Ebola virus infection induces a type I interferon response and the shutdown of key liver functions in human iPSC-derived hepatocytes

Abstract Liver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to the observed inflammatory response. Here, we present an iPSC-derived hepatocyte platform to define the hepato-intrinsic response to EBOV infection. Transcriptomics analysis revealed a delayed host response with minimal transcriptomic changes at one day post infection (dpi) followed by a general downregulation of genes associated with hepatic functions and upregulation of interferon signaling at two and three dpi. Using RNA-FISH, we showed at single cell resolution that IFNβ and CXCL10 were mainly expressed in bystander cells or cells with weak EBOV mRNA signal intensity. We did not observe an inflammatory signature at any timepoint. In conclusion, iPSC-derived hepatocytes are an immune competent platform to study intrinsic responses to EBOV infection that have not been observed in EBOV-infected hepatocarcinoma cell lines.

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Quercetin Blocks Ebola Virus Infection by Counteracting the VP24 Interferon-Inhibitory Function

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00530-20 ◽

2020 ◽

Vol 64 (7) ◽

Cited By ~ 2

Author(s):

Elisa Fanunza ◽

Mathieu Iampietro ◽

Simona Distinto ◽

Angela Corona ◽

Marina Quartu ◽

...

Keyword(s):

Ebola Virus ◽

Hemorrhagic Fever ◽

Type I ◽

Virulence Determinants ◽

Inhibitory Function ◽

Viral Binding ◽

Ebov Infection ◽

Small Set ◽

Tight Correlation ◽

Ebola Virus Infection

ABSTRACT Ebola virus (EBOV) is among the most devastating pathogens causing fatal hemorrhagic fever in humans. The epidemics from 2013 to 2016 resulted in more than 11,000 deaths, and another outbreak is currently ongoing. Since there is no FDA-approved drug so far to fight EBOV infection, there is an urgent need to focus on drug discovery. Considering the tight correlation between the high EBOV virulence and its ability to suppress the type I interferon (IFN-I) system, identifying molecules targeting viral protein VP24, one of the main virulence determinants blocking the IFN response, is a promising novel anti-EBOV therapy approach. Hence, in the effort to find novel EBOV inhibitors, a screening of a small set of flavonoids was performed; it showed that quercetin and wogonin can suppress the VP24 effect on IFN-I signaling inhibition. The mechanism of action of the most active compound, quercetin, showing a half-maximal inhibitory concentration (IC50) of 7.4 μM, was characterized to significantly restore the IFN-I signaling cascade, blocked by VP24, by directly interfering with the VP24 binding to karyopherin-α and thus restoring P-STAT1 nuclear transport and IFN gene transcription. Quercetin significantly blocked viral infection, specifically targeting EBOV VP24 anti-IFN-I function. Overall, quercetin is the first identified inhibitor of the EBOV VP24 anti-IFN function, representing a molecule interacting with a viral binding site that is very promising for further drug development aiming to block EBOV infection at the early steps.

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Quantitative Proteome Analysis of Atg5-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome

mSystems ◽

10.1128/msystems.00481-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 1

Author(s):

Kiran Bala Sharma ◽

Manish Sharma ◽

Suruchi Aggarwal ◽

Amit Kumar Yadav ◽

Shinjini Bhatnagar ◽

...

Keyword(s):

