Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 320
Filtrar
1.
Curr Opin Pharmacol ; 60: 268-274, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34482213

RESUMO

Over 40 filovirus disease outbreaks have been reported since the discovery of the first member of the Filoviridae family, and most of the outbreaks have occurred in Africa. In addition to deaths (primary impacts), there have also been health, social, economic, and political effects (secondary impacts) due to the outbreaks. Two large filovirus disease outbreaks have occurred in West and Central Africa in recent times, and direct and indirect repercussions resulting from the outbreaks underscores the need to strengthen the capacity of health services in disease hotspots.


Assuntos
Ebolavirus , Filoviridae , Doença pelo Vírus Ebola , África , Surtos de Doenças , Doença pelo Vírus Ebola/epidemiologia , Humanos
2.
J Vet Med Sci ; 83(9): 1485-1488, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34275961

RESUMO

I performed metaviromic analysis of publicly available RNA-seq data from reptiles to understand the diversity of filoviruses (family Filoviridae). I identified a coding-complete sequence of a filovirus from the common lancehead (Bothrops atrox (Linnaeus, 1758)), tentatively named Tapajós virus (TAPV). Although the genome organization of TAPV is similar to mammalian filoviruses, our phylogenetic analysis showed that TAPV forms a cluster with a fish filovirus. However, TAPV is still distantly related to all the known filoviruses, suggesting that TAPV can be assigned as a species of a novel genus in Filoviridae. To our knowledge, this is the first report identifying a filovirus in reptiles, and thus contributes to a deeper understanding of the diversity and evolution of filoviruses.


Assuntos
Bothrops , Filoviridae , Animais , Filoviridae/genética , Genoma , Mamíferos , Filogenia
3.
Viruses ; 13(5)2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069246

RESUMO

Filoviruses, including marburgviruses and ebolaviruses, have a single transmembrane glycoprotein (GP) that facilitates their entry into cells. During entry, GP needs to be cleaved by host proteases to expose the receptor-binding site that binds to the endosomal receptor Niemann-Pick C1 (NPC1) protein. The crystal structure analysis of the cleaved GP (GPcl) of Ebola virus (EBOV) in complex with human NPC1 has demonstrated that NPC1 has two protruding loops (loops 1 and 2), which engage a hydrophobic pocket on the head of EBOV GPcl. However, the molecular interactions between NPC1 and the GPcl of other filoviruses remain unexplored. In the present study, we performed molecular modeling and molecular dynamics simulations of NPC1 complexed with GPcls of two ebolaviruses, EBOV and Sudan virus (SUDV), and one marburgvirus, Ravn virus (RAVV). Similar binding structures were observed in the GPcl-NPC1 complexes of EBOV and SUDV, which differed from that of RAVV. Specifically, in the RAVV GPcl-NPC1 complex, the tip of loop 2 was closer to the pocket edge comprising residues at positions 79-88 of GPcl; the root of loop 1 was predicted to interact with P116 and Q144 of GPcl. Furthermore, in the SUDV GPcl-NPC1 complex, the tip of loop 2 was slightly closer to the residue at position 141 than those in the EBOV and RAVV GPcl-NPC1 complexes. These structural differences may affect the size and/or shape of the receptor-binding pocket of GPcl. Our structural models could provide useful information for improving our understanding the differences in host preference among filoviruses as well as contributing to structure-based drug design.


Assuntos
Filoviridae , Modelos Moleculares , Proteína C1 de Niemann-Pick/química , Proteína C1 de Niemann-Pick/metabolismo , Conformação Proteica , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Filoviridae/metabolismo , Ligação Proteica , Relação Estrutura-Atividade
4.
Proc Natl Acad Sci U S A ; 117(48): 30687-30698, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33184176

RESUMO

The SARS-CoV-2 pandemic has made it clear that we have a desperate need for antivirals. We present work that the mammalian SKI complex is a broad-spectrum, host-directed, antiviral drug target. Yeast suppressor screening was utilized to find a functional genetic interaction between proteins from influenza A virus (IAV) and Middle East respiratory syndrome coronavirus (MERS-CoV) with eukaryotic proteins that may be potential host factors involved in replication. This screening identified the SKI complex as a potential host factor for both viruses. In mammalian systems siRNA-mediated knockdown of SKI genes inhibited replication of IAV and MERS-CoV. In silico modeling and database screening identified a binding pocket on the SKI complex and compounds predicted to bind. Experimental assays of those compounds identified three chemical structures that were antiviral against IAV and MERS-CoV along with the filoviruses Ebola and Marburg and two further coronaviruses, SARS-CoV and SARS-CoV-2. The mechanism of antiviral activity is through inhibition of viral RNA production. This work defines the mammalian SKI complex as a broad-spectrum antiviral drug target and identifies lead compounds for further development.


