The Methanolic Extract of Perilla frutescens Robustly Restricts Ebola Virus Glycoprotein-Mediated Entry

Ebola virus (EBOV), one of the most infectious human viruses and a leading cause of viral hemorrhagic fever, imposes a potential public health threat with several recent outbreaks. Despite the difficulties associated with working with this pathogen in biosafety level-4 containment, a protective vaccine and antiviral therapeutic were recently approved. However, the high mortality rate of EBOV infection underscores the necessity to continuously identify novel antiviral strategies to help expand the scope of prophylaxis/therapeutic management against future outbreaks. This includes identifying antiviral agents that target EBOV entry, which could improve the management of EBOV infection. Herein, using EBOV glycoprotein (GP)-pseudotyped particles, we screened a panel of natural medicinal extracts, and identified the methanolic extract of Perilla frutescens (PFME) as a robust inhibitor of EBOV entry. We show that PFME dose-dependently impeded EBOV GP-mediated infection at non-cytotoxic concentrations, and exerted the most significant antiviral activity when both the extract and the pseudoparticles are concurrently present on the host cells. Specifically, we demonstrate that PFME could block viral attachment and neutralize the cell-free viral particles. Our results, therefore, identified PFME as a potent inhibitor of EBOV entry, which merits further evaluation for development as a therapeutic strategy against EBOV infection.

Download Full-text

Viral hemorrhagic fever – a vascular disease?

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613397 ◽

2003 ◽

Vol 89 (06) ◽

pp. 967-972 ◽

Cited By ~ 108

Author(s):

Heinz Feldmann ◽

Hans Schnittler

Keyword(s):

Ebola Virus ◽

Vascular System ◽

Current Knowledge ◽

Hemorrhagic Fever ◽

Host Cells ◽

Fluid Distribution ◽

Target Cells ◽

Viral Hemorrhagic Fever ◽

Virus Infections ◽

Cytokines And Chemokines

SummaryThe syndrome of “viral hemorrhagic fever” in man caused by certain viruses, such as Ebola, Lassa, Dengue, and Crimean-Congo hemorrhagic fever viruses, is often associated with a shock syndrome of undetermined pathogenesis. However, the vascular system, particularly the vascular endothelium, seems to be directly and indirectly targeted by all these viruses. Here we briefly summarize the current knowledge on Marburg and Ebola virus infections, the prototype viral hemorrhagic fever agents, and formulate a working hypothesis for the pathogenesis of viral hemorrhagic fever. Infections with filoviruses show lethality up to 89% and in severe cases lead to a shock syndrome associated with hypotension, coagulation disorders and an imbalance of fluid distribution between the intravascular and extravascular tissue space. The primary target cells for filovi-ruses are mononuclear phagocytotic cells which are activated upon infection and release certain cytokines and chemokines. These mediators indirectly target the endothelium and are thought to play a key role in the pathogenesis of filoviral hemorrhagic fever. In addition, direct infection and subsequent destruction of endothelial cells might contribute to the pathogenesis. Filoviruses, particularly Ebola virus, encode nonstructural glycoproteins which are released from infected host cells. Their function as potential determinants in pathogenicity remains to be investigated.

Download Full-text

Analysis of Resistance of Ebola Virus Glycoprotein-Driven Entry Against MDL28170, An Inhibitor of Cysteine Cathepsins

Pathogens ◽

10.3390/pathogens8040192 ◽

2019 ◽

Vol 8 (4) ◽

pp. 192 ◽

Cited By ~ 3

Author(s):

Markus Hoffmann ◽

Svenja Victoria Kaufmann ◽

Carina Fischer ◽

Wiebke Maurer ◽

Anna-Sophie Moldenhauer ◽

...

Keyword(s):

Viral Entry ◽

Ebola Virus ◽

Mutational Analysis ◽

Cathepsin L ◽

Cysteine Proteases ◽

Host Cells ◽

Single Amino Acid ◽

Limited Information ◽

Cysteine Cathepsins ◽

Ebov Infection

Ebola virus (EBOV) infection can cause severe and frequently fatal disease in human patients. The EBOV glycoprotein (GP) mediates viral entry into host cells. For this, GP depends on priming by the pH-dependent endolysosomal cysteine proteases cathepsin B (CatB) and, to a lesser degree, cathepsin L (CatL), at least in most cell culture systems. However, there is limited information on whether and how EBOV-GP can acquire resistance to CatB/L inhibitors. Here, we addressed this question using replication-competent vesicular stomatitis virus bearing EBOV-GP. Five passages of this virus in the presence of the CatB/CatL inhibitor MDL28170 were sufficient to select resistant viral variants and sequencing revealed that all GP sequences contained a V37A mutation, which, in the context of native GP, is located in the base of the GP surface unit. In addition, some GP sequences harbored mutation S195R in the receptor-binding domain. Finally, mutational analysis demonstrated that V37A but not S195R conferred resistance against MDL28170 and other CatB/CatL inhibitors. Collectively, a single amino acid substitution in GP is sufficient to confer resistance against CatB/CatL inhibitors, suggesting that usage of CatB/CatL inhibitors for antiviral therapy may rapidly select for resistant viral variants.

