RESUMEN
The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.
Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Complejo Antígeno-Anticuerpo/ultraestructura , Enfermedad del Virus de Marburg/inmunología , Marburgvirus/química , Proteínas del Envoltorio Viral/química , Adulto , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Linfocitos B/inmunología , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Marburgvirus/genética , Marburgvirus/inmunología , Modelos Moleculares , Mutación , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of their NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. These structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry.
Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Marburgvirus/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Complejo Antígeno-Anticuerpo/química , Línea Celular , Reacciones Cruzadas , Cristalografía por Rayos X , Drosophila , Ebolavirus/química , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Enfermedad del Virus de Marburg/inmunología , Marburgvirus/genética , Marburgvirus/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Mucinas/química , Alineación de Secuencia , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Ebola (EBOV) and Marburg viruses (MARV) cause severe hemorrhagic fever associated with high mortality rates in humans. A better understanding of filovirus-host interactions that regulate the EBOV and MARV lifecycles can provide biological and mechanistic insight critical for therapeutic development. EBOV glycoprotein (eGP) and MARV glycoprotein (mGP) mediate entry into host cells primarily by actin-dependent macropinocytosis. Here, we identified actin-binding cytoskeletal crosslinking proteins filamin A (FLNa) and B (FLNb) as important regulators of both EBOV and MARV entry. We found that entry of pseudotype psVSV-RFP-eGP, infectious recombinant rVSV-eGP-mCherry, and live authentic EBOV and MARV was inhibited in filamin A knockdown (FLNaKD) cells, but was surprisingly enhanced in filamin B knockdown (FLNbKD) cells. Mechanistically, our findings suggest that differential regulation of macropinocytosis by FLNa and FLNb likely contributes to their specific effects on EBOV and MARV entry. This study is the first to identify the filamin family of proteins as regulators of EBOV and MARV entry. These findings may provide insight into the development of new countermeasures to prevent EBOV and MARV infections.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Marburgvirus , Humanos , Filaminas/genética , Ebolavirus/genética , Actinas , Marburgvirus/genética , GlicoproteínasRESUMEN
Filovirus-infected cells are characterized by typical cytoplasmic inclusion bodies (IBs) located in the perinuclear region. The formation of these IBs is induced mainly by the accumulation of the filoviral nucleoprotein NP, which recruits the other nucleocapsid proteins, the polymerase co-factor VP35, the polymerase L, the transcription factor VP30 and VP24 via direct or indirect protein-protein interactions. Replication of the negative-strand RNA genomes by the viral polymerase L and VP35 occurs in the IBs, resulting in the synthesis of positive-strand genomes, which are encapsidated by NP, thus forming ribonucleoprotein complexes (antigenomic RNPs). These newly formed antigenomic RNPs in turn serve as templates for the synthesis of negative-strand RNA genomes that are also encapsidated by NP (genomic RNPs). Still in the IBs, genomic RNPs mature into tightly packed transport-competent nucleocapsids (NCs) by the recruitment of the viral protein VP24. NCs are tightly coiled left-handed helices whose structure is mainly determined by the multimerization of NP at its N-terminus, and these helices form the inner layer of the NCs. The RNA genome is fixed by 2 lobes of the NP N-terminus and is thus guided by individual NP molecules along the turns of the helix. Direct interaction of the NP C-terminus with the VP35 and VP24 molecules forms the outer layer of the NCs. Once formed, NCs that are located at the border of the IBs recruit actin polymerization machinery to one of their ends to drive their transport to budding sites for their envelopment and final release. Here, we review the current knowledge on the structure, assembly, and transport of filovirus NCs.