Cell Adhesion ◽

Inflammatory Response ◽

Transforming Growth Factor ◽

Proteome Analysis ◽

Interferon Response ◽

Type I ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

The Impact

ABSTRACT Basal autophagy is crucial for maintenance of cellular homeostasis. ATG5 is an essential protein for autophagosome formation, and its depletion has been extensively used as a tool to disrupt autophagy. Here, we characterize the impact of Atg5 deficiency on the cellular proteome of mouse embryonic fibroblasts (MEFs). Using a tandem mass tagging (TMT)-based quantitative proteomics analysis, we observe that 14% of identified proteins show dysregulated levels in atg5−/− MEFs. These proteins were distributed across diverse biological processes, such as cell adhesion, development, differentiation, transport, metabolism, and immune responses. Several of the upregulated proteins were receptors involved in transforming growth factor β (TGF-β) signaling, JAK-STAT signaling, junction adhesion, and interferon/cytokine-receptor interactions and were validated as autophagy substrates. Nearly equal numbers of proteins, including several lysosomal proteins and enzymes, were downregulated, suggesting a complex role of autophagy/ATG5 in regulating their levels. The atg5−/− MEFs had lower levels of key immune sensors and effectors, including Toll-like receptor 2 (TLR2), interferon regulatory factor 3 (IRF3), IRF7, MLKL, and STAT1/3/5/6, which were restored by reexpression of ATG5. While these cells could efficiently mount a type I interferon response to the double-stranded RNA (dsRNA) mimic poly(I·C), they were compromised in their inflammatory response to the bacterial pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and Pam3CSK4. Transcriptional activation and secretion of interleukin-6 (IL-6) in these cells could be recovered by ATG5 expression, supporting the role of autophagy in the TLR2-induced inflammatory response. This study provides a key resource for understanding the effect of autophagy/ATG5 deficiency on the fibroblast proteome. IMPORTANCE Autophagy performs housekeeping functions for cells and maintains a functional mode by degrading damaged proteins and organelles and providing energy under starvation conditions. The process is tightly regulated by the evolutionarily conserved Atg genes, of which Atg5 is one such crucial mediator. Here, we have done a comprehensive quantitative proteome analysis of mouse embryonic fibroblasts that lack a functional autophagy pathway (Atg5 knockout). We observe that 14% of the identified cellular proteome is remodeled, and several proteins distributed across diverse cellular processes with functions in signaling, cell adhesion, development, and immunity show either higher or lower levels under autophagy-deficient conditions. These cells have lower levels of crucial immune proteins that are required to mount a protective inflammatory response. This study will serve as a valuable resource to determine the role of autophagy in modulating specific protein levels in cells.

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Inflammatory and Humoral Immune Response during Ebola Virus Infection in Survivor and Fatal Cases Occurred in Sierra Leone during the 2014–2016 Outbreak in West Africa

Viruses ◽

10.3390/v11040373 ◽

2019 ◽

Vol 11 (4) ◽

pp. 373 ◽

Cited By ~ 8

Author(s):

Francesca Colavita ◽

Mirella Biava ◽

Concetta Castilletti ◽

Simone Lanini ◽

Rossella Miccio ◽

...

Keyword(s):

Immune Response ◽

Inflammatory Response ◽

Sierra Leone ◽

Ebola Virus ◽

Virus Disease ◽

Late Phase ◽

Treatment Center ◽

Humoral Immune ◽

Western Africa ◽

Ebov Infection

Ebola virus (EBOV) infection is characterized by an excessive inflammatory response, a loss of lymphocytes and a general paralysis of the immune system, however pathophysiological mechanisms are not fully understood. In a cohort of 23 fatal and 21 survivors of ebola virus disease (EVD) cases admitted to the Emergency Ebola-Treatment-Center in Goderich (Freetown, Sierra Leone) during the 2014 to 2016 EBOV epidemic in Western Africa, we analyzed the pathway-focused gene expression profile of secreted proteins involved in the immune response and the levels of specific anti-EBOV IgM and IgG from the time of admission till discharge or death. We observed a dysregulated inflammatory response in fatal patients as compared to survivors, mainly consisting of the upregulation of inflammatory mediators, whose extent directly correlated with viremia levels. The upregulation persisted and intensified during the late phase of infection. Relevant differences were also found in humoral immunity, as an earlier and more robust EBOV antibody response was observed in survivor patients.

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Mannosyl Glycodendritic Structure Inhibits DC-SIGN-Mediated Ebola Virus Infection in cis and in trans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.12.3970-3972.2003 ◽

2003 ◽

Vol 47 (12) ◽

pp. 3970-3972 ◽

Cited By ~ 81

Author(s):

Fátima Lasala ◽

Eva Arce ◽

Joaquín R. Otero ◽

Javier Rojo ◽

Rafael Delgado

Keyword(s):

Dendritic Cell ◽

Virus Infection ◽

Adhesion Molecule ◽

Ebola Virus ◽

Cell Entry ◽

Intercellular Adhesion Molecule ◽

Ebov Infection ◽

In Trans ◽

In Cis ◽

Ebola Virus Infection

ABSTRACT We have designed a glycodendritic structure, BH30sucMan, that blocks the interaction between dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and Ebola virus (EBOV) envelope. BH30sucMan inhibits DC-SIGN-mediated EBOV infection at nanomolar concentrations. BH30sucMan may counteract important steps of the infective process of EBOV and, potentially, of microorganisms shown to exploit DC-SIGN for cell entry and infection.

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Investigating the Cellular Transcriptomic Response Induced by the Makona Variant of Ebola Virus in Differentiated THP-1 Cells

Viruses ◽

10.3390/v11111023 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1023 ◽

Cited By ~ 1

Author(s):

Andrew Bosworth ◽

Stuart D. Dowall ◽

Stuart Armstrong ◽

Xuan Liu ◽

Xiaofeng Dong ◽

...

