Identification of Ebola Virus Inhibitors Targeting GP2 Using Principles of Molecular Mimicry

ABSTRACTA key step in the Ebola virus (EBOV) replication cycle involves conformational changes in viral glycoprotein 2 (GP2) which facilitate host-viral membrane fusion and subsequent release of the viral genome. Ebola GP2 plays a critical role in virus entry and has similarities in mechanism and structure to the HIV gp41 protein for which inhibitors have been successfully developed. In this work, a putative binding pocket for the C-terminal heptad repeat in the N-terminal heptad repeat trimer was targeted for identification of small molecules that arrest EBOV-host membrane fusion. Two computational structure-based virtual screens of ∼1.7 M compounds were performed (DOCK program) against a GP2 five-helix bundle, resulting in 165 commercially available compounds purchased for experimental testing. Based on assessment of inhibitory activity, cytotoxicity, and target specificity, four promising candidates emerged with 50% inhibitory concentration values in the 3 to 26 μM range. Molecular dynamics simulations of the two most potent candidates in their DOCK-predicted binding poses indicate that the majority of favorable interactions involve seven highly conserved residues that can be used to guide further inhibitor development and refinement targeting EBOV.IMPORTANCEThe most recent Ebola virus disease outbreak, from 2014 to 2016, resulted in approximately 28,000 individuals becoming infected, which led to over 12,000 causalities worldwide. The particularly high pathogenicity of the virus makes paramount the identification and development of promising lead compounds to serve as inhibitors of Ebola infection. To limit viral load, the virus-host membrane fusion event can be targeted through the inhibition of the class I fusion glycoprotein ofEbolavirus. In the current work, several promising small-molecule inhibitors that target the glycoprotein GP2 were identified through systematic application of structure-based computational and experimental drug design procedures.

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A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

Molecular Biology of the Cell ◽

10.1091/mbc.10.12.4191 ◽

1999 ◽

Vol 10 (12) ◽

pp. 4191-4200 ◽

Cited By ~ 38

Author(s):

David H. Kingsley ◽

Ali Behbahani ◽

Afshin Rashtian ◽

Gary W. Blissard ◽

Joshua Zimmerberg

Keyword(s):

Fusion Protein ◽

Membrane Fusion ◽

Conformational Changes ◽

Fusion Proteins ◽

Critical Role ◽

Low Ph ◽

Heptad Repeat ◽

Viral Fusion ◽

Viral Fusion Protein ◽

Heptad Repeats

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH–induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.

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JOINT ESTIMATION OF THE TRANSMISSIBILITY AND SEVERITY OF EBOLA VIRUS DISEASE IN REAL TIME

Journal of Biological System ◽

10.1142/s0218339017400022 ◽

2017 ◽

Vol 25 (04) ◽

pp. 587-603 ◽

Cited By ~ 2

Author(s):

YUSUKE ASAI ◽

HIROSHI NISHIURA

Keyword(s):

Real Time ◽

Ebola Virus ◽

Virus Disease ◽

Critical Role ◽

Estimation Method ◽

Case Fatality ◽

Ascertainment Bias ◽

Joint Estimation ◽

Ebola Virus Disease ◽

Strong Impact

The effective reproduction number [Formula: see text], the average number of secondary cases that are generated by a single primary case at calendar time [Formula: see text], plays a critical role in interpreting the temporal transmission dynamics of an infectious disease epidemic, while the case fatality risk (CFR) is an indispensable measure of the severity of disease. In many instances, [Formula: see text] is estimated using the reported number of cases (i.e., the incidence data), but such report often does not arrive on time, and moreover, the rate of diagnosis could change as a function of time, especially if we handle diseases that involve substantial number of asymptomatic and mild infections and large outbreaks that go beyond the local capacity of reporting. In addition, CFR is well known to be prone to ascertainment bias, often erroneously overestimated. In this paper, we propose a joint estimation method of [Formula: see text] and CFR of Ebola virus disease (EVD), analyzing the early epidemic data of EVD from March to October 2014 and addressing the ascertainment bias in real time. To assess the reliability of the proposed method, coverage probabilities were computed. When ascertainment effort plays a role in interpreting the epidemiological dynamics, it is useful to analyze not only reported (confirmed or suspected) cases, but also the temporal distribution of deceased individuals to avoid any strong impact of time dependent changes in diagnosis and reporting.

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Ebola Virus Disease – An Emergent Public Health Threat of th the Late 20 Century

NIGERIAN ANNALS OF PURE AND APPLIED SCIENCES ◽

10.46912/napas.1 ◽

2015 ◽

Vol 6 ◽

pp. 1-10

Author(s):

I NS Dozie ◽

B EB Nwoke ◽

A N Amadi ◽

U M Chukwuocha ◽

W U Dozie ◽

...