Assuntos
Antivirais/farmacologia , Coronavirus/efeitos dos fármacos , Filoviridae/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Complexos Multiproteicos/metabolismo , Orthomyxoviridae/efeitos dos fármacos , Linhagem Celular , Genes Supressores , Modelos Moleculares , Terapia de Alvo Molecular , Ligação Proteica , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
5.
J Med Chem ; 63(19): 11085-11099, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-32886512

RESUMO

Filoviridae, including Ebola (EBOV) and Marburg (MARV) viruses, are emerging pathogens that pose a serious threat to public health. No agents have been approved to treat filovirus infections, representing a major unmet medical need. The selective estrogen receptor modulator (SERM) toremifene was previously identified from a screen of FDA-approved drugs as a potent EBOV viral entry inhibitor, via binding to EBOV glycoprotein (GP). A focused screen of ER ligands identified ridaifen-B as a potent dual inhibitor of EBOV and MARV. Optimization and reverse-engineering to remove ER activity led to a novel compound 30 (XL-147) showing potent inhibition against infectious EBOV Zaire (0.09 µM) and MARV (0.64 µM). Mutagenesis studies confirmed that inhibition of EBOV viral entry is mediated by the direct interaction with GP. Importantly, compound 30 displayed a broad-spectrum antifilovirus activity against Bundibugyo, Tai Forest, Reston, and Menglà viruses and is the first submicromolar antiviral agent reported for some of these strains, therefore warranting further development as a pan-filovirus inhibitor.


Assuntos
Antivirais/farmacologia , Filoviridae/efeitos dos fármacos , Receptores de Estrogênio/efeitos dos fármacos , Antivirais/química , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Filoviridae/fisiologia , Humanos , Ligantes , Fusão de Membrana/efeitos dos fármacos , Modelos Biológicos , Relação Estrutura-Atividade
6.
Antiviral Res ; 183: 104932, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32946918

RESUMO

Ebolaviruses and marburgviruses, members of the family Filoviridae, are known to cause fatal diseases often associated with hemorrhagic fever. Recent outbreaks of Ebola virus disease in West African countries and the Democratic Republic of the Congo have made clear the urgent need for the development of therapeutics and vaccines against filoviruses. Using replication-incompetent vesicular stomatitis virus (VSV) pseudotyped with the Ebola virus (EBOV) envelope glycoprotein (GP), we screened a chemical compound library to obtain new drug candidates that inhibit filoviral entry into target cells. We discovered a biaryl sulfonamide derivative that suppressed in vitro infection mediated by GPs derived from all known human-pathogenic filoviruses. To determine the inhibitory mechanism of the compound, we monitored each entry step (attachment, internalization, and membrane fusion) using lipophilic tracer-labeled ebolavirus-like particles and found that the compound efficiently blocked fusion between the viral envelope and the endosomal membrane during cellular entry. However, the compound did not block the interaction of GP with the Niemann-Pick C1 protein, which is believed to be the receptor of filoviruses. Using replication-competent VSVs pseudotyped with EBOV GP, we selected escape mutants and identified two EBOV GP amino acid residues (positions 47 and 66) important for the interaction with this compound. Interestingly, these amino acid residues were located at the base region of the GP trimer, suggesting that the compound might interfere with the GP conformational change required for membrane fusion. These results suggest that this biaryl sulfonamide derivative is a novel fusion inhibitor and a possible drug candidate for the development of a pan-filovirus therapeutic.


Assuntos
Filoviridae/efeitos dos fármacos , Sulfonamidas/química , Sulfonamidas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Chlorocebus aethiops , Descoberta de Drogas , Ebolavirus/efeitos dos fármacos , Filoviridae/classificação , Infecções por Filoviridae/tratamento farmacológico , Infecções por Filoviridae/virologia , Células HEK293 , Doença pelo Vírus Ebola/tratamento farmacológico , Humanos , Doença do Vírus de Marburg/tratamento farmacológico , Marburgvirus/efeitos dos fármacos , Receptores Virais/metabolismo , Células Vero
7.
Arch Virol ; 165(10): 2165-2176, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32740830