Keyword(s):

Inflammatory Response ◽

Ebola Virus ◽

Virus Disease ◽

Monocytic Cell ◽

Monocytic Cell Line ◽

Transcriptomic Response ◽

Human Monocytic Cell Line ◽

Human Monocytic Cell ◽

Transcriptomic Changes ◽

Reston Virus

Recent studies have shown that transcriptomic analysis of blood samples taken from patients with acute Ebola virus disease (EVD) during the 2013–2016 West African outbreak was suggestive that a severe inflammatory response took place in acutely ill patients. The significant knowledge gained from studying the Makona variant, a cause of the largest known EVD outbreak, may be applicable to other species of ebolavirus, and other variants of the Ebola virus (EBOV) species. To investigate the ability of Makona to initiate an inflammatory response in human macrophages and characterise the host response in a similar manner to previously characterised EBOV variants, the human monocytic cell line THP-1 was differentiated into macrophage-like cells and infected with Makona. RNA-Seq and quantitative proteomics were used to identify and quantify host mRNA and protein abundance during infection. Data from infection with Reston virus (RESTV) were used as comparators to investigate changes that may be specific to, or enhanced in, Makona infection in relation to a less pathogenic species of ebolavirus.. This study found demonstrable induction of the inflammatory response, and increase in the activation state of THP-1 macrophages infected with Makona. NFκB and inflammation-associated transcripts displayed significant changes in abundance, reflective of what was observed in human patients during the 2013–2016 EBOV outbreak in West Africa, and demonstrated that transcriptomic changes found in Makona-infected cells were similar to that observed in Reston virus infection and that have been described in previous studies of other variants of EBOV.

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Ebolaviruses Associated with Differential Pathogenicity Induce Distinct Host Responses in Human Macrophages

Journal of Virology ◽

10.1128/jvi.00179-17 ◽

2017 ◽

Vol 91 (11) ◽

Cited By ~ 25

Author(s):

Judith Olejnik ◽

Adriana Forero ◽

Laure R. Deflubé ◽

Adam J. Hume ◽

Whitney A. Manhart ◽

...

Keyword(s):

Host Response ◽

Ebola Virus ◽

Severe Disease ◽

Health Concern ◽

Type I ◽

Proinflammatory Response ◽

Tlr4 Signaling ◽

Human Macrophages ◽

Ebov Infection ◽

Reston Virus

ABSTRACT Ebola virus (EBOV) and Reston virus (RESTV) are members of the Ebolavirus genus which greatly differ in their pathogenicity. While EBOV causes a severe disease in humans characterized by a dysregulated inflammatory response and elevated cytokine and chemokine production, there are no reported disease-associated human cases of RESTV infection, suggesting that RESTV is nonpathogenic for humans. The underlying mechanisms determining the pathogenicity of different ebolavirus species are not yet known. In this study, we dissected the host response to EBOV and RESTV infection in primary human monocyte-derived macrophages (MDMs). As expected, EBOV infection led to a profound proinflammatory response, including strong induction of type I and type III interferons (IFNs). In contrast, RESTV-infected macrophages remained surprisingly silent. Early activation of IFN regulatory factor 3 (IRF3) and NF-κB was observed in EBOV-infected, but not in RESTV-infected, MDMs. In concordance with previous results, MDMs treated with inactivated EBOV and Ebola virus-like particles (VLPs) induced NF-κB activation mediated by Toll-like receptor 4 (TLR4) in a glycoprotein (GP)-dependent manner. This was not the case in cells exposed to live RESTV, inactivated RESTV, or VLPs containing RESTV GP, indicating that RESTV GP does not trigger TLR4 signaling. Our results suggest that the lack of immune activation in RESTV-infected MDMs contributes to lower pathogenicity by preventing the cytokine storm observed in EBOV infection. We further demonstrate that inhibition of TLR4 signaling abolishes EBOV GP-mediated NF-κB activation. This finding indicates that limiting the excessive TLR4-mediated proinflammatory response in EBOV infection should be considered as a potential supportive treatment option for EBOV disease. IMPORTANCE Emerging infectious diseases are a major public health concern, as exemplified by the recent devastating Ebola virus (EBOV) outbreak. Different ebolavirus species are associated with widely varying pathogenicity in humans, ranging from asymptomatic infections for Reston virus (RESTV) to severe disease with fatal outcomes for EBOV. In this comparative study of EBOV- and RESTV-infected human macrophages, we identified key differences in host cell responses. Consistent with previous data, EBOV infection is associated with a proinflammatory signature triggered by the surface glycoprotein (GP), which can be inhibited by blocking TLR4 signaling. In contrast, infection with RESTV failed to stimulate a strong host response in infected macrophages due to the inability of RESTV GP to stimulate TLR4. We propose that disparate proinflammatory host signatures contribute to the differences in pathogenicity reported for ebolavirus species and suggest that proinflammatory pathways represent an intriguing target for the development of novel therapeutics.