Keyword(s):

Ebola Virus ◽

Virus Disease ◽

Immune Therapy ◽

Ebola Virus Disease ◽

Personal Hygiene ◽

Enzyme Linked Immunosorbent Assay ◽

Severe Bleeding ◽

Electrolyte Balance ◽

Viral Glycoprotein ◽

In The Wild

Ebola virus disease is one of the new emerged infectious diseases of the late 20 century. It is a severe, often fatal illness in humans marked by severe bleeding (haemorrhage), organ failure and with fatality rates of between 50% and 90%. Ebola virus is native to Africa and is previously characterized by outbreaks in isolated and remote communities in the rainforest. The 2014 Ebola outbreak is reported in four West African countries namely, Guinea, Liberia, Sierra Leone, and Nigeria. Ebola virus disease (EVD) is caused by members of the genus Ebolavirus with five (5) recognized species namely Zaire Ebolavirus, Sudan Ebolavirus, Ivory Coast Ebolavirus, Reston Ebolavirus, and Bundibugyo Ebolavirus, all of which belongs to the family, Filoviridae. The transmission of Ebola virus involve two major steps; firstly from suspected natural hosts or reservoir believed to be fruit bats to animals in the wild and secondly, from animals in the wild to humans. Human-to-human transmission occurs through direct contact (through broken skin or mucous membranes) with the blood, secretions, organs or other bodily fluids (vomit, faeces, urine, breast milk, semen, and sweat) of infected persons. Although the clinical course of infection with an incubation period of between 2 to 21 days is well known, the specific mechanisms underlying the pathogenicity of Ebola virus have not been clearly understood. Several lines of evidence suggest that the viral glycoprotein (GP) plays a key role in the manifestation of Ebola virus infection. EVD can be diagnosed in the laboratory by reverse transcriptase polymerase chain reaction (RT-PCR) assay, antibody-capture enzyme-linked immunosorbent assay (ELISA), antigen detection tests, serum neutralization tests and virus isolation by cell culture. Currently, there are no approved drugs or vaccines to treat or prevent Ebola. Treatment consists of supportive therapy to maintain electrolyte balance. However experimental vaccines and antiviral drugs are undergoing development and clinical trials. The potential treatment of Ebola Haemorrhagic fever patients with passive immune therapy (i.e. blood transfusion) from convalescent patients is being explored. Prevention of EVD consists of avoiding close contact with gravely ill patients, improvement of personal hygiene especially hand hygiene, strict barrier nursing techniques including the use of personal protective equipment and safe burial of the dead.

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Statins Suppress Ebola Virus Infectivity by Interfering with Glycoprotein Processing

mBio ◽

10.1128/mbio.00660-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 19

Author(s):

Punya Shrivastava-Ranjan ◽

Mike Flint ◽

Éric Bergeron ◽

Anita K. McElroy ◽

Payel Chatterjee ◽

...

Keyword(s):

Ebola Virus ◽

Virus Disease ◽

Statin Treatment ◽

Ebola Virus Disease ◽

Health Concern ◽

Virus Infectivity ◽

Viral Glycoprotein ◽

Antiviral Effects ◽

Ebov Infection ◽

Glycoprotein Processing

ABSTRACTEbola virus (EBOV) infection is a major public health concern due to high fatality rates and limited effective treatments. Statins, widely used cholesterol-lowering drugs, have pleiotropic mechanisms of action and were suggested as potential adjunct therapy for Ebola virus disease (EVD) during the 2013–2016 outbreak in West Africa. Here, we evaluated the antiviral effects of statin (lovastatin) on EBOV infectionin vitro. Statin treatment decreased infectious EBOV production in primary human monocyte-derived macrophages and in the hepatic cell line Huh7. Statin treatment did not interfere with viral entry, but the viral particles released from treated cells showed reduced infectivity due to inhibition of viral glycoprotein processing, as evidenced by decreased ratios of the mature glycoprotein form to precursor form. Statin-induced inhibition of infectious virus production and glycoprotein processing was reversed by exogenous mevalonate, the rate-limiting product of the cholesterol biosynthesis pathway, but not by low-density lipoprotein. Finally, statin-treated cells produced EBOV particles devoid of the surface glycoproteins required for virus infectivity. Our findings demonstrate that statin treatment inhibits EBOV infection and suggest that the efficacy of statin treatment should be evaluated in appropriate animal models of EVD.IMPORTANCETreatments targeting Ebola virus disease (EVD) are experimental, expensive, and scarce. Statins are inexpensive generic drugs that have been used for many years for the treatment of hypercholesterolemia and have a favorable safety profile. Here, we show the antiviral effects of statins on infectious Ebola virus (EBOV) production. Our study reveals a novel molecular mechanism in which statin regulates EBOV particle infectivity by preventing glycoprotein processing and incorporation into virus particles. Additionally, statins have anti-inflammatory and immunomodulatory effects. Since inflammation and dysregulation of the immune system are characteristic features of EVD, statins could be explored as part of EVD therapeutics.

