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1.
PLoS Biol ; 18(2): e3000626, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32040508

RESUMO

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, removing the glycan cap and exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. NPC1 binding to cleaved GP1 is required for entry. How this interaction translates to GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a bulk fluorescence dequenching assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding synergistically induce conformational changes in GP2 and permit virus-liposome lipid mixing. Acidic pH and Ca2+ shifted the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. Glycan cap cleavage on GP1 enabled GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the postfusion 6-helix bundle; NPC1 binding further promoted transition to the irreversible conformation. Thus, the glycan cap of GP1 may allosterically protect against inactivation of EBOV by premature triggering of GP2.


Assuntos
Ebolavirus/fisiologia , Fusão de Membrana , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Regulação Alostérica , Cálcio/metabolismo , Ebolavirus/química , Ebolavirus/genética , Ebolavirus/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas do Envelope Viral/genética , Internalização do Vírus
2.
PLoS Negl Trop Dis ; 14(1): e0007952, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961874

RESUMO

The significance of the integral membrane protein Niemann-Pick C1 (NPC1) in the ebolavirus entry process has been determined using various cell lines derived from humans, non-human primates and fruit bats. Fruit bats have long been purported as the potential reservoir host for ebolaviruses, however several studies provide evidence that Mops condylurus, an insectivorous microbat, is also an ebolavirus reservoir. NPC1 receptor expression in the context of ebolavirus replication in microbat cells remains unstudied. In order to study Ebola virus (EBOV) cellular entry and replication in M. condylurus, we derived primary and immortalized cell cultures from 12 different organs. The NPC1 receptor expression was characterized by confocal microscopy and flow cytometry comparing the expression levels of M. condylurus primary and immortalized cells, HeLa cells, human embryonic kidney cells and cells from a European microbat species. EBOV replication kinetics was studied for four representative cell cultures using qRT-PCR. The aim was to elucidate the suitability of primary and immortalized cells from different tissues for studying NPC1 receptor expression levels and their potential influence on EBOV replication. The NPC1 receptor expression level in M. condylurus primary cells differed depending on the organ they were derived from and was for most cell types significantly lower than in human cell lines. Immortalized cells showed for most cell types higher expression levels than their corresponding primary cells. Concluding from our infection experiments with EBOV we suggest a potential correlation between NPC1 receptor expression level and virus replication rate in vitro.


Assuntos
Quirópteros/genética , Reservatórios de Doenças/virologia , Ebolavirus/fisiologia , Proteína C1 de Niemann-Pick/genética , Proteína C1 de Niemann-Pick/metabolismo , Receptores Virais/genética , Animais , Quirópteros/metabolismo , Quirópteros/virologia , Humanos , Receptores Virais/metabolismo , Internalização do Vírus
3.
PLoS Negl Trop Dis ; 13(12): e0007819, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31825972

RESUMO

BACKGROUND: Ebolavirus (EBOV) outbreaks, while sporadic, cause tremendous morbidity and mortality. No therapeutics or vaccines are currently licensed; however, a vaccine has shown promise in clinical trials. A critical step towards development of effective therapeutics is a better understanding of factors that govern host susceptibility to this pathogen. As macrophages are an important cell population targeted during virus replication, we explore the effect of cytokine polarization on macrophage infection. METHODS/MAIN FINDINGS: We utilized a BSL2 EBOV model virus, infectious, recombinant vesicular stomatitis virus encoding EBOV glycoprotein (GP) (rVSV/EBOV GP) in place of its native glycoprotein. Macrophages polarized towards a M2-like anti-inflammatory state by combined IL-4 and IL-13 treatment were more susceptible to rVSV/EBOV GP, but not to wild-type VSV (rVSV/G), suggesting that EBOV GP-dependent entry events were enhanced by these cytokines. Examination of RNA expression of known surface receptors that bind and internalize filoviruses demonstrated that IL-4/IL-13 stimulated expression of the C-type lectin receptor DC-SIGN in human macrophages and addition of the competitive inhibitor mannan abrogated IL-4/IL-13 enhanced infection. Two murine DC-SIGN-like family members, SIGNR3 and SIGNR5, were upregulated by IL-4/IL-13 in murine macrophages, but only SIGNR3 enhanced virus infection in a mannan-inhibited manner, suggesting that murine SIGNR3 plays a similar role to human DC-SIGN. In vivo IL-4/IL-13 administration significantly increased virus-mediated mortality in a mouse model and transfer of ex vivo IL-4/IL-13-treated murine peritoneal macrophages into the peritoneal cavity of mice enhanced pathogenesis. SIGNIFICANCE: These studies highlight the ability of macrophage polarization to influence EBOV GP-dependent virus replication in vivo and ex vivo, with M2a polarization upregulating cell surface receptor expression and thereby enhancing virus replication. Our findings provide an increased understanding of the host factors in macrophages governing susceptibility to filoviruses and identify novel murine receptors mediating EBOV entry.