RESUMO

The PI3K/Akt signalling pathway is a crucial signalling cascade that regulates transcription, protein translation, cell growth, proliferation, cell survival, and metabolism. During viral infection, viruses exploit a variety of cellular pathways, including the well-known PI3K/Akt signalling pathway. Conversely, cells rely on this pathway to stimulate an antiviral response. The PI3K/Akt pathway is manipulated by a number of viruses, including DNA and RNA viruses and retroviruses. The aim of this review is to provide up-to-date information about the role of the PI3K-Akt pathway in infection with members of five different families of negative-sense ssRNA viruses. This pathway is hijacked for viral entry, regulation of endocytosis, suppression of premature apoptosis, viral protein expression, and replication. Although less common, the PI3K/Akt pathway can be downregulated as an immunomodulatory strategy or as a mechanism for inducing autophagy. Moreover, the cell activates this pathway as an antiviral strategy for interferon and cytokine production, among other strategies. Here, we present new data concerning the role of this pathway in infection with the paramyxovirus Newcastle disease virus (NDV). Our data seem to indicate that NDV uses the PI3K/Akt pathway to delay cell death and increase cell survival as a means of improving its replication. The interference of negative-sense ssRNA viruses with this essential pathway might have implications for the development of antiviral therapies.


Assuntos
Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Fosfatidilinositol 3-Quinase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Infecções por Vírus de RNA/genética , Apoptose/genética , Autofagia/genética , Autofagia/imunologia , Citocinas/genética , Citocinas/imunologia , Endocitose/genética , Endocitose/imunologia , Filoviridae/genética , Filoviridae/metabolismo , Filoviridae/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Interferons/genética , Interferons/imunologia , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Orthomyxoviridae/patogenicidade , Paramyxoviridae/genética , Paramyxoviridae/metabolismo , Paramyxoviridae/patogenicidade , Fosfatidilinositol 3-Quinase/imunologia , Pneumovirinae/genética , Pneumovirinae/metabolismo , Pneumovirinae/patogenicidade , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-akt/imunologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Rhabdoviridae/genética , Rhabdoviridae/metabolismo , Rhabdoviridae/patogenicidade , Transdução de Sinais , Proteínas Virais/genética , Proteínas Virais/imunologia , Internalização do Vírus , Replicação Viral
8.
Eur J Med Chem ; 204: 112595, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32707357

RESUMO

Filoviruses, including Ebolavirus (EBOV), Marburgvirus (MARV) and Cuevavirus, cause hemorrhagic fevers in humans with up to 90% mortality rates. In the 2014-2016 West Africa Ebola epidemic, there are 15,261 laboratory confirmed cases and 11,325 total deaths. The lack of effective vaccines and medicines for the prevention and treatment of filovirus infection in humans stresses the urgency to develop antiviral therapeutics against filovirus-associated diseases. Our previous study identified a histamine receptor antagonist compound CP19 as an entry inhibitor against both EBOV and MARV. The preliminary structure-activity relationship (SAR) studies of CP19 showed that its piperidine, coumarin and linker were related with its antiviral activities. In this study, we performed detailed SAR studies on these groups with synthesized CP19 derivatives. We discovered that 1) the piperidine group could be optimized with heterocycles, 2) the substitution groups of C3 and C4 of coumarin should be relatively large hydrophobic groups and 3) the linker part should be least substituted. Based on the SAR analysis, we synthesized compound 32 as a potent entry inhibitor of EBOV and MARV (IC50 = 0.5 µM for EBOV and 1.5 µM for MARV). The mutation studies of Ebola glycoprotein and molecular docking studies showed that the coumarin and its substituted groups of compound 32 bind to the pocket of Ebola glycoprotein in a similar way to the published entry inhibitor compound 118a. However, the carboxamide group of compound 32 does not have strong interaction with N61 as compound 118a does. The coumarin skeleton structure and the binding model of compound 32 elucidated by this study could be utilized to guide further design and optimization of entry inhibitors targeting the filovirus glycoproteins.


Assuntos
Cumarínicos/química , Cumarínicos/farmacologia , Desenho de Fármacos , Filoviridae/efeitos dos fármacos , Filoviridae/fisiologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus/efeitos dos fármacos , Antivirais/química , Antivirais/farmacologia , Filoviridae/metabolismo , Terapia de Alvo Molecular , Piperidinas/química , Relação Estrutura-Atividade
9.
J Virol ; 94(16)2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32493822