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Conformational changes in the Ebola virus membrane fusion machine induced by pH, Ca2+, and receptor binding

PLoS Biology ◽

10.1371/journal.pbio.3000626 ◽

2020 ◽

Vol 18 (2) ◽

pp. e3000626 ◽

Cited By ~ 12

Author(s):

Dibyendu Kumar Das ◽

Uriel Bulow ◽

William E. Diehl ◽

Natasha D. Durham ◽

Fernando Senjobe ◽

...

Keyword(s):

Membrane Fusion ◽

Receptor Binding ◽

Conformational Changes ◽

Ebola Virus

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Evolving Strategy and Incident Management Systems in Hard to Reach Areas and Fragile Security Settings: The Case of Ebola Response in the Democratic Republic of Congo

Prehospital and Disaster Medicine ◽

10.1017/s1049023x19000505 ◽

2019 ◽

Vol 34 (s1) ◽

pp. s16-s16

Author(s):

Nda Konan Michel Yao ◽

Tambwe Bathe Ndjoloko

Keyword(s):

Ebola Virus ◽

Democratic Republic Of Congo ◽

Virus Disease ◽

Critical Role ◽

Response Strategy ◽

Incident Management ◽

Effective Response ◽

Case Study Methodology ◽

Geographical Access ◽

Flexible Response

Introduction:DRC Ministry of Health declared the 9th outbreak of Ebola Virus Disease (EVD) in the Equator province on May 8, 2018, that ended on July 25, 2018. There were 54 cases with 38 confirmed, 33 deaths (61%), and 21 survivors in three “zones de santé” (districts). On August 1, 2018, the 10th EVD outbreak of the country was declared in the Ituri and North Kivu provinces. This one is the most important outbreak ever experienced. By November 18, 2018, 373 cases were reported with 326 confirmed and 214 deaths (58%) in two provinces including 14 “zones de santé.” While the 9th outbreak occurred in hard-to-reach areas, the 10th is occurring in fragile security settings, requiring specific strategic/operational approaches.Aim:To describe strategic and operational approaches including IMSs used to address these deadly outbreaks.Methods:A case study methodology using response strategy documents and observations was used, coupled with the use of operation review exercises.Results:The response strategy evolved continuously taking into account the epidemiological context, including geographical spread. It also took into account cultural, political, and sociological (community resistances) sensitivities. Conditions of pre-existing health system and services were considered. The prevailing security context (armed groups) was taken into account. The evolving situation impacted implementation of response areas including critical interventions like setting up confirmation and treatment centres, rapid response teams, and IMS structures. Areas of response were reviewed continuously, including response structures with further decentralization, outreach, or locally delegated interventions to ensure geographical access and continuity in response services.Discussion:Response areas to deal with EVD outbreaks are well known. However, an effective response requires a continuous adjustment of the strategy and a flexible response structures with related IMSs based on regular deep situation analysis. Social sciences still have a critical role to play for that purpose.

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A Conserved Region in the F2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation

Journal of Virology ◽

10.1128/jvi.00366-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 8303-8314 ◽

Cited By ~ 29

Author(s):

Amanda E. Gardner ◽

Rebecca E. Dutch

Keyword(s):

Membrane Fusion ◽

Cell Fusion ◽

Critical Role ◽

Simian Virus ◽

Hendra Virus ◽

Heptad Repeat ◽

F Protein ◽

Conserved Region ◽

Heptad Repeats ◽

Cell Cell

ABSTRACT Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F1 and F2. Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F1 (CBF1) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F2 subunit (CBF2). To analyze the functions of CBF2, alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF2 mutations resulted in folding and expression defects. However, the CBF2 mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF2 Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF2 I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF2 in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion.