Assuntos
Ebolavirus/fisiologia , Interações Hospedeiro-Patógeno , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , Internalização do Vírus , Animais , Modelos Animais de Doenças , Feminino , Doença pelo Vírus Ebola/patologia , Doença pelo Vírus Ebola/virologia , Masculino , Camundongos Endogâmicos C57BL
4.
PLoS Negl Trop Dis ; 13(11): e0007890, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31751347

RESUMO

Recent outbreaks of the Ebola virus (EBOV) have focused attention on the dire need for antivirals to treat these patients. We identified pyronaridine tetraphosphate as a potential candidate as it is an approved drug in the European Union which is currently used in combination with artesunate as a treatment for malaria (EC50 between 420 nM-1.14 µM against EBOV in HeLa cells). Range-finding studies in mice directed us to a single 75 mg/kg i.p. dose 1 hr after infection which resulted in 100% survival and statistically significantly reduced viremia at study day 3 from a lethal challenge with mouse-adapted EBOV (maEBOV). Further, an EBOV window study suggested we could dose pyronaridine 2 or 24 hrs post-exposure to result in similar efficacy. Analysis of cytokine and chemokine panels suggests that pyronaridine may act as an immunomodulator during an EBOV infection. Our studies with pyronaridine clearly demonstrate potential utility for its repurposing as an antiviral against EBOV and merits further study in larger animal models with the added benefit of already being used as a treatment against malaria.


Assuntos
Antimaláricos/administração & dosagem , Antivirais/administração & dosagem , Doença pelo Vírus Ebola/prevenção & controle , Naftiridinas/administração & dosagem , Animais , Antimaláricos/efeitos adversos , Antimaláricos/farmacocinética , Antivirais/efeitos adversos , Antivirais/farmacocinética , Citocinas/imunologia , Reposicionamento de Medicamentos , Ebolavirus/efeitos dos fármacos , Ebolavirus/genética , Ebolavirus/fisiologia , Feminino , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Naftiridinas/efeitos adversos , Naftiridinas/farmacocinética
5.
BMC Infect Dis ; 19(1): 981, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31752717

RESUMO

BACKGROUND: The ninth outbreak of Ebola Virus Disease (EVD) in the Democratic Republic of the Congo occurred in Équateur Province from 8 May-24 July 2018. A system of health facility (HF)-based active case finding (ACF) was implemented in Mbandaka, a regional capital with four confirmed EVD cases, following completion of contact tracing. The goal of this HF-based ACF system was to look for undetected EVD cases among patients that visited HFs beginning one week prior to the system's implementation. METHODS: From 23 June - 24 July 2018, ACF teams visited HFs in Mbandaka and reviewed all medical records as far back as 17 June for any consultations meeting the suspected EVD case definition. The teams then assessed whether to validate these as suspected EVD cases based on factors such as recovery, epidemiological links, and their clinical judgement. ACF teams also assessed HFs' awareness of EVD symptoms and the process for alerting suspected cases. We calculated descriptive statistics regarding the characteristics of reviewed consultations, alert cases, and visited HFs. We also used univariate and multivariate random effects logistic regression models to evaluate the impact of repeated ACF visits to the same HF on the staff's awareness of EVD. RESULTS: ACF teams reviewed 37,746 consultations, of which 690 met the definition of a suspected case of EVD. Two were validated as suspected EVD cases and transferred to the Ebola Treatment Unit for testing; both tested negative. Repeated ACF visits to the same HF were significantly associated with improved EVD awareness (p < 0.001) in univariate and multivariate analyses. CONCLUSION: HF-based ACF during EVD outbreaks may improve EVD awareness and reveal many individuals meeting the suspected case definition. However, many who meet this definition may not have EVD, depending on the population size covered by ACF and amount of ongoing EVD transmission. Given the burdensome procedure of testing suspected EVD cases, future HF-based ACF systems would benefit from improved clarity on which patients require further testing.