Download Full-text

Enhancement of Ebola Virus Infection via Ficolin-1 Interaction with the Mucin Domain of GP Glycoprotein

Journal of Virology ◽

10.1128/jvi.00232-16 ◽

2016 ◽

Vol 90 (11) ◽

pp. 5256-5269 ◽

Cited By ~ 12

Author(s):

Anne-Laure Favier ◽

Evelyne Gout ◽

Olivier Reynard ◽

Olivier Ferraris ◽

Jean-Philippe Kleman ◽

...

Keyword(s):

Virus Infection ◽

Ebola Virus ◽

Mannose Binding Lectin ◽

Innate Immune ◽

Host Cells ◽

Target Cells ◽

Viral Glycoprotein ◽

Mannose Binding ◽

Ebov Infection ◽

Binding Lectin

ABSTRACTEbola virus infection requires the surface viral glycoprotein to initiate entry into the target cells. The trimeric glycoprotein is a highly glycosylated viral protein which has been shown to interact with host C-type lectin receptors and the soluble complement recognition protein mannose-binding lectin, thereby enhancing viral infection. Similarly to mannose-binding lectin, ficolins are soluble effectors of the innate immune system that recognize particular glycans at the pathogen surface. In this study, we demonstrate that ficolin-1 interacts with the Zaire Ebola virus (EBOV) glycoprotein, and we characterized this interaction by surface plasmon resonance spectroscopy. Ficolin-1 was shown to bind to the viral glycoprotein with a high affinity. This interaction was mediated by the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of the viral glycoprotein. Using a ficolin-1 control mutant devoid of sialic acid-binding capacity, we identified sialylated moieties of the mucin domain to be potential ligands on the glycoprotein. In cell culture, using both pseudotyped viruses and EBOV, ficolin-1 was shown to enhance EBOV infection independently of the serum complement. We also observed that ficolin-1 enhanced EBOV infection on human monocyte-derived macrophages, described to be major viral target cells,. Competition experiments suggested that although ficolin-1 and mannose-binding lectin recognized different carbohydrate moieties on the EBOV glycoprotein, the observed enhancement of the infection likely depended on a common cellular receptor/partner. In conclusion, ficolin-1 could provide an alternative receptor-mediated mechanism for enhancing EBOV infection, thereby contributing to viral subversion of the host innate immune system.IMPORTANCEA specific interaction involving ficolin-1 (M-ficolin), a soluble effector of the innate immune response, and the glycoprotein (GP) of EBOV was identified. Ficolin-1 enhanced virus infection instead of tipping the balance toward its elimination. An interaction between the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of Ebola virus GP occurred. In this model, the enhancement of infection was shown to be independent of the serum complement. The facilitation of EBOV entry into target host cells by the interaction with ficolin-1 and other host lectins shunts virus elimination, which likely facilitates the survival of the virus in infected host cells and contributes to the virus strategy to subvert the innate immune response.

Download Full-text

Ebola Virus Uptake into Polarized Cells from the Apical Surface

Viruses ◽

10.3390/v11121117 ◽

2019 ◽

Vol 11 (12) ◽

pp. 1117 ◽

Cited By ~ 1

Author(s):

Meng Hu ◽

Fei Wang ◽

Wei Li ◽

Xiaowei Zhang ◽

Zhiping Zhang ◽

...