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Conformational Dynamics Related to Membrane Fusion Observed in Single Ebola GP Molecules

Proceedings ◽

10.3390/proceedings2020050049 ◽

2020 ◽

Vol 50 (1) ◽

pp. 49

Author(s):

Dibyendu Kumar Das ◽

Uriel Bulow ◽

Natasha D. Durham ◽

Ramesh Govindan ◽

James B. Munro

Keyword(s):

Membrane Fusion ◽

Single Molecule ◽

Conformational Changes ◽

Ebola Virus ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Acidic Ph ◽

Cellular Environment ◽

Niemann Pick

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following the endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. The NPC1 binding to the cleaved GP1 is required for entry, but how this interaction translates to the GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a virus-liposome hemifusion assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding act synergistically to induce conformational changes in GP2 that drive lipid mixing. Acidic pH and Ca2+ shift the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. GP1 cleavage and NPC1 binding enable GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the post-fusion 6-helix bundle. Thus, the GP senses the cellular environment to protect against triggering prior to the arrival of EBOV in a permissive cellular compartment.

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Spring-Loaded Heptad Repeat Residues Regulate the Expression and Activation of Paramyxovirus Fusion Protein

Journal of Virology ◽

10.1128/jvi.02464-06 ◽

2007 ◽

Vol 81 (7) ◽

pp. 3130-3141 ◽

Cited By ~ 25

Author(s):

Laura E. Luque ◽

Charles J. Russell

Keyword(s):

Protein Expression ◽

Membrane Fusion ◽

Conformational Changes ◽

Structural Changes ◽

Tertiary Structure ◽

Sendai Virus ◽

Coiled Coil ◽

Viral Envelope ◽

Heptad Repeat ◽

F Protein

ABSTRACT During viral entry, the paramyxovirus fusion (F) protein fuses the viral envelope to a cellular membrane. Similar to other class I viral fusion glycoproteins, the F protein has two heptad repeat regions (HRA and HRB) that are important in membrane fusion and can be targeted by antiviral inhibitors. Upon activation of the F protein, HRA refolds from a spring-loaded, crumpled structure into a coiled coil that inserts a hydrophobic fusion peptide into the target membrane and binds to the HRB helices to form a fusogenic hairpin. To investigate how F protein conformational changes are regulated, we mutated in the Sendai virus F protein a highly conserved 10-residue sequence in HRA that undergoes major structural changes during protein refolding. Nine of the 15 mutations studied caused significant defects in F protein expression, processing, and fusogenicity. Conversely, the remaining six mutations enhanced the fusogenicity of the F protein, most likely by helping spring the HRA coil. Two of the residues that were neither located at “a” or “d” positions in the heptad repeat nor conserved among the paramyxoviruses were key regulators of the folding and fusion activity of the F protein, showing that residues not expected to be important in coiled-coil formation may play important roles in regulating membrane fusion. Overall, the data support the hypothesis that regions in the F protein that undergo dramatic changes in secondary and tertiary structure between the prefusion and hairpin conformations regulate F protein expression and activation.

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Diagnosis of Ebola Virus Disease: Past, Present, and Future

Clinical Microbiology Reviews ◽

10.1128/cmr.00003-16 ◽

2016 ◽

Vol 29 (4) ◽

pp. 773-793 ◽

Cited By ~ 86

Author(s):

M. Jana Broadhurst ◽

Tim J. G. Brooks ◽

Nira R. Pollock

Keyword(s):

West Africa ◽

Ebola Virus ◽

Virus Disease ◽

Critical Role ◽

Diagnostic Testing ◽

Laboratory Diagnosis ◽

Ebola Virus Disease ◽

Outbreak Response ◽

Culture Techniques ◽

Viral Culture

SUMMARYLaboratory diagnosis of Ebola virus disease plays a critical role in outbreak response efforts; however, establishing safe and expeditious testing strategies for this high-biosafety-level pathogen in resource-poor environments remains extremely challenging. Since the discovery of Ebola virus in 1976 via traditional viral culture techniques and electron microscopy, diagnostic methodologies have trended toward faster, more accurate molecular assays. Importantly, technological advances have been paired with increasing efforts to support decentralized diagnostic testing capacity that can be deployed at or near the point of patient care. The unprecedented scope of the 2014-2015 West Africa Ebola epidemic spurred tremendous innovation in this arena, and a variety of new diagnostic platforms that have the potential both to immediately improve ongoing surveillance efforts in West Africa and to transform future outbreak responses have reached the field. In this review, we describe the evolution of Ebola virus disease diagnostic testing and efforts to deploy field diagnostic laboratories in prior outbreaks. We then explore the diagnostic challenges pervading the 2014-2015 epidemic and provide a comprehensive examination of novel diagnostic tests that are likely to address some of these challenges moving forward.

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