Asunto(s)
Ebolavirus , Cuerpos de Inclusión Viral , Marburgvirus , Humanos , Ebolavirus/genética , Marburgvirus/genética , Nucleocápside/metabolismo , Ribonucleoproteínas/metabolismo , ARN/metabolismoRESUMEN
Marburg virus (MARV) infection results in severe viral hemorrhagic fever with mortalities up to 90%, and there is a pressing need for effective therapies. Here, we established a small interfering RNA (siRNA) conjugate platform that enabled successful subcutaneous delivery of siRNAs targeting the MARV nucleoprotein. We identified a hexavalent mannose ligand with high affinity to macrophages and dendritic cells, which are key cellular targets of MARV infection. This ligand enabled successful siRNA conjugate delivery to macrophages both in vitro and in vivo. The delivered hexa-mannose-siRNA conjugates rendered substantial target gene silencing in macrophages when supported by a mannose functionalized endosome release polymer. This hexa-mannose-siRNA conjugate was further evaluated alongside our hepatocyte-targeting GalNAc-siRNA conjugate, to expand targeting of infected liver cells. In MARV-Angola-infected guinea pigs, these platforms offered limited survival benefit when used as individual agents. However, in combination, they achieved up to 100% protection when dosed 24 h post infection. This novel approach, using two different ligands to simultaneously deliver siRNA to multiple cell types relevant to infection, provides a convenient subcutaneous route of administration for treating infection by these dangerous pathogens. The mannose conjugate platform has potential application to other diseases involving macrophages and dendritic cells.
Asunto(s)
Enfermedad del Virus de Marburg , Marburgvirus , Virosis , Animales , Cobayas , ARN Interferente Pequeño/genética , Manosa , Ligandos , ARN Bicatenario , Marburgvirus/genética , Enfermedad del Virus de Marburg/metabolismo , Enfermedad del Virus de Marburg/prevención & controlRESUMEN
Filoviruses encode viral protein 24 (VP24) which effectively inhibit the innate immune responses in infected cells. Here we systematically analysed the effects of nine mammalian filovirus VP24 proteins on interferon (IFN)-induced immune response. We transiently expressed Ebola, Bombali, Bundibugyo, Reston, Sudan and Taï Forest ebolavirus (EBOV, BOMV, BDBV, RESTV, SUDV, TAFV, respectively), Lloviu virus (LLOV), Mengla dianlovirus (MLAV) and Marburgvirus (MARV) VP24 proteins and analysed their ability to inhibit IFN-α-induced activation of myxovirus resistance protein 1 (MxA) and interferon-induced transmembrane protein 3 (IFITM3) promoters. In addition, we analysed the expression of endogenous MxA protein in filovirus VP24-expressing cells. Eight filovirus VP24 proteins, including the VP24s of the recently discovered MLAV, BOMV and LLOV, inhibited IFN-induced MxA and IFITM3 promoter activation. MARV VP24 was the only protein with no inhibitory effect on the activation of either promoter. Endogenous MxA protein expression was impaired in cells transiently expressing VP24s with the exception of MARV VP24. We mutated nuclear localization signal (NLS) of two highly pathogenic filoviruses (EBOV and SUDV) and two putatively non-pathogenic filoviruses (BOMV and RESTV), and showed that the inhibitory effect on IFN-induced expression of MxA was dependent on functional cluster 3 of VP24 nuclear localization signal. Our findings suggest that filovirus VP24 proteins are both genetically and functionally conserved, and that VP24 proteins of most filovirus species are capable of inhibiting IFN-induced antiviral gene expression thereby efficiently downregulating the host innate immune responses.