RESUMO

Ebola virus (EBOV) entry requires internalization into host cells and extensive trafficking through the endolysosomal network in order to reach late endosomal/lysosomal compartments that contain triggering factors for viral membrane fusion. These triggering factors include low-pH-activated cellular cathepsin proteases, which cleave the EBOV glycoprotein (GP), exposing a domain which binds to the filoviral receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). Here, we report that trafficking of EBOV to NPC1 requires expression of the homotypic fusion and protein sorting (HOPS) tethering complex as well as its regulator, UV radiation resistance-associated gene (UVRAG). Using an inducible clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we demonstrated that depletion of HOPS subunits as well as UVRAG impairs entry by all pathogenic filoviruses. UVRAG depletion resulted in reduced delivery of EBOV virions to NPC1+ cellular compartments. Furthermore, we show that deletion of a domain on UVRAG known to be required for interaction with the HOPS complex results in impaired EBOV entry. Taken together, our studies demonstrate that EBOV requires both expression of and coordination between the HOPS complex and UVRAG in order to mediate efficient viral entry.IMPORTANCE Ebola viruses (EBOV) and other filoviruses cause sporadic and unpredictable outbreaks of highly lethal diseases. The lack of FDA-approved therapeutics, particularly ones with panfiloviral specificity, highlights the need for continued research efforts to understand aspects of the viral life cycle that are common to all filoviruses. As such, viral entry is of particular interest, as all filoviruses must reach cellular compartments containing the viral receptor Niemann-Pick C1 to enter cells. Here, we present an inducible CRISPR/Cas9 method to rapidly and efficiently generate knockout cells in order to interrogate the roles of a broad range of host factors in viral entry. Using this approach, we showed that EBOV entry depends on both the homotypic fusion and protein sorting (HOPS) tethering complex in coordination with UV radiation resistance-associated gene (UVRAG). Importantly, we demonstrate that the HOPS complex and UVRAG are required by all pathogenic filoviruses, representing potential targets for panfiloviral therapeutics.


Assuntos
Ebolavirus/metabolismo , Proteína C1 de Niemann-Pick/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Ebolavirus/genética , Ebolavirus/patogenicidade , Endossomos/metabolismo , Filoviridae/genética , Infecções por Filoviridae/genética , Infecções por Filoviridae/metabolismo , Glicoproteínas/metabolismo , Doença pelo Vírus Ebola/metabolismo , Interações Hospedeiro-Patógeno , Glicoproteínas de Membrana/metabolismo , Transporte Proteico/genética , Transporte Proteico/fisiologia , Receptores Virais/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas do Envelope Viral/genética , Internalização do Vírus/efeitos dos fármacos
10.
Am J Pathol ; 190(9): 1867-1880, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32479821

RESUMO

The most commonly reported symptom of post-Ebola virus disease syndrome in survivors is arthralgia, yet involvement of the joints in acute or convalescent Ebola virus infection is not well characterized in human patients or animal models. Through immunohistochemistry, we found that the lining synovial intima of the stifle (knee) is a target for acute infection by Ebola virus/Kikwit, Ebola virus/Makona-C05, and Marburg virus/Angola in the rhesus macaque model. Furthermore, histologic analysis, immunohistochemistry, RNAscope in situ hybridization, and transmission electron microscopy showed that synoviocytes of the stifle, shoulder, and hip are a target for mouse-adapted Ebola virus/Yambuku-Mayinga infection during acute disease in rhesus macaques. A time course of infection study with Ebola virus/Kikwit found that the large joint synovium became immunopositive beginning on postinfection day 6. In total, the synovium of 28 of 30 rhesus macaques with terminal filovirus disease had evidence of infection (64 of 96 joints examined). On the basis of immunofluorescence, infected cell types included CD68+ type A (macrophage-like) synoviocytes and CD44+ type B (fibroblast-like) synoviocytes. Cultured primary human fibroblast-like synoviocytes were permissive to infection with Ebola and Marburg viruses in vitro. Because synovial joints include immune privileged sites, these findings are significant for future investigations of filovirus pathogenesis and persistence as well as arthralgias in acute and convalescent filovirus disease.


Assuntos
Infecções por Filoviridae/virologia , Sinoviócitos/virologia , Animais , Células Cultivadas , Filoviridae , Humanos , Macaca mulatta
11.
Artigo em Inglês | MEDLINE | ID: mdl-32513799

