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1.
Cell ; 174(4): 938-952.e13, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30096313

RESUMO

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Ebolavirus/imunologia , Epitopos/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Glicoproteínas de Membrana/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Feminino , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Resultado do Tratamento
2.
Biophys J ; 118(10): 2470-2488, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32348724

RESUMO

The structural characterization of modular proteins containing long intrinsically disordered regions intercalated with folded domains is complicated by their conformational diversity and flexibility and requires the integration of multiple experimental approaches. Nipah virus (NiV) phosphoprotein, an essential component of the viral RNA transcription/replication machine and a component of the viral arsenal that hijacks cellular components and counteracts host immune responses, is a prototypical model for such modular proteins. Curiously, the phosphoprotein of NiV is significantly longer than the corresponding protein of other paramyxoviruses. Here, we combine multiple biophysical methods, including x-ray crystallography, NMR spectroscopy, and small angle x-ray scattering, to characterize the structure of this protein and provide an atomistic representation of the full-length protein in the form of a conformational ensemble. We show that full-length NiV phosphoprotein is tetrameric, and we solve the crystal structure of its tetramerization domain. Using NMR spectroscopy and small angle x-ray scattering, we show that the long N-terminal intrinsically disordered region and the linker connecting the tetramerization domain to the C-terminal X domain exchange between multiple conformations while containing short regions of residual secondary structure. Some of these transient helices are known to interact with partners, whereas others represent putative binding sites for yet unidentified proteins. Finally, using NMR spectroscopy and isothermal titration calorimetry, we map a region of the phosphoprotein, comprising residues between 110 and 140 and common to the V and W proteins, that binds with weak affinity to STAT1 and confirm the involvement of key amino acids of the viral protein in this interaction. This provides new, to our knowledge, insights into how the phosphoprotein and the nonstructural V and W proteins of NiV perform their multiple functions.


Assuntos
Vírus Nipah , Fosfoproteínas , Conformação Proteica , Proteínas Virais , Replicação Viral
3.
Emerg Infect Dis ; 26(1): 104-113, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31855143

RESUMO

We conducted an in-depth characterization of the Nipah virus (NiV) isolate previously obtained from a Pteropus lylei bat in Cambodia in 2003 (CSUR381). We performed full-genome sequencing and phylogenetic analyses and confirmed CSUR381 is part of the NiV-Malaysia genotype. In vitro studies revealed similar cell permissiveness and replication of CSUR381 (compared with 2 other NiV isolates) in both bat and human cell lines. Sequence alignments indicated conservation of the ephrin-B2 and ephrin-B3 receptor binding sites, the glycosylation site on the G attachment protein, as well as the editing site in phosphoprotein, suggesting production of nonstructural proteins V and W, known to counteract the host innate immunity. In the hamster animal model, CSUR381 induced lethal infections. Altogether, these data suggest that the Cambodia bat-derived NiV isolate has high pathogenic potential and, thus, provide insight for further studies and better risk assessment for future NiV outbreaks in Southeast Asia.


Assuntos
Quirópteros/virologia , Infecções por Henipavirus/veterinária , Vírus Nipah/patogenicidade , Animais , Camboja , Genoma Viral/genética , Infecções por Henipavirus/epidemiologia , Infecções por Henipavirus/virologia , Humanos , Vírus Nipah/genética , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Sequenciamento Completo do Genoma
4.
PLoS Pathog ; 13(9): e1006610, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28957419