Keyword(s):

Viral Entry ◽

Ebola Virus ◽

Apical Cell ◽

Hemorrhagic Fever ◽

Vero Cells ◽

Cell Polarization ◽

Apical Surface ◽

Intracellular Receptor ◽

Ebov Infection ◽

Niemann Pick

Ebola virus (EBOV) causes severe hemorrhagic fever with high mortality rates. EBOV can infect many types of cells. During severe EBOV infection, polarized epithelial and endothelial cells are damaged, which promotes vascular instability and dysregulation. However, the mechanism causing these symptoms is largely unknown. Here, we studied virus infection in polarized Vero C1008 cells grown on semipermeable Transwell by using EGFP-labeled Ebola virus-like particles (VLPs). Our results showed that Ebola VLPs preferred to enter polarized Vero cells from the apical cell surface. Furthermore, we showed that the EBOV receptors TIM-1 and Axl were distributed apically, which could be responsible for mediating efficient apical viral entry. Macropinocytosis and intracellular receptor Niemann–Pick type C1 (NPC1) had no polarized distribution, although they played roles in virus entry. This study provides a new view of EBOV uptake and cell polarization, which facilitates a further understanding of EBOV infection and pathogenesis.

Download Full-text

Altered microRNA Transcriptome in Cultured Human Liver Cells upon Infection with Ebola Virus

International Journal of Molecular Sciences ◽

10.3390/ijms22073792 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3792

Author(s):

Idrissa Diallo ◽

Jeffrey Ho ◽

Benoit Laffont ◽

Jonathan Laugier ◽

Abderrahim Benmoussa ◽

...

Keyword(s):

Human Liver ◽

Ebola Virus ◽

Hemorrhagic Fever ◽

Regulatory Elements ◽

Liver Cells ◽

Molecular Signature ◽

Small Rna Sequencing ◽

Human Liver Cell ◽

Ebov Infection ◽

The Impact

Ebola virus (EBOV) is a virulent pathogen, notorious for inducing life-threatening hemorrhagic fever, that has been responsible for several outbreaks in Africa and remains a public health threat. Yet, its pathogenesis is still not completely understood. Although there have been numerous studies on host transcriptional response to EBOV, with an emphasis on the clinical features, the impact of EBOV infection on post-transcriptional regulatory elements, such as microRNAs (miRNAs), remains largely unexplored. MiRNAs are involved in inflammation and immunity and are believed to be important modulators of the host response to viral infection. Here, we have used small RNA sequencing (sRNA-Seq), qPCR and functional analyses to obtain the first comparative miRNA transcriptome (miRNome) of a human liver cell line (Huh7) infected with one of the following three EBOV strains: Mayinga (responsible for the first Zaire outbreak in 1976), Makona (responsible for the West Africa outbreak in 2013–2016) and the epizootic Reston (presumably innocuous to humans). Our results highlight specific miRNA-based immunity pathways and substantial differences between the strains beyond their clinical manifestation and pathogenicity. These analyses shed new light into the molecular signature of liver cells upon EBOV infection and reveal new insights into miRNA-based virus attack and host defense strategy.

Download Full-text

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication

Journal of Virology ◽

10.1128/jvi.00833-17 ◽

2017 ◽

Vol 91 (18) ◽

Cited By ~ 31

Author(s):

Preeti Bharaj ◽

Colm Atkins ◽

Priya Luthra ◽

Maria Isabel Giraldo ◽

Brian E. Dawes ◽

...

Keyword(s):

Virus Replication ◽

Ubiquitin Ligase ◽

Ebola Virus ◽

E3 Ubiquitin Ligase ◽

Hemorrhagic Fever ◽

Viral Polymerase ◽

Highly Pathogenic ◽

Polyubiquitin Chains ◽

Ebov Infection ◽

Potent Antagonist

ABSTRACT Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV.

Download Full-text

Early Transcriptional Changes within Liver, Adrenal Gland, and Lymphoid Tissues Significantly Contribute to Ebola Virus Pathogenesis in Cynomolgus Macaques

Journal of Virology ◽

10.1128/jvi.00250-20 ◽

2020 ◽

Vol 94 (11) ◽

Author(s):

Allen Jankeel ◽

Andrea R. Menicucci ◽

Courtney Woolsey ◽

Karla A. Fenton ◽

Norma Mendoza ◽

...

Keyword(s):