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Role of NLRs in the Regulation of Type I Interferon Signaling, Host Defense and Tolerance to Inflammation

International Journal of Molecular Sciences ◽

10.3390/ijms22031301 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1301

Author(s):

Ioannis Kienes ◽

Tanja Weidl ◽

Nora Mirza ◽

Mathias Chamaillard ◽

Thomas A. Kufer

Keyword(s):

Viral Infections ◽

Type I Interferon ◽

Tissue Injury ◽

Interferon Response ◽

Type I ◽

Interferon Signaling ◽

Microbial Products ◽

Interferon Responses

Type I interferon signaling contributes to the development of innate and adaptive immune responses to either viruses, fungi, or bacteria. However, amplitude and timing of the interferon response is of utmost importance for preventing an underwhelming outcome, or tissue damage. While several pathogens evolved strategies for disturbing the quality of interferon signaling, there is growing evidence that this pathway can be regulated by several members of the Nod-like receptor (NLR) family, although the precise mechanism for most of these remains elusive. NLRs consist of a family of about 20 proteins in mammals, which are capable of sensing microbial products as well as endogenous signals related to tissue injury. Here we provide an overview of our current understanding of the function of those NLRs in type I interferon responses with a focus on viral infections. We discuss how NLR-mediated type I interferon regulation can influence the development of auto-immunity and the immune response to infection.

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Epromoters function as a hub to recruit key transcription factors required for the inflammatory response

Nature Communications ◽

10.1038/s41467-021-26861-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

David Santiago-Algarra ◽

Charbel Souaid ◽

Himanshu Singh ◽

Lan T. M. Dao ◽

Saadat Hussain ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Inflammatory Response ◽

Gene Clusters ◽

Regulatory Elements ◽

Interferon Response ◽

Type I ◽

Gene Promoters ◽

Bona Fide ◽

Distal Gene

AbstractGene expression is controlled by the involvement of gene-proximal (promoters) and distal (enhancers) regulatory elements. Our previous results demonstrated that a subset of gene promoters, termed Epromoters, work as bona fide enhancers and regulate distal gene expression. Here, we hypothesized that Epromoters play a key role in the coordination of rapid gene induction during the inflammatory response. Using a high-throughput reporter assay we explored the function of Epromoters in response to type I interferon. We find that clusters of IFNa-induced genes are frequently associated with Epromoters and that these regulatory elements preferentially recruit the STAT1/2 and IRF transcription factors and distally regulate the activation of interferon-response genes. Consistently, we identified and validated the involvement of Epromoter-containing clusters in the regulation of LPS-stimulated macrophages. Our findings suggest that Epromoters function as a local hub recruiting the key TFs required for coordinated regulation of gene clusters during the inflammatory response.

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Activation of RIG-I-mediated antiviral signaling triggers autophagy through the MAVS-TRAF6-Beclin-1 signaling axis

10.1101/340323 ◽

2018 ◽

Cited By ~ 1

Author(s):

Na-Rae Lee ◽

Junsu Ban ◽

Noh-Jin Lee ◽

Chae-Min Yi ◽

Jiyoon Choi ◽

...

Keyword(s):

Type I Interferon ◽

Sendai Virus ◽

Feedback Mechanism ◽

Innate Immune ◽

Beclin 1 ◽

Interferon Response ◽

Type I ◽

Interferon Signaling ◽

Negative Feedback Mechanism ◽

Signaling Axis

AbstractAutophagy has been implicated in innate immune responses against various intracellular pathogens. Recent studies have reported that autophagy can be triggered by pathogen recognizing sensors, including Toll-like receptors and cyclic guanosine monophosphate-adenosine monophosphate synthase, to participate in innate immunity. In the present study, we examined whether the RIG-I signaling pathway, which detects viral infections by recognizing viral RNA, triggers the autophagic process. The introduction of polyI:C into the cytoplasm, or Sendai virus infection, significantly induced autophagy in normal cells but not in RIG-I-deficient cells. PolyI:C transfection or Sendai virus infection induced autophagy in the cells lacking type-I interferon signaling. This demonstrated that the effect was not due to interferon signaling. RIG-I-mediated autophagy diminished by the deficiency of mitochondrial antiviral signaling protein (MAVS) or tumor necrosis factor receptor-associated factor (TRAF)6, showing that the RIG-I-MAVS-TRAF6 signaling axis was critical for RIG-I-mediated autophagy. We also found that Beclin-1 was translocated to the mitochondria, and it interacted with TRAF6 upon RIG-I activation. Furthermore, Beclin-1 underwent K63-polyubiquitination upon RIG-I activation, and the ubiquitination decreased in TRAF6-deficient cells. This suggests that the RIG-I-MAVS-TRAF6 axis induced K63-linked polyubiquitination of Beclin-1, which has been implicated in triggering autophagy. Collectively, the results of this study show that the recognition of viral infection by RIG-I is capable of inducing autophagy to control viral replication. As deficient autophagy increases the type-I interferon response, the induction of autophagy by the RIG-I pathway might also contribute to preventing an excessive interferon response as a negative-feedback mechanism.ImportanceMammalian cells utilize various innate immune sensors to detect pathogens. Among those sensors, RIG-I recognizes viral RNA to detect intracellular viral replication. Although cells experience diverse physiological changes upon viral infection, studies to understand the role of RIG-I signaling have focused on the induction of type-I interferon. Autophagy is a process that sequesters cytosolic regions and degrades the contents to maintain cellular homeostasis. Autophagy participates in the immune system, and has been known to be triggered by some innate immune sensors, such as TLR4 and cGAS. We demonstrated that autophagy can be triggered by the activation of RIG-I. In addition, we also proved that MAVS-TRAF6 downstream signaling is crucial for the process. Beclin-1, a key molecule in autophagy, is translocated to mitochondria, where it undergoes K63-ubiquitination in a TRAF6-dependent manner upon RIG-I activation. As autophagy negatively regulates RIG-I-mediated signaling, the RIG-I-mediated activation of autophagy may function as a negative-feedback mechanism.

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Suppression of Type I Interferon Signaling by E1A via RuvBL1/Pontin

Journal of Virology ◽

10.1128/jvi.02484-16 ◽

2017 ◽

Vol 91 (8) ◽

Cited By ~ 8

Author(s):

Oladunni Olanubi ◽

Jasmine Rae Frost ◽

Sandi Radko ◽

Peter Pelka

Keyword(s):

Gene Expression ◽

Cellular Response ◽

Type I Interferon ◽

Early Gene ◽

Interferon Response ◽

Type I ◽

Dependent Manner ◽

Interferon Signaling ◽

Interferon Pathway ◽

Regulated Promoters

ABSTRACT Suppression of interferon signaling is of paramount importance to a virus. Interferon signaling significantly reduces or halts the ability of a virus to replicate; therefore, viruses have evolved sophisticated mechanisms that suppress activation of the interferon pathway or responsiveness of the infected cell to interferon. Adenovirus has multiple modes of inhibiting the cellular response to interferon. Here, we report that E1A, previously shown to regulate interferon signaling in multiple ways, inhibits interferon-stimulated gene expression by modulating RuvBL1 function. RuvBL1 was previously shown to affect type I interferon signaling. E1A binds to RuvBL1 and is recruited to RuvBL1-regulated promoters in an interferon-dependent manner, preventing their activation. Depletion of RuvBL1 impairs adenovirus growth but does not appear to significantly affect viral protein expression. Although RuvBL1 has been shown to play a role in cell growth, its depletion had no effect on the ability of the virus to replicate its genome or to drive cells into S phase. E1A was found to bind to RuvBL1 via the C terminus of E1A, and this interaction was important for suppression of interferon-stimulated gene transcriptional activation and recruitment of E1A to interferon-regulated promoters. Here, we report the identification of RuvBL1 as a new target for adenovirus in its quest to suppress the interferon response. IMPORTANCE For most viruses, suppression of the interferon signaling pathway is crucial to ensure a successful replicative cycle. Human adenovirus has evolved several different mechanisms that prevent activation of interferon or the ability of the cell to respond to interferon. The viral immediate-early gene E1A was previously shown to affect interferon signaling in several different ways. Here, we report a novel mechanism reliant on RuvBL1 that E1A uses to prevent activation of interferon-stimulated gene expression following infection or interferon treatment. This adds to the growing knowledge of how viruses are able to inhibit interferon and identifies a novel target used by adenovirus for modulation of the cellular interferon pathway.

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