Asunto(s)
Ebolavirus , Marburgvirus , Animales , Señales de Localización Nuclear , Inmunidad Innata , Interferón-alfa , Antivirales , Marburgvirus/genética , Proteínas de la Matriz Viral , MamíferosRESUMEN
Marburg virus (MARV) and Ebola virus (EBOV) of the Filoviridae family are the most lethal viruses in terms of mortality rate. However, the development of antiviral treatment is hampered by the requirement for biosafety level-4 (BSL-4) containment. The establishment of BSL-2 pseudotyped viruses can provide important tools for the study of filoviruses. This chapter summarizes general information on the filoviruses and then focuses on the construction of replication-deficient pseudotyped MARV and EBOV (e.g., lentivirus system and vesicular stomatitis virus system). It also details the potential applications of the pseudotyped viruses, including neutralization antibody detection, the study of infection mechanisms, the evaluation of antibody-dependent enhancement, virus entry inhibitor screening, and glycoprotein mutation analysis.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Marburgvirus , Humanos , Ebolavirus/genética , Marburgvirus/genética , Pseudotipado Viral , Antivirales/farmacología , Glicoproteínas , Fiebre Hemorrágica Ebola/prevención & controlRESUMEN
The filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5' and 3' untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3' UTRs are prone to editing by adenosine deaminase acting on RNA 1 (ADAR1). A reporter mRNA approach, in which different 5' or 3' UTRs were inserted into luciferase-encoding mRNAs, demonstrates that MARV 3' UTRs yield different levels of reporter gene expression, suggesting modulation of translation. The modulation occurs in cells unable to produce microRNAs (miRNAs) and can be recapitulated in a MARV minigenome assay. Deletion mutants identified negative regulatory regions at the ends of the MARV nucleoprotein (NP) and large protein (L) 3' UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3' UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3' UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3' UTR, but not the edited or deletion mutant NP 3' UTRs, induces a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the viral protein of 40 kDa (VP40) 3' UTR, VP40 3' UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3' UTR edits also enhanced translation, but neither the wild-type nor the edited 3' UTRs induced IFN. These findings implicate filoviral mRNA 3' UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1. IMPORTANCE UTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and ADAR1 editing has been uncertain. This study demonstrates that MARV and EBOV 3' UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3' UTRs relieves the negative effects of the UTRs. These data indicate that filovirus 3' UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity.
Asunto(s)
Regiones no Traducidas 3' , Adenosina Desaminasa/metabolismo , Ebolavirus/genética , Marburgvirus/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular , Ebolavirus/metabolismo , Genes Reporteros , Humanos , Interferón Tipo I/biosíntesis , Marburgvirus/metabolismo , MicroARNs/genética , Mutación , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Polirribosomas/metabolismo , Edición de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
The ability of viruses in the Filoviridae family (Ebola virus [EBOV] and Marburg virus [MARV]) to cause severe human disease and their pandemic potential makes all emerging filoviral pathogens a concern to humanity. Menglà virus (MLAV) belonging to the new genus Dianlovirus was recently discovered in the liver of bats from Menglà County, Yunnan Province, China. The capacity of MLAV to utilize NPC1 as an endosomal receptor, to transduce mammalian cells, and suppress IFN response suggests that this potential pathogen could cause human illness. Despite great effort by researchers, only the viral genome has been recovered and isolation of live MLAV had been unsuccessful. Here using a pseudovirus model baring the MLAV glycoprotein (GP), we studied the protease dependence of the MLAV-GP, and the ability of small molecules and antibodies to inhibit MLAV viral entry. Like EBOV and MARV, the MLAV-GP requires proteolytic processing but like MARV it does not depend on cathepsin B activity for viral entry. Furthermore, previously discovered small-molecule inhibitors and antibodies are MLAV inhibitors and show the possibility of developing these inhibitors as broad-spectrum filovirus antivirals. Overall, the findings in the study confirmed that MLAV viral entry is biologically distinct but has similarities to MARV.