RESUMO

Several cationic amphiphilic drugs (CADs) have been found to inhibit cell entry of filoviruses and other enveloped viruses. Structurally unrelated CADs may have antiviral activity, yet the underlying common mechanism and structure-activity relationship are incompletely understood. We aimed to understand how widespread antiviral activity is among CADs and which structural and physico-chemical properties are linked to entry inhibition. We measured inhibition of Marburg virus pseudoparticle (MARVpp) cell entry by 45 heterogeneous and mostly FDA-approved CADs and cytotoxicity in EA.hy926 cells. We analyzed correlation of antiviral activity with four chemical properties: pKa, hydrophobicity (octanol/water partitioning coefficient; ClogP), molecular weight, and distance between the basic group and hydrophobic ring structures. Additionally, we quantified drug-induced phospholipidosis (DIPL) of a CAD subset by flow cytometry. Structurally similar compounds (derivatives) and those with similar chemical properties but unrelated structures (analogues) to those of strong inhibitors were obtained by two in silico similarity search approaches and tested for antiviral activity. Overall, 11 out of 45 (24%) CADs inhibited MARVpp by 40% or more. The strongest antiviral compounds were dronedarone, triparanol, and quinacrine. Structure-activity relationship studies revealed highly significant correlations between antiviral activity, hydrophobicity (ClogP > 4), and DIPL. Moreover, pKa and intramolecular distance between hydrophobic and hydrophilic moieties correlated with antiviral activity but to a lesser extent. We also showed that in contrast to analogues, derivatives had antiviral activity similar to that of the seed compound dronedarone. Overall, one-quarter of CADs inhibit MARVpp entry in vitro, and antiviral activity of CADs mostly relies on their hydrophobicity yet is promoted by the individual structure.


Assuntos
Filoviridae , Marburgvirus , Preparações Farmacêuticas , Antivirais/farmacologia , Internalização do Vírus
12.
J Virol ; 94(13)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32295912

RESUMO

Menglà virus (MLAV), identified in Rousettus bats, is a phylogenetically distinct member of the family Filoviridae Because the filoviruses Ebola virus (EBOV) and Marburg virus (MARV) modulate host innate immunity, MLAV VP35, VP40, and VP24 proteins were compared with their EBOV and MARV homologs for innate immune pathway modulation. In human and Rousettus cells, MLAV VP35 behaved like EBOV and MARV VP35s, inhibiting virus-induced activation of the interferon beta (IFN-ß) promoter and interferon regulatory factor 3 (IRF3) phosphorylation. MLAV VP35 also interacted with PACT, a host protein engaged by EBOV VP35 to inhibit RIG-I signaling. MLAV VP35 also inhibits PKR activation. MLAV VP40 was demonstrated to inhibit type I IFN-induced gene expression in human and bat cells. It blocked STAT1 tyrosine phosphorylation induced either by type I IFN or overexpressed Jak1, paralleling MARV VP40. MLAV VP40 also inhibited virus-induced IFN-ß promoter activation, a property shared by MARV VP40 and EBOV VP24. A Jak kinase inhibitor did not recapitulate this inhibition in the absence of viral proteins. Therefore, inhibition of Jak-STAT signaling is insufficient to explain inhibition of IFN-ß promoter activation. MLAV VP24 did not inhibit IFN-induced gene expression or bind karyopherin α proteins, properties of EBOV VP24. MLAV VP24 differed from MARV VP24 in that it failed to interact with Keap1 or activate an antioxidant response element reporter gene due to the absence of a Keap1-binding motif. These functional observations support a closer relationship of MLAV to MARV than to EBOV but also are consistent with MLAV belonging to a distinct genus.IMPORTANCE EBOV and MARV, members of the family Filoviridae, are highly pathogenic zoonotic viruses that cause severe disease in humans. Both viruses use several mechanisms to modulate the host innate immune response, and these likely contribute to the severity of disease. Here, we demonstrate that MLAV, a filovirus newly discovered in a bat, suppresses antiviral type I interferon responses in both human and bat cells. Inhibitory activities are possessed by MLAV VP35 and VP40, which parallels how MARV blocks IFN responses. However, whereas MARV activates cellular antioxidant responses through an interaction between its VP24 protein and host protein Keap1, MLAV VP24 lacks a Keap1-binding motif and fails to activate this cytoprotective response. These data indicate that MLAV possesses immune-suppressing functions that could facilitate human infection. They also support the placement of MLAV in a different genus than either EBOV or MARV.