Assuntos
Notificação de Doenças/métodos , Doença pelo Vírus Ebola/virologia , República Democrática do Congo/epidemiologia , Surtos de Doenças , Ebolavirus/genética , Ebolavirus/isolamento & purificação , Ebolavirus/fisiologia , Feminino , Instalações de Saúde/estatística & dados numéricos , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/epidemiologia , Humanos , Modelos Logísticos , Masculino
6.
Rev Sci Tech ; 38(1): 113-122, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31564736

RESUMO

The natural ecology of Ebola virus infection remains enigmatic. No clear reservoir species has been confirmed but there is evidence of infection in a wide spectrum of mammals, including humans, non-human primates, domestic and wild ungulates and a variety of bat species, both frugivorous and insectivorous. Humans and most other species examined appear to be spillover hosts and suffer disease. Bats are the exception and are tolerant to infection in some laboratory studies. Some surveys show a low prevalence of antibodies against Zaire Ebola virus (ZEBOV) strains in bats during human outbreaks and inter-epidemic periods, and this order of mammals is considered to be the likely reservoir for the virus. Other putative sources include insects but this hypothesis is unproven in the field or laboratory. Moreover, some potential sources, such as aquatic species, have yet to be investigated. There are a number of environmental, human behavioural and ecological risk factors proposed with respect to spillover and spread. In the West African outbreak, which was unprecedented in scale and geographic spread, the source of the spillover remains unproven, although an association exists between the proposed index case and a colony of insectivorous bats. In all but a few Ebola virus disease events, spillover has only been superficially investigated and this was also the case in the West African epidemic. The authors suggest that, to address risks at the human-animal-environmental interface, using a One Health approach, more effort is needed to investigate spillover factors at the time of a ZEBOV epidemic, in addition to conducting inter-epidemic surveys in peridomestic environments. The true prevalence of ZEBOV infection in any species of bats remains unknown. Large-scale, expensive, non-randomised surveys, with low sampling numbers per species, are unlikely to provide evidence for Ebola virus reservoirs or to improve our epidemiological understanding.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , África Ocidental/epidemiologia , Animais , Quirópteros/virologia , Surtos de Doenças/prevenção & controle , Reservatórios de Doenças , Ebolavirus/fisiologia , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/virologia , Humanos
7.
Nat Commun ; 10(1): 4531, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615986

RESUMO

Recent outbreaks of animal-borne emerging infectious diseases have likely been precipitated by a complex interplay of changing ecological, epidemiological and socio-economic factors. Here, we develop modelling methods that capture elements of each of these factors, to predict the risk of Ebola virus disease (EVD) across time and space. Our modelling results match previously-observed outbreak patterns with high accuracy, and suggest further outbreaks could occur across most of West and Central Africa. Trends in the underlying drivers of EVD risk suggest a 1.75 to 3.2-fold increase in the endemic rate of animal-human viral spill-overs in Africa by 2070, given current modes of healthcare intervention. Future global change scenarios with higher human population growth and lower rates of socio-economic development yield a fourfold higher likelihood of epidemics occurring as a result of spill-over events. Our modelling framework can be used to target interventions designed to reduce epidemic risk for many zoonotic diseases.


