The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains

Departments of Pathology and.
Journal of Virology (Impact Factor: 4.65). 04/2013; 87(13). DOI: 10.1128/JVI.03316-12
Source: PubMed

ABSTRACT Ebola virus (EBOV) infections are characterized by deficient T lymphocyte responses, T lymphocyte apoptosis and lymphopenia, in the absence of direct infection of lymphocytes. In contrast, dendritic cells (DC) are infected but fail to mature appropriately, thereby impairing the T cell response. We investigated the contribution of EBOV proteins in modulating DC maturation by generating recombinant viruses expressing enhanced green fluorescent protein and carrying mutations affecting several potentially immunomodulating domains. These included envelope glycoprotein (GP) domains as well as innate response antagonist domains (IRADs) previously identified in the VP24 and VP35 proteins. GP expressed by an unrelated vector, but not the wild-type EBOV, was found to strongly induce DC maturation, and infections with recombinant EBOV carrying mutations disabling GP functional domains did not restore DC maturation. In contrast, each of the viruses carrying mutations disabling any IRAD in VP35 induced a dramatic upregulation of DC maturation markers. This was dependent on infection, but not interaction with GP. Disabling of IRADs also resulted in a several hundred fold increase in secretion of cytokines and chemokines. Furthermore, these mutations induced formation of homotypic DC clusters, which represent close correlates of their maturation, and presumably facilitate transfer of antigen from migratory DC to lymph node DC. Thus, an individual IRAD is insufficient to suppress DC maturation; rather, the suppression of DC maturation and the "immune paralysis" observed during EBOV infections results from a cooperative effect of two or more individual IRADs.

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    ABSTRACT: During Ebola virus (EBOV) infection a significant amount of surface glycoprotein GP is shed from infected cells in a soluble form due to cleavage by cellular metalloprotease TACE. Shed GP and non-structural secreted glycoprotein sGP, both expressed from the same GP gene, have been detected in the blood of human patients and experimentally infected animals. In this study we demonstrate that shed GP could play a particular role during EBOV infection. In effect it binds and activates non-infected dendritic cells and macrophages inducing the secretion of pro- and anti-inflammatory cytokines (TNFα, IL1β, IL6, IL8, IL12p40, and IL1-RA, IL10). Activation of these cells by shed GP correlates with the increase in surface expression of co-stimulatory molecules CD40, CD80, CD83 and CD86. Contrary to shed GP, secreted sGP activates neither DC nor macrophages while it could bind DCs. In this study, we show that shed GP activity is likely mediated through cellular toll-like receptor 4 (TLR4) and is dependent on GP glycosylation. Treatment of cells with anti-TLR4 antibody completely abolishes shed GP-induced activation of cells. We also demonstrate that shed GP activity is negated upon addition of mannose-binding sera lectin MBL, a molecule known to interact with sugar arrays present on the surface of different microorganisms. Furthermore, we highlight the ability of shed GP to affect endothelial cell function both directly and indirectly, demonstrating the interplay between shed GP, systemic cytokine release and increased vascular permeability. In conclusion, shed GP released from virus-infected cells could activate non-infected DCs and macrophages causing the massive release of pro- and anti-inflammatory cytokines and effect vascular permeability. These activities could be at the heart of the excessive and dysregulated inflammatory host reactions to infection and thus contribute to high virus pathogenicity.
    PLoS Pathogens 11/2014; 10(11):e1004509. DOI:10.1371/journal.ppat.1004509 · 8.14 Impact Factor
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    ABSTRACT: Zaire ebolavirus (EBOV) VP35 is a double-stranded RNA (dsRNA) binding protein that inhibits RIG-I signaling and interferon (IFN)-α/β responses by both dsRNA-binding dependent and independent mechanisms. VP35 also suppresses DC maturation. Here, we define the pathways and mechanisms through which VP35 impairs DC maturation. Wild-type VP35 (VP35-WT) and two well-characterized VP35 mutants (F239A and R322A) which independently ablate dsRNA-binding and RIG-I inhibition were delivered to primary human monocyte-derived DCs using a lentivirus-based expression system. VP35-WT suppressed not only IFN-α/β but also proinflammatory responses following stimulation of MDDCs with activators of RIG-I-like receptor (RLR) signaling, including RIG-I activators such as Sendai virus (SeV) or 5'-triphosphate RNA or MDA5 activators such as encephalomyocarditis virus (EMCV) or poly(I:C). F239A and R322A exhibited greatly reduced suppression of IFN-α/β and proinflammatory cytokine production following treatment of DCs with RLR agonists. VP35-WT also blocked the upregulation of DC maturation markers and the stimulation of allogeneic T cell responses upon SeV infection, whereas the mutants did not. In contrast to the RLR activators, VP35-WT and the mutant VP35s impaired IFN-β production induced by Toll-Like Receptor (TLR) 3 or TLR4 agonists but failed to inhibit proinflammatory cytokine production induced by TLR2, TLR3, or TLR4 agonists. Further, VP35 did not prevent LPS-induced upregulation of surface markers of MDDC maturation and did not prevent LPS-triggered allogeneic T cell stimulation. Therefore, VP35 is a general antagonist of DC responses to RLR activation. However, TLR agonists can circumvent many of the inhibitory effects of VP35. This suggests strategies to counteract VP35 immune evasion functions.
    Journal of Virology 08/2014; 88(21). DOI:10.1128/JVI.02163-14 · 4.65 Impact Factor
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    ABSTRACT: March 2014, the World Health Organization declared the outbreak of Ebola viral disease (EVD) in Guinea with extensions into Liberia. This is the first known outbreak in an urban settlement. [1] By mid August 2014, 10 cases have been reported so far in Nigeria with two deaths. The first case and death was from a visitor who flew in from Liberia. Ebola conjures fear. Palpable fear, panic and uncertainty seem to have taken over particularly the West African sub-region. This fear derives mainly from paucity of factual knowledge about it, reinforced by the realization that there is currently no vaccine or treatment for this fatal illness with case fatality rate of up to 90%. [2],[3],[4],[5] Further concern exists about the potential for exporting the virus from the outbreak regions to other countries, as well as the possibility of employing the virus as a bioweapon. [6] However, it must be noted that EVD is rare and ordinarily would not be of so much public health significance but for the sensationalistic reportage and lack of effective prophylactic or therapeutic measures. [7] Such reportage has been rife in Nigeria that panic has taken over and permeated every fabric of the Nigerian society. African customs and traditional practices are threatened. Charlatans and spiritualist dish out unscientific and unsubstantiated remedies and claims to cure. Unfortunately, the unwary public has fallen to this in fear and panic. The most celebrated was the employment of warm salt baths and salt drinks for prevention of EVD, to which some citizens hearkened with resultant untoward medical consequences.

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Oct 21, 2014