Assuntos
Infecções por Filoviridae/fisiopatologia , Filoviridae/genética , Animais , Quirópteros/imunologia , Quirópteros/virologia , Ebolavirus , Filoviridae/metabolismo , Filoviridae/patogenicidade , Células HEK293 , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/imunologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Marburgvirus , Fator 2 Relacionado a NF-E2/metabolismo , Fator de Transcrição STAT1 , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
13.
Cell Rep ; 30(13): 4540-4550.e3, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32234486

RESUMO

Ebola virus causes severe hemorrhagic fever, often leading to death in humans. The trimeric fusion glycoprotein (GP) is the sole target for neutralizing antibodies and is the major focus of vaccine development. Soluble GP ectodomains are unstable and mostly monomeric when not fused to a heterologous trimerization domain. Here, we report structure-based designs of Ebola and Marburg GP trimers based on a stabilizing mutation in the hinge loop in refolding region 1 and substitution of a partially buried charge at the interface of the GP1 and GP2 subunits. The combined substitutions (T577P and K588F) substantially increased trimer expression for Ebola GP proteins. We determined the crystal structure of stabilized GP from the Makona Zaire ebolavirus strain without a trimerization domain or complexed ligand. The structure reveals that the stabilized GP adopts the same trimeric prefusion conformation, provides insight into triggering of GP conformational changes, and should inform future filovirus vaccine development.


Assuntos
Filoviridae/metabolismo , Glicoproteínas/química , Multimerização Proteica , Substituição de Aminoácidos , Linhagem Celular , Cristalografia por Raios X , Ebolavirus/metabolismo , Glicoproteínas/genética , Humanos , Marburgvirus/metabolismo , Modelos Moleculares , Mutação/genética , Perfusão , Domínios Proteicos , Estabilidade Proteica , Relação Estrutura-Atividade
14.
Virus Genes ; 56(2): 150-167, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32076918

RESUMO

The viruses historically implicated or currently considered as candidates for misuse in bioterrorist events are poxviruses, filoviruses, bunyaviruses, orthomyxoviruses, paramyxoviruses and a number of arboviruses causing encephalitis, including alpha- and flaviviruses. All these viruses are of concern for public health services when they occur in natural outbreaks or emerge in unvaccinated populations. Recent events and intelligence reports point to a growing risk of dangerous biological agents being used for nefarious purposes. Public health responses effective in natural outbreaks of infectious disease may not be sufficient to deal with the severe consequences of a deliberate release of such agents. One important aspect of countermeasures against viral biothreat agents are the antiviral treatment options available for use in post-exposure prophylaxis. These issues were adressed by the organizers of the 16th Medical Biodefense Conference, held in Munich in 2018, in a special session on the development of drugs to treat infections with viruses currently perceived as a threat to societies or associated with a potential for misuse as biothreat agents. This review will outline the state-of-the-art methods in antivirals research discussed and provide an overview of antiviral compounds in the pipeline that are already approved for use or still under development.


Assuntos
Antivirais/uso terapêutico , Arbovírus/efeitos dos fármacos , Bioterrorismo/prevenção & controle , Viroses/tratamento farmacológico , Arbovírus/patogenicidade , Filoviridae/efeitos dos fármacos , Filoviridae/patogenicidade , Humanos , Orthobunyavirus/efeitos dos fármacos , Orthobunyavirus/patogenicidade , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/patogenicidade , Paramyxovirinae/efeitos dos fármacos , Paramyxovirinae/patogenicidade , Poxviridae/efeitos dos fármacos , Poxviridae/patogenicidade , Viroses/virologia
15.
Biochem Biophys Res Commun ; 525(2): 392-397, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32093889

RESUMO

The family Filoviridae contains many important human viruses, including Marburg virus (MARV) and Ebola virus (EBOV). Menglà virus (MLAV), a newly discovered filovirus, is considered a potential human pathogen. The VP30 C-terminal domain (CTD) of these filoviruses plays an essential role in virion assembly. In common with other filoviruses, MLAV VP30 CTD mainly exists as a dimer in solution. In this work, we determined the crystal structure of recombinant MLAV VP30 CTD monomer, verifying that C-terminal helix-7 (H7) is critical for the dimerization process. This study provides a preliminary model for investigation of MLAV VP30 CTD as an anti-filovirus drug development target.


Assuntos
Infecções por Filoviridae/virologia , Filoviridae/química , Proteínas Virais/química , Animais , Cristalografia por Raios X , Descoberta de Drogas , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Multimerização Proteica
16.
J Infect Dis ; 221(Suppl 4): S375-S382, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32034942