Assuntos
Doenças Transmissíveis Emergentes/virologia , Ebolavirus/fisiologia , Meio Ambiente , Doença pelo Vírus Ebola/virologia , Fatores Socioeconômicos , Zoonoses/virologia , África/epidemiologia , Animais , Doenças Transmissíveis Emergentes/epidemiologia , Surtos de Doenças/prevenção & controle , Epidemias/prevenção & controle , Doença pelo Vírus Ebola/epidemiologia , Humanos , Fatores de Risco , Zoonoses/epidemiologia
8.
Emerg Microbes Infect ; 8(1): 1511-1523, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31631785

RESUMO

Interferons (IFNs) control viral infections by inducing expression of IFN-stimulated genes (ISGs) that restrict distinct steps of viral replication. We report herein that gamma-interferon-inducible lysosomal thiol reductase (GILT), a lysosome-associated ISG, restricts the infectious entry of selected enveloped RNA viruses. Specifically, we demonstrated that GILT was constitutively expressed in lung epithelial cells and fibroblasts and its expression could be further induced by type II interferon. While overexpression of GILT inhibited the entry mediated by envelope glycoproteins of SARS coronavirus (SARS-CoV), Ebola virus (EBOV) and Lassa fever virus (LASV), depletion of GILT enhanced the entry mediated by these viral envelope glycoproteins. Furthermore, mutations that impaired the thiol reductase activity or disrupted the N-linked glycosylation, a posttranslational modification essential for its lysosomal localization, largely compromised GILT restriction of viral entry. We also found that the induction of GILT expression reduced the level and activity of cathepsin L, which is required for the entry of these RNA viruses in lysosomes. Our data indicate that GILT is a novel antiviral ISG that specifically inhibits the entry of selected enveloped RNA viruses in lysosomes via disruption of cathepsin L metabolism and function and may play a role in immune control and pathogenesis of these viruses.


Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/imunologia , Febre Lassa/imunologia , Vírus Lassa/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/imunologia , Vírus da SARS/fisiologia , Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Catepsina L/genética , Catepsina L/imunologia , Linhagem Celular , Ebolavirus/genética , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/virologia , Humanos , Febre Lassa/genética , Febre Lassa/virologia , Vírus Lassa/genética , Lisossomos/genética , Lisossomos/imunologia , Lisossomos/virologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Vírus da SARS/genética , Síndrome Respiratória Aguda Grave/genética , Síndrome Respiratória Aguda Grave/virologia , Proteínas do Envelope Viral/genética , Replicação Viral
9.
Emerg Microbes Infect ; 8(1): 1347-1360, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31516086

RESUMO

The polymerase complex of Ebola virus (EBOV) is the functional unit for transcription and replication of viral genome. Nucleoprotein (NP) is a multifunctional protein with high RNA binding affinity and recruits other viral proteins to form functional polymerase complex. In our study, we investigated host proteins associated with EBOV polymerase complex using NP as bait in a transcription and replication competent minigenome system by mass spectrometry analysis and identified SET and MYND domain-containing protein 3 (SMYD3) as a novel host protein which was required for the replication of EBOV. SMYD3 specifically interacted with NP and was recruited to EBOV inclusion bodies through NP. The depletion of SMYD3 dramatically suppressed EBOV mRNA production. A mimic of non-phosphorylated VP30, which is a transcription activator, could partially rescue the viral mRNA production downregulated by the depletion of SMYD3. In addition, SMYD3 promoted NP-VP30 interaction in a dose-dependent manner. These results revealed that SMYD3 was a novel host factor recruited by NP to supporting EBOV mRNA transcription through increasing the binding of VP30 to NP. Thus, our study provided a new understanding of mechanism underlying the transcription of EBOV genome, and a novel anti-EBOV drug design strategy by targeting SMYD3.