Asunto(s)
Ebolavirus , Marburgvirus , Animales , China , Ebolavirus/genética , Glicoproteínas/genética , Humanos , Mamíferos , Marburgvirus/genética , Internalización del VirusRESUMEN
The question as to whether RNA viruses produce bona fide microRNAs (miRNAs) during infection has been the focus of intense research and debate. Recently, several groups using computational prediction methods have independently reported possible miRNA candidates produced by Ebola virus (EBOV). Additionally, efforts to detect these predicted RNA products in samples from infected animals and humans have produced positive results. However, these studies and their conclusions are predicated on the assumption that these RNA products are actually processed through, and function within, the miRNA pathway. In the present study, we performed the first rigorous assessment of the ability of filoviruses to produce miRNA products during infection of both human and bat cells. Using next-generation sequencing, we detected several candidate miRNAs from both EBOV and the closely related Marburg virus (MARV). Focusing our validation efforts on EBOV, we found evidence contrary to the idea that these small RNA products function as miRNAs. The results of our study are important because they highlight the potential pitfalls of relying on computational methods alone for virus miRNA discovery.IMPORTANCE Here, we report the discovery, via deep sequencing, of numerous noncoding RNAs (ncRNAs) derived from both EBOV and MARV during infection of both bat and human cell lines. In addition to identifying several novel ncRNAs from both viruses, we identified two EBOV ncRNAs in our sequencing data that were near-matches to computationally predicted viral miRNAs reported in the literature. Using molecular and immunological techniques, we assessed the potential of EBOV ncRNAs to function as viral miRNAs. Importantly, we found little evidence supporting this hypothesis. Our work is significant because it represents the first rigorous assessment of the potential for EBOV to encode viral miRNAs and provides evidence contrary to the existing paradigm regarding the biological role of computationally predicted EBOV ncRNAs. Moreover, our work highlights further avenues of research regarding the nature and function of EBOV ncRNAs.
Asunto(s)
Ebolavirus/metabolismo , MicroARNs/metabolismo , Interferencia de ARN , ARN Viral/metabolismo , Animales , Línea Celular , Quirópteros , Ebolavirus/genética , Humanos , Marburgvirus/genética , Marburgvirus/metabolismo , MicroARNs/genética , ARN Viral/genéticaRESUMEN
Interaction between filovirus glycoprotein (GP) and the Niemann-Pick C1 (NPC1) protein is essential for membrane fusion during virus entry. Some single-nucleotide polymorphism (SNPs) in two surface-exposed loops of NPC1 are known to reduce viral infectivity. However, the dependence of differences in entry efficiency on SNPs remains unclear. Using vesicular stomatitis virus pseudotyped with Ebola and Marburg virus GPs, we investigated the cell-to-cell spread of viruses in cultured cells expressing NPC1 or SNP derivatives. Eclipse and virus-producing phases were assessed by in vitro infection experiments, and we developed a mathematical model describing spatial-temporal virus spread. This mathematical model fit the plaque radius data well from day 2 to day 6. Based on the estimated parameters, we found that SNPs causing the P424A and D508N substitutions in NPC1 most effectively reduced the entry efficiency of Ebola and Marburg viruses, respectively. Our novel approach could be broadly applied to other virus plaque assays.
Asunto(s)
Ebolavirus/fisiología , Marburgvirus/fisiología , Modelos Biológicos , Internalización del Virus , Línea Celular , Ebolavirus/genética , Ebolavirus/patogenicidad , Humanos , Marburgvirus/genética , Marburgvirus/patogenicidad , Mutación , Polimorfismo de Nucleótido Simple , Ensayo de Placa ViralRESUMEN
BACKGROUND: Marburg virus (MARV) and Ebola virus (EBOV) are acute infections with high case fatality rates. It is of great significance for epidemic monitoring and prevention and control of infectious diseases by the development of a rapid, specific, and sensitive quantitative PCR method to detect two pathogens simultaneously. METHODS: Primers and TaqMan probes were designed according to highly conserved sequences of these viruses. Sensitivity, specificity, linear range, limit of detection, and the effects of hemolysis and lipid on real-time qPCR were evaluated. RESULTS: The linearity of the curve allowed quantification of nucleic acid concentrations in range from 103 to 109 copies/ml per reaction (MARV and EBOV). The limit of detection of EBOV was 40 copies/ml, and MARV was 100 copies/ml. It has no cross-reaction with other pathogens such as hepatitis b virus (HBV), hepatitis c virus (HCV), human papillomavirus (HPV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), cytomegalovirus (CMV), and human immunodeficiency virus (HIV). Repeatability analysis of the two viruses showed that their coefficient of variation (CV) was less than 5.0%. The above results indicated that fluorescence quantitative PCR could detect EBOV and MARV sensitively and specifically. CONCLUSIONS: The TaqMan probe-based multiplex fluorescence quantitative PCR assays could detect EBOV and MARV sensitively specifically and simultaneously.