RESUMO

Bat-borne zoonotic pathogens belonging to the family Paramxyoviridae, including Nipah and Hendra viruses, and the family Filoviridae, including Ebola and Marburg viruses, can cause severe disease and high mortality rates on spillover into human populations. Surveillance efforts for henipaviruses and filoviruses have been largely restricted to the Old World; however, recent studies suggest a potentially broader distribution for henipaviruses and filoviruses than previously recognized. In the current study, we screened for henipaviruses and filoviruses in New World bats collected across 4 locations in Trinidad near the coast of Venezuela. Bat tissue samples were screened using previously established reverse-transcription polymerase chain reaction assays. Serum were screened using a multiplex immunoassay to detect antibodies reactive with the envelope glycoprotein of viruses in the genus Henipavirus and the family Filoviridae. Serum samples were also screened by means of enzyme-linked immunosorbent assay for antibodies reactive with Nipah G and F glycoproteins. Of 84 serum samples, 28 were reactive with ≥1 henipavirus glycoprotein by ≥1 serological method, and 6 serum samples were reactive against ≥1 filovirus glycoproteins. These data provide evidence of potential circulation of viruses related to the henipaviruses and filoviruses in New World bats.


Assuntos
Quirópteros/virologia , Infecções por Filoviridae/veterinária , Filoviridae , Infecções por Henipavirus/veterinária , Henipavirus , Animais , Quirópteros/sangue , Quirópteros/classificação , Infecções por Filoviridae/epidemiologia , Infecções por Filoviridae/virologia , Infecções por Henipavirus/epidemiologia , Infecções por Henipavirus/virologia , Testes Sorológicos , Trinidad e Tobago/epidemiologia
17.
PLoS Pathog ; 16(1): e1008231, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31905227

RESUMO

Ebola (EBOV) and Marburg (MARV) are members of the Filoviridae family, which continue to emerge and cause sporadic outbreaks of hemorrhagic fever with high mortality rates. Filoviruses utilize their VP40 matrix protein to drive virion assembly and budding, in part, by recruitment of specific WW-domain-bearing host proteins via its conserved PPxY Late (L) domain motif. Here, we screened an array of 115 mammalian, bacterially expressed and purified WW-domains using a PPxY-containing peptide from MARV VP40 (mVP40) to identify novel host interactors. Using this unbiased approach, we identified Yes Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ) as novel mVP40 PPxY interactors. YAP and TAZ function as downstream transcriptional effectors of the Hippo signaling pathway that regulates cell proliferation, migration and apoptosis. We demonstrate that ectopic expression of YAP or TAZ along with mVP40 leads to significant inhibition of budding of mVP40 VLPs in a WW-domain/PPxY dependent manner. Moreover, YAP colocalized with mVP40 in the cytoplasm, and inhibition of mVP40 VLP budding was more pronounced when YAP was localized predominantly in the cytoplasm rather than in the nucleus. A key regulator of YAP nuclear/cytoplasmic localization and function is angiomotin (Amot); a multi-PPxY containing protein that strongly interacts with YAP WW-domains. Interestingly, we found that expression of PPxY-containing Amot rescued mVP40 VLP egress from either YAP- or TAZ-mediated inhibition in a PPxY-dependent manner. Importantly, using a stable Amot-knockdown cell line, we found that expression of Amot was critical for efficient egress of mVP40 VLPs as well as egress and spread of authentic MARV in infected cell cultures. In sum, we identified novel negative (YAP/TAZ) and positive (Amot) regulators of MARV VP40-mediated egress, that likely function in part, via competition between host and viral PPxY motifs binding to modular host WW-domains. These findings not only impact our mechanistic understanding of virus budding and spread, but also may impact the development of new antiviral strategies.


Assuntos
Filoviridae/fisiologia , Marburgvirus/fisiologia , Mimetismo Molecular , Proteínas Proto-Oncogênicas c-yes/metabolismo , Proteínas da Matriz Viral/fisiologia , Liberação de Vírus , Sítios de Ligação , Membrana Celular/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Domínios PDZ , Domínios Proteicos , Proteínas Recombinantes de Fusão/metabolismo
18.
Cell Rep ; 30(2): 308-319.e5, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31940478

RESUMO

Fruit bats are suspected to be natural hosts of filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV). Interestingly, however, previous studies suggest that these viruses have different tropisms depending on the bat species. Here, we show a molecular basis underlying the host-range restriction of filoviruses. We find that bat-derived cell lines FBKT1 and ZFBK13-76E show preferential susceptibility to EBOV and MARV, respectively, whereas the other bat cell lines tested are similarly infected with both viruses. In FBKT1 and ZFBK13-76E, unique amino acid (aa) sequences are found in the Niemann-Pick C1 (NPC1) protein, one of the cellular receptors interacting with the filovirus glycoprotein (GP). These aa residues, as well as a few aa differences between EBOV and MARV GPs, are crucial for the differential susceptibility to filoviruses. Taken together, our findings indicate that the heterogeneity of bat NPC1 orthologs is an important factor controlling filovirus species-specific host tropism.