Ebola Virus ◽

Adrenal Glands ◽

Case Fatality ◽

Hemorrhagic Fever ◽

Organ Damage ◽

Host Responses ◽

Lymphoid Tissues ◽

Cynomolgus Macaques ◽

Ebov Infection ◽

Transcriptional Changes

ABSTRACT Ebola virus (EBOV) continues to pose a significant threat to human health, as evidenced by the 2013–2016 epidemic in West Africa and the ongoing outbreak in the Democratic Republic of the Congo. EBOV causes hemorrhagic fever, organ damage, and shock culminating in death, with case fatality rates as high as 90%. This high lethality combined with the paucity of licensed medical countermeasures makes EBOV a critical human pathogen. Although EBOV infection results in significant damage to the liver and the adrenal glands, little is known about the molecular signatures of injury in these organs. Moreover, while changes in peripheral blood cells are becoming increasingly understood, the host responses within organs and lymphoid tissues remain poorly characterized. To address this knowledge gap, we tracked longitudinal transcriptional changes in tissues collected from EBOV-Makona-infected cynomolgus macaques. Following infection, both liver and adrenal glands exhibited significant and early downregulation of genes involved in metabolism, coagulation, hormone synthesis, and angiogenesis; upregulated genes were associated with inflammation. Analysis of lymphoid tissues showed early upregulation of genes that play a role in innate immunity and inflammation and downregulation of genes associated with cell cycle and adaptive immunity. Moreover, transient activation of innate immune responses and downregulation of humoral immune responses in lymphoid tissues were confirmed with flow cytometry. Together, these data suggest that the liver, adrenal gland, and lymphatic organs are important sites of EBOV infection and that dysregulating the function of these vital organs contributes to the development of Ebola virus disease. IMPORTANCE Ebola virus (EBOV) remains a high-priority pathogen since it continues to cause outbreaks with high case fatality rates. Although it is well established that EBOV results in severe organ damage, our understanding of tissue injury in the liver, adrenal glands, and lymphoid tissues remains limited. We begin to address this knowledge gap by conducting longitudinal gene expression studies in these tissues, which were collected from EBOV-infected cynomolgus macaques. We report robust and early gene expression changes within these tissues, indicating they are primary sites of EBOV infection. Furthermore, genes involved in metabolism, coagulation, and adaptive immunity were downregulated, while inflammation-related genes were upregulated. These results indicate significant tissue damage consistent with the development of hemorrhagic fever and lymphopenia. Our study provides novel insight into EBOV-host interactions and elucidates how host responses within the liver, adrenal glands, and lymphoid tissues contribute to EBOV pathogenesis.

Download Full-text

Ebola Virus and Severe Acute Respiratory Syndrome Coronavirus Display Late Cell Entry Kinetics: Evidence that Transport to NPC1+Endolysosomes Is a Rate-Defining Step

Journal of Virology ◽

10.1128/jvi.03398-14 ◽

2014 ◽

Vol 89 (5) ◽

pp. 2931-2943 ◽

Cited By ~ 67

Author(s):

Rebecca M. Mingo ◽

James A. Simmons ◽

Charles J. Shoemaker ◽

Elizabeth A. Nelson ◽

Kathryn L. Schornberg ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Ebola Virus ◽

Cathepsin L ◽

Hemorrhagic Fever ◽

Endocytic Pathway ◽

Host Cells ◽

Effective Vaccine ◽

Severe Respiratory Distress ◽

Cathepsin Activity ◽

Do So

ABSTRACTEbola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1+LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity.IMPORTANCEEbola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1+LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1+LE/Lys, as a therapeutic target for SARS and EBOV.

Download Full-text

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.

Download Full-text

The Roles of Ebola Virus Soluble Glycoprotein in Replication, Pathogenesis, and Countermeasure Development

Viruses ◽

10.3390/v11110999 ◽

2019 ◽

Vol 11 (11) ◽

pp. 999 ◽

Cited By ~ 3

Author(s):

Wenjun Zhu ◽

Logan Banadyga ◽

Karla Emeterio ◽

Gary Wong ◽

Xiangguo Qiu

Keyword(s):

Immune Response ◽

Envelope Protein ◽

Ebola Virus ◽

Hemorrhagic Fever ◽

Antiviral Drugs ◽

Viral Envelope ◽

Multiple Roles ◽

Its Sequence ◽

Viral Envelope Protein ◽

Ebov Infection

Ebola virus (EBOV) is a highly lethal pathogen that has caused several outbreaks of severe hemorrhagic fever in humans since its emergence in 1976. The EBOV glycoprotein (GP1,2) is the sole viral envelope protein and a major component of immunogenicity; it is encoded by the GP gene along with two truncated versions: soluble GP (sGP) and small soluble GP (ssGP). sGP is, in fact, the primary product of the GP gene, and it is secreted in abundance during EBOV infection. Since sGP shares large portions of its sequence with GP1,2, it has been hypothesized that sGP may subvert the host immune response by inducing antibodies against sGP rather than GP1,2. Several reports have shown that sGP plays multiple roles that contribute to the complex pathogenesis of EBOV. In this review, we focus on sGP and discuss its possible roles with regards to the pathogenesis of EBOV and the development of specific antiviral drugs.

Download Full-text