Asunto(s)
Ebolavirus/genética , Fiebre Hemorrágica Ebola/diagnóstico , Enfermedad del Virus de Marburg/diagnóstico , Marburgvirus/genética , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Fiebre Hemorrágica Ebola/virología , Humanos , Enfermedad del Virus de Marburg/virología , Curva ROCRESUMEN
We detected Marburg virus RNA in rectal swab samples from Egyptian rousette bats in South Africa in 2017. This finding signifies that fecal contamination of natural bat habitats is a potential source of infection for humans. Identified genetic sequences are closely related to Ravn virus, implying wider distribution of Marburg virus in Africa.
Asunto(s)
Quirópteros , Enfermedad del Virus de Marburg , Marburgvirus , Animales , Humanos , Enfermedad del Virus de Marburg/epidemiología , Marburgvirus/genética , Sudáfrica/epidemiologíaRESUMEN
We detected Marburg virus genome in Egyptian fruit bats (Rousettus aegyptiacus) captured in Zambia in September 2018. The virus was closely related phylogenetically to the viruses that previously caused Marburg outbreaks in the Democratic Republic of the Congo. This finding demonstrates that Zambia is at risk for Marburg virus disease.
Asunto(s)
Quirópteros/virología , Enfermedad del Virus de Marburg/virología , Marburgvirus , Animales , Genes Virales , Humanos , Enfermedad del Virus de Marburg/diagnóstico , Enfermedad del Virus de Marburg/epidemiología , Marburgvirus/clasificación , Marburgvirus/genética , Marburgvirus/aislamiento & purificación , Filogenia , Prevalencia , Vigilancia en Salud Pública , ARN Viral , Zambia/epidemiologíaRESUMEN
The filoviruses Marburg virus (MARV) and Ebola virus (EBOV) cause hemorrhagic fever in humans and nonhuman primates, with high case fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here, we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling the full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions, mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation of a cluster of amino acids at positions 46 through 53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, coimmunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule, 1E7-03, targeting a noncatalytic site of the enzyme that previously was shown to increase EBOV VP30 phosphorylation was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at a cluster of phosphorylated amino acids from Ser-46 to Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35, and dramatically reduced replication of infectious MARV particles. Thus, MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds. IMPORTANCE The largest outbreak of MARV occurred in Angola in 2004 to 2005 and had a 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires a fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member of Filoviridae family, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of the MARV genome demonstrated differences from those of EBOV. The identified phosphorylation sites appeared to inhibit transcription and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of the MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility developing antivirals based on compounds targeting PP1.
Asunto(s)
Marburgvirus/crecimiento & desarrollo , Nucleoproteínas/metabolismo , Proteínas Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación Viral/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Genoma Viral/genética , Células HEK293 , Humanos , Indoles/farmacología , Marburgvirus/genética , Espectrometría de Masas , Fosforilación , ARN Viral/genética , Transcripción Genética/genética , Urea/análogos & derivados , Urea/farmacología , Células Vero , Proteínas Virales/genéticaRESUMEN
Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Autofagia/fisiología , Ebolavirus/metabolismo , Fiebre Hemorrágica Ebola/transmisión , Enfermedad del Virus de Marburg/transmisión , Marburgvirus/metabolismo , Proteínas de la Matriz Viral/metabolismo , Liberación del Virus/genética , Animales , Autofagia/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Cricetinae , Ebolavirus/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte , Células HEK293 , Células HeLa , Fiebre Hemorrágica Ebola/patología , Fiebre Hemorrágica Ebola/virología , Humanos , Enfermedad del Virus de Marburg/patología , Enfermedad del Virus de Marburg/virología , Marburgvirus/genética , Ubiquitina-Proteína Ligasas Nedd4 , Prolina/análogos & derivados , Prolina/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers.