h following the initial injection of IFN-alpha in eleven patients with chronic hepatitis C. Hourly assessments of plasma concentrations of adrenocorticotropic hormone, cortisol and interleukin-6 were also obtained. Symptoms of depression and fatigue were measured at baseline and after 4 and 12 weeks of IFN-alpha treatment. Acute administration of IFN-alpha significantly increased the percentage of lymphocytes staining positive for intracellular phosphorylated p38 (p-p38). IFN-alpha-induced increases in p-p38 were significantly greater in patients that developed clinically significant depressive symptoms [Montgomery-Asberg Depression Rating Scale (MADRS) score≥15] during the first 12 weeks of IFN-alpha treatment. Increases in the percentage of p-p38-positive lymphocytes following the first IFN-alpha injection also highly correlated with depression severity at weeks 4 (r=0.85, p=0.001) and 12 (r=0.70, p=0.018). Similar relationships were observed for fatigue. Examination of relationships between p-p38 induction and factors previously reported to predict IFN-alpha-induced depressive symptoms revealed strong associations of p-p38 with baseline MADRS (r=0.82, p=0.002) and cortisol responses to the initial injection of IFN-alpha (r=0.91, p=0.000). Taken together, these findings indicate that sensitivity of p38 MAPK signaling pathways to immune stimulation is associated with depressive symptoms during chronic IFN-alpha treatment.
    Brain Behavior and Immunity 02/2011; 25(6):1094-8. DOI:10.1016/j.bbi.2011.02.015 · 5.89 Impact Factor
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