Assuntos
Ebolavirus/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Interações Hospedeiro-Patógeno , Proteínas do Nucleocapsídeo/metabolismo , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Transcrição Genética , Células HEK293 , Humanos , Espectrometria de Massas , Ligação Proteica , Mapeamento de Interação de Proteínas
10.
Elife ; 82019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31411558

RESUMO

Beneficial and deleterious mutations cause the fitness of lineages to vary across a phylogeny and thereby shape its branching structure. While standard phylogenetic models do not allow mutations to feedback and shape trees, birth-death models can account for this feedback by letting the fitness of lineages depend on their type. To date, these multi-type birth-death models have only been applied to cases where a lineage's fitness is determined by a single character state. We extend these models to track sequence evolution at multiple sites. This approach remains computationally tractable by tracking the genotype and fitness of lineages probabilistically in an approximate manner. Although approximate, we show that we can accurately estimate the fitness of lineages and site-specific mutational fitness effects from phylogenies. We apply this approach to estimate the population-level fitness effects of mutations in Ebola and influenza virus, and compare our estimates with in vitro fitness measurements for these mutations.


Assuntos
Adaptação Biológica , Ebolavirus/genética , Evolução Molecular , Aptidão Genética , Modelos Genéticos , Orthomyxoviridae/genética , Ebolavirus/fisiologia , Genótipo , Orthomyxoviridae/fisiologia
11.
Nat Microbiol ; 4(9): 1558-1570, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31160823

RESUMO

Several Ebola viruses cause outbreaks of lethal haemorrhagic fever in humans, but developing therapies tackle only Zaire Ebola virus. Dendritic cells (DCs) are targets of this infection in vivo. Here, we found that Ebola virus entry into activated DCs requires the sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169), which recognizes sialylated gangliosides anchored to viral membranes. Blockage of the Siglec-1 receptor by anti-Siglec-1 monoclonal antibodies halted Ebola viral uptake and cytoplasmic entry, offering cross-protection against other ganglioside-containing viruses such as human immunodeficiency virus type 1.


Assuntos
Anticorpos Monoclonais/farmacologia , Citoplasma/virologia , Ebolavirus/fisiologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Gangliosídeos/metabolismo , HIV-1/fisiologia , Doença pelo Vírus Ebola/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Lipopolissacarídeos/farmacologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Vírion/metabolismo
12.
PLoS Negl Trop Dis ; 13(6): e0006983, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31242184

RESUMO

BACKGROUND: T cell immunoglobulin mucin domain-1 (TIM-1) is a phosphatidylserine (PS) receptor, mediating filovirus entry into cells through interactions with PS on virions. TIM-1 expression has been implicated in Ebola virus (EBOV) pathogenesis; however, it remains unclear whether this is due to TIM-1 serving as a filovirus receptor in vivo or, as others have suggested, TIM-1 induces a cytokine storm elicited by T cell/virion interactions. Here, we use a BSL2 model virus that expresses EBOV glycoprotein to demonstrate the importance of TIM-1 as a virus receptor late during in vivo infection. METHODOLOGY/PRINCIPAL FINDINGS: Infectious, GFP-expressing recombinant vesicular stomatitis virus encoding either full length EBOV glycoprotein (EBOV GP/rVSV) or mucin domain deleted EBOV glycoprotein (EBOV GPΔO/rVSV) was used to assess the role of TIM-1 during in vivo infection. GFP-expressing rVSV encoding its native glycoprotein G (G/rVSV) served as a control. TIM-1-sufficient or TIM-1-deficient BALB/c interferon α/ß receptor-/- mice were challenged with these viruses. While G/rVSV caused profound morbidity and mortality in both mouse strains, TIM-1-deficient mice had significantly better survival than TIM-1-expressing mice following EBOV GP/rVSV or EBOV GPΔO/rVSV challenge. EBOV GP/rVSV or EBOV GPΔO/rVSV in spleen of infected animals was high and unaffected by expression of TIM-1. However, infectious virus in serum, liver, kidney and adrenal gland was reduced late in infection in the TIM-1-deficient mice, suggesting that virus entry via this receptor contributes to virus load. Consistent with higher virus loads, proinflammatory chemokines trended higher in organs from infected TIM-1-sufficient mice compared to the TIM-1-deficient mice, but proinflammatory cytokines were more modestly affected. To assess the role of T cells in EBOV GP/rVSV pathogenesis, T cells were depleted in TIM-1-sufficient and -deficient mice and the mice were challenged with virus. Depletion of T cells did not alter the pathogenic consequences of virus infection. CONCLUSIONS: Our studies provide evidence that at late times during EBOV GP/rVSV infection, TIM-1 increased virus load and associated mortality, consistent with an important role of this receptor in virus entry. This work suggests that inhibitors which block TIM-1/virus interaction may serve as effective antivirals, reducing virus load at late times during EBOV infection.


Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/virologia , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Receptores Virais/metabolismo , Internalização do Vírus , Animais , Ebolavirus/genética , Feminino , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Receptor Celular 1 do Vírus da Hepatite A/deficiência , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores Virais/deficiência , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Genética Reversa , Vesiculovirus/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
13.
Viruses ; 11(4)2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018511

RESUMO

Ebola virus (EBOV) is a filovirus that has become a global public health threat in recent years. EBOV is the causative agent of a severe, often fatal hemorrhagic fever. A productive viral infection relies on the successful recruitment of host factors for various stages of the viral life cycle. To date, several investigations have discovered specific host-pathogen interactions for various EBOV proteins. However, relatively little is known about the EBOV nucleoprotein (NP) with regard to host interactions. In the present study, we aimed to elucidate NP-host protein-protein interactions (PPIs). Affinity purification-mass spectrometry (AP-MS) was used to identify candidate NP cellular interactors. Candidate interactors RUVBL1 and RUVBL2, partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) superfamily, were confirmed to interact with NP in co-immunoprecipitation (co-IP) and immunofluorescence (IF) experiments. Functional studies using a minigenome system revealed that the siRNA-mediated knockdown of RUVBL1 but not RUVBL2 moderately decreased EBOV minigenome activity. Super resolution structured illumination microscopy (SIM) was used to identify an association between NP and components of the R2TP complex, which includes RUVBL1, RUVBL2, RPAP3, and PIH1D1, suggesting a potential role for the R2TP complex in capsid formation. Moreover, the siRNA-mediated knockdown of RPAP3 and subsequent downregulation of PIH1D1 was shown to have no effect on minigenome activity, further suggesting a role in capsid formation. Overall, we identify RUVBL1 and RUVBL2 as novel interactors of EBOV NP and for the first time report EBOV NP recruitment of the R2TP complex, which may provide novel targets for broad-acting anti-EBOV therapeutics.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Ebolavirus/fisiologia , Interações Hospedeiro-Patógeno , Nucleoproteínas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , DNA Helicases/genética , Ebolavirus/genética , Técnicas de Silenciamento de Genes , Genoma Viral , Humanos , Nucleoproteínas/genética , Ligação Proteica , RNA Interferente Pequeno
14.
Viruses ; 11(3)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30832223

RESUMO

Filoviruses, such as Ebola virus (EBOV) and Marburg virus, are causative agents of unpredictable outbreaks of severe hemorrhagic fevers in humans and non-human primates. For infection, filoviral particles need to be internalized and delivered to intracellular vesicles containing cathepsin proteases and the viral receptor Niemann-Pick C1. Previous studies have shown that EBOV triggers macropinocytosis of the viral particles in a glycoprotein (GP)-dependent manner, but the molecular events required for filovirus internalization remain mostly unknown. Here we report that the diacylglycerol kinase inhibitor, R-59-022, blocks EBOV GP-mediated entry into Vero cells and bone marrow-derived macrophages. Investigation of the mode of action of the inhibitor revealed that it blocked an early step in entry, more specifically, the internalization of the viral particles via macropinocytosis. Finally, R-59-022 blocked viral entry mediated by a panel of pathogenic filovirus GPs and inhibited growth of replicative Ebola virus. Taken together, our studies suggest that R-59-022 could be used as a tool to investigate macropinocytic uptake of filoviruses and could be a starting point for the development of pan-filoviral therapeutics.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Filoviridae/efeitos dos fármacos , Filoviridae/fisiologia , Pirimidinonas/farmacologia , Tiazóis/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Ebolavirus/fisiologia , Células HEK293 , Humanos , Macrófagos/virologia , Marburgvirus/fisiologia , Pinocitose/efeitos dos fármacos , Receptores Virais , Células Vero , Replicação Viral/efeitos dos fármacos
15.
Viruses ; 11(3)2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30893774

RESUMO

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the Filoviridae family. The recent large outbreak of EVD in Western Africa (2013⁻2016) highlighted the worldwide threat represented by the disease and its impact on global public health and the economy. The development of highly needed anti-Ebola virus antivirals has been so far hampered by the shortage of tools to study their life cycle in vitro, allowing to screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to study Ebola virus entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus-like particles, tremendously boosted both our knowledge of the viral life cycle and the identification of promising antiviral compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD.


Assuntos
Descoberta de Drogas , Reposicionamento de Medicamentos , Ebolavirus/efeitos dos fármacos , Ebolavirus/fisiologia , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/farmacologia , Surtos de Doenças/prevenção & controle , Desenho de Fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Humanos , Camundongos , Vacinas de Partículas Semelhantes a Vírus
16.
Viruses ; 11(3)2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30893855

RESUMO

Filoviruses infect a wide range of cell types with the exception of lymphocytes. The intracellular proteins cathepsin B and L, two-pore channel 1 and 2, and bona fide receptor Niemann⁻Pick Disease C1 (NPC1) are essential for the endosomal phase of cell entry. However, earlier steps of filoviral infection remain poorly characterized. Numerous plasma membrane proteins have been implicated in attachment but it is still unclear which ones are sufficient for productive entry. To define a minimal set of host factors required for filoviral glycoprotein-driven cell entry, we screened twelve cell lines and identified the nonlymphocytic cell line SH-SY5Y to be specifically resistant to filovirus infection. Heterokaryons of SH-SY5Y cells fused to susceptible cells were susceptible to filoviruses, indicating that SH-SY5Y cells do not express a restriction factor but lack an enabling factor critical for filovirus entry. However, all tested cell lines expressed functional intracellular factors. Global gene expression profiling of known cell surface entry factors and protein expression levels of analyzed attachment factors did not reveal any correlation between susceptibility and expression of a specific host factor. Using binding assays with recombinant filovirus glycoprotein, we identified cell attachment as the step impaired in filovirus entry in SH-SY5Y cells. Individual overexpression of attachment factors T-cell immunoglobulin and mucin domain 1 (TIM-1), Axl, Mer, or dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) rendered SH-SY5Y cells susceptible to filovirus glycoprotein-driven transduction. Our study reveals that a lack of attachment factors limits filovirus entry and provides direct experimental support for a model of filoviral cell attachment where host factor usage at the cell surface is highly promiscuous.


Assuntos
Membrana Celular/virologia , Filoviridae/fisiologia , Interações Hospedeiro-Patógeno , Receptores Virais/genética , Proteínas Virais/genética , Internalização do Vírus , Células A549 , Proteínas de Transporte/genética , Linhagem Celular , Ebolavirus/genética , Ebolavirus/fisiologia , Filoviridae/genética , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética
17.
Nat Commun ; 10(1): 105, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631063

RESUMO

The 2013-2016 Ebola virus (EBOV) disease epidemic demonstrated the grave consequences of filovirus epidemics in the absence of effective therapeutics. Besides EBOV, two additional ebolaviruses, Sudan (SUDV) and Bundibugyo (BDBV) viruses, as well as multiple variants of Marburg virus (MARV), have also caused high fatality epidemics. Current experimental EBOV monoclonal antibodies (mAbs) are ineffective against SUDV, BDBV, or MARV. Here, we report that a cocktail of two broadly neutralizing ebolavirus mAbs, FVM04 and CA45, protects nonhuman primates (NHPs) against EBOV and SUDV infection when delivered four days post infection. This cocktail when supplemented by the anti-MARV mAb MR191 exhibited 100% efficacy in MARV-infected NHPs. These findings provide a solid foundation for clinical development of broadly protective immunotherapeutics for use in future filovirus epidemics.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Ebolavirus/imunologia , Infecções por Filoviridae/imunologia , Marburgvirus/imunologia , Doenças dos Primatas/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Ebolavirus/classificação , Ebolavirus/efeitos dos fármacos , Ebolavirus/fisiologia , Infecções por Filoviridae/terapia , Infecções por Filoviridae/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Imunoterapia/métodos , Marburgvirus/efeitos dos fármacos , Marburgvirus/fisiologia , Doenças dos Primatas/terapia , Doenças dos Primatas/virologia , Primatas , Resultado do Tratamento
18.
Viruses ; 11(1)2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30609802

RESUMO

The filoviruses Ebolavirus and Marburgvirus are among the deadliest viral pathogens known to infect humans, causing emerging diseases with fatality rates of up to 90% during some outbreaks. The replication cycles of these viruses are comprised of numerous complex molecular processes and interactions with their human host, with one key feature being the means by which nascent virions exit host cells to spread to new cells and ultimately to a new host. This review focuses on our current knowledge of filovirus egress and the viral and host factors and processes that are involved. Within the virus, these factors consist of the major matrix protein, viral protein 40 (VP40), which is necessary and sufficient for viral particle release, and nucleocapsid and glycoprotein that interact with VP40 to promote egress. In the host cell, some proteins are hijacked by filoviruses in order to enhance virion budding capacity that include members of the family of E3 ubiquitin ligase and the endosomal sorting complexes required for transport (ESCRT) pathway, while others such as tetherin inhibit viral egress. An understanding of these molecular interactions that modulate viral particle egress provides an important opportunity to identify new targets for the development of antivirals to prevent and treat filovirus infections.


Assuntos
Ebolavirus/fisiologia , Interações entre Hospedeiro e Microrganismos , Marburgvirus/fisiologia , Liberação de Vírus , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Camundongos , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
19.
Zoonoses Public Health ; 66(3): 288-295, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30677236

RESUMO

The ecology of Ebola virus (EBV) remains largely unknown, but the previous detection of viral RNA and anti-EBV antibodies in African bats suggests that they might play a role in the EBV reservoir. Moreover, African bats also carry other potentially zoonotic agents such as Henipah-like viruses, coronaviruses and lyssaviruses. Today only little information is available on interactions between humans and bats. The objective of our exploratory study was to describe the extent and modes of contacts between humans and bats in southern Cameroon, considered as an area at risk for future EBV outbreaks. The survey was conducted in 11 villages of four distinct rural areas in southern Cameroon. A total of 135 respondents were interviewed using semi-structured questionnaires, between February and May 2017. The study showed that direct contacts between bats and humans are relatively common. Bat bushmeat appeared to be an occasional meat resource; 40% of respondents consume bats with a median annual consumption of three, and 28% of respondents hunt them. About 22% of the respondents reported children catching bats. Indirect contact also appeared to be common; 55% of hunters use caves as shelters and 67% of interviewees eat fruits previously chewed by bats. Bat consumption varied significantly between regions (from 0% to 87%) and between pygmies and bantus in the extreme south-east of Cameroon. The study revealed considerable diversity in practices among interviewees, most of them being subsistence cultivators and relying on self-hunted bushmeat. Geographical diversity of contacts and perceptions regarding bats in Cameroon emphasizes the need to adjust zoonotic pathogen surveillance and education campaigns to the specificities of the communities and their context of interaction with wildlife.


Assuntos
Quirópteros/virologia , Ebolavirus/fisiologia , Doença pelo Vírus Ebola/virologia , Zoonoses , Animais , Camarões/epidemiologia , Coleta de Dados , Conhecimentos, Atitudes e Prática em Saúde , Doença pelo Vírus Ebola/transmissão , Humanos , Inquéritos e Questionários
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