Assuntos
Filoviridae/genética , Proteína C1 de Niemann-Pick/metabolismo , Tropismo/genética , Sequência de Aminoácidos , Animais , Quirópteros , Humanos , Modelos Moleculares
19.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941778

RESUMO

Southern China is a hot spot of emerging infectious diseases, in which diverse species of bats dwell, a large group of flying mammals considered natural reservoirs for zoonotic viruses. Recently, divergent filoviruses (FiVs) have been identified in bats within this region, which pose a potential risk to public health, but the true infection situation in bats remains largely unclear. Here, 689 archived bat serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA), Western blotting, and neutralization assay to investigate the seroprevalence and cross-reactivity of four divergent FiVs and two other viruses (rabies virus and Tuhoko pararubulavirus 1) of different families within the order Mononegavirales Results showed no cross-antigenicity between FiVs and other mononegaviruses but different cross-reactivity among the FiVs themselves. The total FiV seroreactive rate was 36.3% (250/689), with infection by the indigenous Chinese FiV DH04 or an antigenically related one being the most widely and the most highly prevalent. Further viral metagenomic analysis of fruit bat tissues also identified the gene sequence of a novel FiV. These results indicate the likely prevalence of other so far unidentified FiVs within the Chinese bat population, with frugivorous Rousettus leschenaultii and Eonycteris spelaea bats and insectivorous Myotis horsfieldii and Miniopterus schreibersii bats being their major reservoirs.IMPORTANCE Bats are natural hosts of many FiVs, from which diverse FiVs were serologically or virologically detected in Africa, Europe, and East Asia. Recently, very divergent FiVs were identified in the Chinese bat population, but their antigenic relationship with other known FiVs remains unknown. Here, we conducted serological characterization and investigation of Chinese indigenous FiVs and prototypes of other viruses in bats. Results indicated that Chinese indigenous FiVs are antigenically distant to other FiVs, and infection of novel or multiple FiVs occurred in Chinese bats, with FiV DH04 or an antigenically related one being the most widely and the most highly prevalent. Additionally, besides Rousettus leschenaultii and Eonycteris spelaea bats, the insectivorous Myotis horsfieldii and M. schreibersii bats are highly preferential hosts of FiVs. Seroreactive and viral metagenomic results indicated that more as yet unknown bat-borne FiVs circulate in Southern China, and to uncover them further, investigation and timely surveillance is needed.


Assuntos
Anticorpos Antivirais/sangue , Quirópteros/virologia , Infecções por Filoviridae/veterinária , Filoviridae/imunologia , Animais , China , Quirópteros/sangue , Coinfecção , Reservatórios de Doenças/veterinária , Reservatórios de Doenças/virologia , Ensaio de Imunoadsorção Enzimática , Filoviridae/classificação , Metagenômica , Testes de Neutralização , Paramyxoviridae , Infecções por Paramyxoviridae/sangue , Infecções por Paramyxoviridae/veterinária , Filogenia , Rhabdoviridae , Infecções por Rhabdoviridae/sangue , Infecções por Rhabdoviridae/veterinária , Estudos Soroepidemiológicos
20.
Emerg Microbes Infect ; 9(1): 124-128, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31913767

RESUMO

A serological survey of 2,430 archived serum samples collected between 1997 and 2012 was conducted to retrospectively determine the prevalence of Marburg virus in five African countries. Serum samples were screened for neutralizing antibodies in a pseudotype micro-neutralization assay and confirmed by enzyme-linked immunosorbent assay (ELISA). Surprisingly, a seroprevalence for Marburg virus of 7.5 and 6.3% was found in Cameroon and Ghana, respectively, suggesting the circulation of filoviruses or related viruses outside of known endemic areas that remain undetected by current surveillance efforts. However, due to the lack of validated assays and appropriate positive controls, these results must be considered preliminary.


Assuntos
Anticorpos Antivirais/sangue , Filoviridae/imunologia , Doença do Vírus de Marburg/sangue , Doença do Vírus de Marburg/epidemiologia , Marburgvirus/imunologia , Animais , Camarões/epidemiologia , Ensaio de Imunoadsorção Enzimática , Filoviridae/genética , Infecções por Filoviridae/sangue , Infecções por Filoviridae/epidemiologia , Infecções por Filoviridae/virologia , Gana/epidemiologia , Humanos , Doença do Vírus de Marburg/virologia , Marburgvirus/genética , Estudos Retrospectivos , Estudos Soroepidemiológicos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...