RESUMO

IFITMs are broad antiviral factors that block incoming virions in endosomal vesicles, protecting target cells from infection. In the case of HIV-1, we and others reported the existence of an additional antiviral mechanism through which IFITMs lead to the production of virions of reduced infectivity. However, whether this second mechanism of inhibition is unique to HIV or extends to other viruses is currently unknown. To address this question, we have analyzed the susceptibility of a broad spectrum of viruses to the negative imprinting of the virion particles infectivity by IFITMs. The results we have gathered indicate that this second antiviral property of IFITMs extends well beyond HIV and we were able to identify viruses susceptible to the three IFITMs altogether (HIV-1, SIV, MLV, MPMV, VSV, MeV, EBOV, WNV), as well as viruses that displayed a member-specific susceptibility (EBV, DUGV), or were resistant to all IFITMs (HCV, RVFV, MOPV, AAV). The swapping of genetic elements between resistant and susceptible viruses allowed us to point to specificities in the viral mode of assembly, rather than glycoproteins as dominant factors of susceptibility. However, we also show that, contrarily to X4-, R5-tropic HIV-1 envelopes confer resistance against IFITM3, suggesting that viral receptors add an additional layer of complexity in the IFITMs-HIV interplay. Lastly, we show that the overall antiviral effects ascribed to IFITMs during spreading infections, are the result of a bimodal inhibition in which IFITMs act both by protecting target cells from incoming viruses and in driving the production of virions of reduced infectivity. Overall, our study reports for the first time that the negative imprinting of the virion particles infectivity is a conserved antiviral property of IFITMs and establishes IFITMs as a paradigm of restriction factor capable of interfering with two distinct phases of a virus life cycle.


Assuntos
Antígenos de Diferenciação/metabolismo , Vírion , Replicação Viral , Linhagem Celular , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Internalização do Vírus
5.
J Infect Dis ; 218(suppl_5): S666-S671, 2018 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-30239745

RESUMO

The West African outbreak of Ebola virus (EBOV) infection during 2013-2016 highlighted the need for development of field-applicable therapeutic drugs for this infection. Here we report that mannoside glycolipid conjugates (MGCs) consisting of a trimannose head and a lipophilic chain assembled by a linker inhibit EBOV infection not only of human monocyte-derived dendritic cells and macrophages, but also of a number of susceptible cells. Analysis of the mode of action leads us to conclude that MGCs act directly on cells, notably by preventing virus endocytosis.


Assuntos
Antivirais/farmacologia , Ebolavirus/efeitos dos fármacos , Glicolipídeos/farmacologia , Manosídeos/uso terapêutico , Animais , Chlorocebus aethiops , Ebolavirus/fisiologia , Humanos , Células Vero , Internalização do Vírus/efeitos dos fármacos
6.
J Virol ; 90(11): 5256-5269, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-26984723

RESUMO

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


Assuntos
Ebolavirus/metabolismo , Lectinas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Proteínas do Sistema Complemento/metabolismo , Ebolavirus/química , Ebolavirus/genética , Células HEK293 , Humanos , Macrófagos/virologia , Lectina de Ligação a Manose/metabolismo , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Células Vero , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Ficolinas
7.
PLoS Pathog ; 10(11): e1004509, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25412102

RESUMO

During Ebola virus (EBOV) infection a significant amount of surface glycoprotein GP is shed from infected cells in a soluble form due to cleavage by cellular metalloprotease TACE. Shed GP and non-structural secreted glycoprotein sGP, both expressed from the same GP gene, have been detected in the blood of human patients and experimentally infected animals. In this study we demonstrate that shed GP could play a particular role during EBOV infection. In effect it binds and activates non-infected dendritic cells and macrophages inducing the secretion of pro- and anti-inflammatory cytokines (TNFα, IL1ß, IL6, IL8, IL12p40, and IL1-RA, IL10). Activation of these cells by shed GP correlates with the increase in surface expression of co-stimulatory molecules CD40, CD80, CD83 and CD86. Contrary to shed GP, secreted sGP activates neither DC nor macrophages while it could bind DCs. In this study, we show that shed GP activity is likely mediated through cellular toll-like receptor 4 (TLR4) and is dependent on GP glycosylation. Treatment of cells with anti-TLR4 antibody completely abolishes shed GP-induced activation of cells. We also demonstrate that shed GP activity is negated upon addition of mannose-binding sera lectin MBL, a molecule known to interact with sugar arrays present on the surface of different microorganisms. Furthermore, we highlight the ability of shed GP to affect endothelial cell function both directly and indirectly, demonstrating the interplay between shed GP, systemic cytokine release and increased vascular permeability. In conclusion, shed GP released from virus-infected cells could activate non-infected DCs and macrophages causing the massive release of pro- and anti-inflammatory cytokines and effect vascular permeability. These activities could be at the heart of the excessive and dysregulated inflammatory host reactions to infection and thus contribute to high virus pathogenicity.


Assuntos
Células Dendríticas/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas Virais/imunologia , Animais , Antígenos CD/imunologia , Citocinas/imunologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Cobaias , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Macrófagos/patologia , Macrófagos/virologia , Receptor 4 Toll-Like/imunologia
8.
J Infect Dis ; 212 Suppl 2: S372-8, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26232760

RESUMO

Ebola virus is the etiological agent of a severe hemorrhagic fever with a high mortality rate. As the only protein exposed on the surface of viral particles, the spike glycoprotein GP is the unique target for neutralizing monoclonal antibodies. In this study, we demonstrate the strong neutralization capacity of the monoclonal antibody #3327 and characterize its activity. GP residues that are required for recognition and neutralization were found to be located both in the internal fusion loop and in the receptor-binding domain. Analysis of Ebola virus entry in the presence of #3327 allows us to hypothesize that this antibody binds to the virus particle before internalization and endosomal processing of GP and likely prevents the final viral fusion step. Importantly, #3327 is able to block entry of virions bearing GP that contain the Q508 escape mutation common to a number of virus-neutralizing antibodies, and therefore provides future perspectives for treatment strategies against Ebola virus infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Animais , Linhagem Celular , Chlorocebus aethiops , Glicoproteínas/imunologia , Células HEK293 , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Ligação Proteica/imunologia , Células Vero , Vírion/imunologia , Internalização do Vírus
9.
J Infect Dis ; 212 Suppl 2: S199-203, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25941332

RESUMO

Ebola virus (EBOV) is responsible for a severe fever with a high mortality rate. The diverse nature of the attachment of the virus to the cell surface, the initial step of virus entry, raises questions concerning the kinetics of the virus internalization process. We investigated EBOV entry kinetics using the activity of a particular monoclonal antibody that neutralizes virus infectivity. We demonstrate that inoculation of cells with EBOV results in an asynchronous entry process, as revealed by the ability of the virus to remain in a cell-bound state for an extended period of time before it is internalized.


Assuntos
Ebolavirus/fisiologia , Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/virologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Humanos , Cinética , Células Vero , Internalização do Vírus
10.
J Infect Dis ; 212 Suppl 2: S226-33, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26138826

RESUMO

Synthesis of the surface glycoprotein GP of Ebola virus (EBOV) is dependent on transcriptional RNA editing, whereas direct expression of the GP gene results in synthesis of nonstructural secreted glycoprotein sGP. In this study, we investigate the role of RNA editing in the pathogenicity of EBOV using a guinea pig model and recombinant guinea pig-adapted EBOV containing mutations at the editing site, allowing expression of surface GP without the need for RNA editing, and also preventing synthesis of sGP. We demonstrate that the elimination of the editing site leads to EBOV attenuation in vivo, explained by lower virus spread caused by the higher virus cytotoxicity and, most likely, by an increased ability of the host defense systems to recognize and eliminate virus-infected cells. We also demonstrate that expression of sGP does not affect pathogenicity of EBOV in guinea pigs. In conclusion, data obtained indicate that downregulation of the level of surface GP expression through a mechanism of GP gene RNA editing plays an important role in the high pathogenicity of EBOV.


Assuntos
Ebolavirus/genética , Genes Virais/genética , Doença pelo Vírus Ebola/virologia , Edição de RNA/genética , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Fatores de Virulência/genética , Animais , Linhagem Celular , Regulação para Baixo/genética , Ebolavirus/patogenicidade , Regulação Viral da Expressão Gênica/genética , Cobaias , Glicoproteínas de Membrana/genética , Mutação/genética
11.
J Infect Dis ; 212 Suppl 2: S191-8, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25838269

RESUMO

Synthesis of Ebola virus (EBOV) surface glycoprotein (GP) is dependent on transcriptional RNA editing. Northern blot analysis of EBOV-infected cells using GP-gene-specific probes reveals that, in addition to full-length GP messenger RNAs (mRNAs), a shorter RNA is also synthesized, representing >40% of the total amount of GP mRNA. Sequence analysis demonstrates that this RNA is a truncated version of the full-length GP mRNA that is polyadenylated at the editing site and thus lacks a stop codon. An absence of detectable levels of protein synthesis in cellulo is consistent with the existence of tight regulation of the translation of such mRNA. However, nonstop GP mRNA was shown to be only slightly less stable than the same mRNA containing a stop codon, against the general belief in nonstop decay mechanisms aimed at detecting and destroying mRNAs lacking a stop codon. In conclusion, we demonstrate that the editing site indeed serves as a cryptic transcription termination/polyadenylation site, which rarely also functions to edit GP mRNA for expression of surface GP. This new data suggest that the downregulation of surface GP expression is even more dramatic than previously thought, reinforcing the importance of the GP gene editing site for EBOV replication and pathogenicity.


Assuntos
Ebolavirus/genética , Glicoproteínas/genética , Poliadenilação/genética , Edição de RNA/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Códon de Terminação/genética , Regulação para Baixo/genética , Células HEK293 , Humanos , RNA Mensageiro/genética , Células Vero
12.
J Infect Dis ; 212 Suppl 2: S322-8, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26092855

RESUMO

The surface glycoprotein (GP) is responsible for Ebola virus (EBOV) attachment and membrane fusion during virus entry. Surface expression of highly glycosylated GP causes marked cytotoxicity via masking of a wide range of cellular surface molecules, including integrins. Considerable amounts of surface GP are shed from virus-infected cells in a soluble truncated form by tumor necrosis factor α-converting enzyme. In this study, the role of GP shedding was investigated using a reverse genetics approach by comparing recombinant viruses possessing amino acid substitutions at the GP shedding site. Virus with an L635V substitution showed a substantial decrease in shedding, whereas a D637V substitution resulted in a striking increase in the release of shed GP. Variations in shedding efficacy correlated with observed differences in the amounts of shed GP in the medium, GP present in virus-infected cells, and GP present on virions. An increase in shedding appeared to be associated with a reduction in viral cytotoxicity, and, vice versa, the virus that shed less was more cytotoxic. An increase in shedding also resulted in a reduction in viral infectivity, whereas a decrease in shedding efficacy enhanced viral growth characteristics in vitro. Differences in shedding efficacy and, as a result, differences in the amount of mature GP available for incorporation into budding virions did not equate to differences in overall release of viral particles. Likewise, data suggest that the resulting differences in the amount of mature GP on the cell surface led to variations in the GP content of released particles and, as a consequence, in infectivity. In conclusion, fine-tuning of the levels of EBOV GP expressed at the surface of virus-infected cells via GP shedding plays an important role in EBOV replication by orchestrating the balance between optimal virion GP content and cytotoxicity caused by GP.


Assuntos
Ebolavirus/metabolismo , Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/virologia , Glicoproteínas de Membrana/metabolismo , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Ebolavirus/genética , Glicoproteínas de Membrana/genética , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/genética , Vírion/metabolismo , Vírion/patogenicidade , Virulência/genética , Internalização do Vírus , Replicação Viral/genética
13.
J Infect Dis ; 212 Suppl 2: S368-71, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25732811

RESUMO

The current unprecedented outbreak of Ebola virus (EBOV) disease in West Africa has demonstrated the urgent need for a vaccine. Here, we describe the evaluation of an EBOV vaccine candidate based on Kunjin replicon virus-like particles (KUN VLPs) encoding EBOV glycoprotein with a D637L mutation (GP/D637L) in nonhuman primates. Four African green monkeys (Cercopithecus aethiops) were injected subcutaneously with a dose of 10(9) KUN VLPs per animal twice with an interval of 4 weeks, and animals were challenged 3 weeks later intramuscularly with 600 plaque-forming units of Zaire EBOV. Three animals were completely protected against EBOV challenge, while one vaccinated animal and the control animal died from infection. We suggest that KUN VLPs encoding GP/D637L represent a viable EBOV vaccine candidate.


Assuntos
Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Replicon/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vírus do Nilo Ocidental/imunologia , África Ocidental , Animais , Chlorocebus aethiops , Glicoproteínas/imunologia , Imunização/métodos , Primatas , Proteínas Virais/imunologia
14.
Med Sci (Paris) ; 31(2): 143-50, 2015 Feb.
Artigo em Francês | MEDLINE | ID: mdl-25744260

RESUMO

Filoviruses are responsible for highly lethal infections. Those viruses are found in intertropical areas of Africa and Asia where they circulate in their supposed natural reservoir, fruit bats. During filovirus outbreaks and depending on the strains, various modifications in hemostasis have been observed in patients. The disseminated intravascular coagulation identified in these infections is multicausal and involves both viral factors and abnormal physiological responses. In this review we will describe the mechanisms responsible for these disturbances and we will highlight some aspects of the basis of filovirus high pathogenicity.


Assuntos
Coagulação Intravascular Disseminada/etiologia , Infecções por Filoviridae/sangue , Córtex Suprarrenal/patologia , Animais , Quirópteros/virologia , Doenças Transmissíveis Emergentes/sangue , Doenças Transmissíveis Emergentes/complicações , Citocinas/metabolismo , Reservatórios de Doenças , Células Endoteliais/patologia , Filoviridae/fisiologia , Infecções por Filoviridae/epidemiologia , Infecções por Filoviridae/patologia , Infecções por Filoviridae/veterinária , Infecções por Filoviridae/virologia , Haplorrinos , Hepatócitos/patologia , Interações Hospedeiro-Patógeno , Humanos , Necrose , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Tromboplastina/antagonistas & inibidores , Tromboplastina/fisiologia , Carga Viral , Proteínas Virais/fisiologia
15.
Arch Virol ; 159(4): 821-30, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24122154

RESUMO

The International Committee on Taxonomy of Viruses (ICTV) Filoviridae Study Group prepares proposals on the classification and nomenclature of filoviruses to reflect current knowledge or to correct disagreements with the International Code of Virus Classification and Nomenclature (ICVCN). In recent years, filovirus taxonomy has been corrected and updated, but parts of it remain controversial, and several topics remain to be debated. This article summarizes the decisions and discussion of the currently acting ICTV Filoviridae Study Group since its inauguration in January 2012.


Assuntos
Classificação/métodos , Filoviridae/classificação , Terminologia como Assunto , Humanos
16.
Arch Virol ; 159(5): 1229-37, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24190508

RESUMO

Specific alterations (mutations, deletions, insertions) of virus genomes are crucial for the functional characterization of their regulatory elements and their expression products, as well as a prerequisite for the creation of attenuated viruses that could serve as vaccine candidates. Virus genome tailoring can be performed either by using traditionally cloned genomes as starting materials, followed by site-directed mutagenesis, or by de novo synthesis of modified virus genomes or parts thereof. A systematic nomenclature for such recombinant viruses is necessary to set them apart from wild-type and laboratory-adapted viruses, and to improve communication and collaborations among researchers who may want to use recombinant viruses or create novel viruses based on them. A large group of filovirus experts has recently proposed nomenclatures for natural and laboratory animal-adapted filoviruses that aim to simplify the retrieval of sequence data from electronic databases. Here, this work is extended to include nomenclature for filoviruses obtained in the laboratory via reverse genetics systems. The previously developed template for natural filovirus genetic variant naming, (/)///-, is retained, but we propose to adapt the type of information added to each field for cDNA clone-derived filoviruses. For instance, the full-length designation of an Ebola virus Kikwit variant rescued from a plasmid developed at the US Centers for Disease Control and Prevention could be akin to "Ebola virus H.sapiens-rec/COD/1995/Kikwit-abc1" (with the suffix "rec" identifying the recombinant nature of the virus and "abc1" being a placeholder for any meaningful isolate designator). Such a full-length designation should be used in databases and the methods section of publications. Shortened designations (such as "EBOV H.sap/COD/95/Kik-abc1") and abbreviations (such as "EBOV/Kik-abc1") could be used in the remainder of the text, depending on how critical it is to convey information contained in the full-length name. "EBOV" would suffice if only one EBOV strain/variant/isolate is addressed.


Assuntos
Filoviridae/classificação , Filoviridae/genética , Vírus Reordenados/classificação , Vírus Reordenados/genética , Genoma Viral
17.
J Virol ; 86(19): 10766-75, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22837207

RESUMO

Nipah virus (NiV) is a highly pathogenic, negative-strand RNA paramyxovirus that has recently emerged from flying foxes to cause serious human disease. We have analyzed the role of the nonstructural NiV C protein in viral immunopathogenesis using recombinant virus lacking the expression of NiV C (NiVΔC). While wild-type NiV was highly pathogenic in the hamster animal model, NiVΔC was strongly attenuated. Replication of NiVΔC was followed by the production of NiV-specific antibodies and associated with higher recruitment of inflammatory cells and less intensive histopathological lesions in different organs than in wild-type-NiV-infected animals. To analyze the molecular basis of NiVΔC attenuation, we studied early changes in gene expression in infected primary human endothelial cells, a major cellular target of NiV infection. The transcriptomic approach revealed the striking difference between wild-type and mutant NiV in the expression of genes involved in immunity, with the particularly interesting differential patterns of proinflammatory cytokines. Compared to wild-type virus, NiVΔC induced increased expression of interleukin 1 beta (IL-1ß), IL-8, CXCL2, CXCL3, CXCL6, CCL20, and beta interferon. Furthermore, the expression of NiV C in stably transfected cells decreased the production of the same panel of cytokines, revealing a role of the C protein in the regulation of cytokine balance. Together, these results suggest that NiV C regulates expression of proinflammatory cytokines, therefore providing a signal responsible for the coordination of leukocyte recruitment and the chemokine-induced immune response and controlling the lethal outcome of the infection.


Assuntos
Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Proteínas Virais/genética , Proteínas Virais/fisiologia , Animais , Cricetinae , Citocinas/metabolismo , Células Endoteliais/citologia , Células Endoteliais/virologia , Regulação Viral da Expressão Gênica , Células HEK293 , Humanos , Inflamação , Mesocricetus , Microcirculação , Vírus Nipah/metabolismo , Proteínas Recombinantes/química , Fatores de Tempo , Veias Umbilicais/citologia , Virulência
18.
Biomolecules ; 13(3)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36979390

RESUMO

The protein C is a small viral protein encoded in an overlapping frame of the P gene in the subfamily Orthoparamyxovirinae. This protein, expressed by alternative translation initiation, is a virulence factor that regulates viral transcription, replication, and production of defective interfering RNA, interferes with the host-cell innate immunity systems and supports the assembly of viral particles and budding. We expressed and purified full-length and an N-terminally truncated C protein from Tupaia paramyxovirus (TupV) C protein (genus Narmovirus). We solved the crystal structure of the C-terminal part of TupV C protein at a resolution of 2.4 Å and found that it is structurally similar to Sendai virus C protein, suggesting that despite undetectable sequence conservation, these proteins are homologous. We characterized both truncated and full-length proteins by SEC-MALLS and SEC-SAXS and described their solution structures by ensemble models. We established a mini-replicon assay for the related Nipah virus (NiV) and showed that TupV C inhibited the expression of NiV minigenome in a concentration-dependent manner as efficiently as the NiV C protein. A previous study found that the Orthoparamyxovirinae C proteins form two clusters without detectable sequence similarity, raising the question of whether they were homologous or instead had originated independently. Since TupV C and SeV C are representatives of these two clusters, our discovery that they have a similar structure indicates that all Orthoparamyxovirine C proteins are homologous. Our results also imply that, strikingly, a STAT1-binding site is encoded by exactly the same RNA region of the P/C gene across Paramyxovirinae, but in different reading frames (P or C), depending on which cluster they belong to.


Assuntos
Vírus Nipah , Espalhamento a Baixo Ângulo , Difração de Raios X , Vírus Nipah/genética , Vírus Nipah/metabolismo , Imunidade Inata , RNA/metabolismo
19.
J Virol ; 85(17): 8502-13, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21697477

RESUMO

With the exception of Reston and Lloviu viruses, filoviruses (marburgviruses, ebolaviruses, and "cuevaviruses") cause severe viral hemorrhagic fevers in humans. Filoviruses use a class I fusion protein, GP(1,2), to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP(1,2), ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP). All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini. We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP(1,2)-mediated cell entry of pseudotyped retroviruses. Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP(1,2), as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs. Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP(1,2)-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect. These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.


Assuntos
Antivirais/metabolismo , Ebolavirus/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/metabolismo , Marburgvirus/efeitos dos fármacos , Proteínas Virais/metabolismo , Internalização do Vírus/efeitos dos fármacos , Animais , Produtos Biológicos/metabolismo , Linhagem Celular , Ebolavirus/fisiologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Marburgvirus/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/genética
20.
Nat Med ; 11(7): 786-90, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15937495

RESUMO

Vaccines and therapies are urgently needed to address public health needs stemming from emerging pathogens and biological threat agents such as the filoviruses Ebola virus (EBOV) and Marburg virus (MARV). Here, we developed replication-competent vaccines against EBOV and MARV based on attenuated recombinant vesicular stomatitis virus vectors expressing either the EBOV glycoprotein or MARV glycoprotein. A single intramuscular injection of the EBOV or MARV vaccine elicited completely protective immune responses in nonhuman primates against lethal EBOV or MARV challenges. Notably, vaccine vector shedding was not detectable in the monkeys and none of the animals developed fever or other symptoms of illness associated with vaccination. The EBOV vaccine induced humoral and apparent cellular immune responses in all vaccinated monkeys, whereas the MARV vaccine induced a stronger humoral than cellular immune response. No evidence of EBOV or MARV replication was detected in any of the protected animals after challenge. Our data suggest that these vaccine candidates are safe and highly efficacious in a relevant animal model.


Assuntos
Ebolavirus/imunologia , Marburgvirus/imunologia , Vacinas Atenuadas/imunologia , Vacinas Combinadas/imunologia , Vacinas Virais/imunologia , Animais , Formação de Anticorpos , Reações Cruzadas , Vacinas contra Ebola/imunologia , Vacinas contra Ebola/farmacologia , Primatas , Vacinas Atenuadas/genética , Vacinas Atenuadas/farmacologia , Vacinas Combinadas/genética , Vacinas Combinadas/farmacologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/farmacologia , Vírus da Estomatite Vesicular Indiana/genética , Vacinas Virais/genética , Vacinas Virais/farmacologia , Viremia/imunologia , Viremia/virologia , Replicação Viral
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