RESUMEN
The development of virus-like particle (VLP) based vaccines for human papillomavirus, hepatitis B and hepatitis E viruses represented a breakthrough in vaccine development. However, for dengue and COVID-19, technical complications, such as an incomplete understanding of the requirements for protective immunity, but also limitations in processes to manufacture VLP vaccines for enveloped viruses to large scale, have hampered VLP vaccine development. Selecting the right adjuvant is also an important consideration to ensure that a VLP vaccine induces protective antibody and T cell responses. For diseases like COVID-19 and dengue fever caused by RNA viruses that exist as families of viral variants with the potential to escape vaccine-induced immunity, the development of more efficacious vaccines is also necessary. Here, we describe the development and characterisation of novel VLP vaccine candidates using SARS-CoV-2 and dengue virus (DENV), containing the major viral structural proteins, as protypes for a novel approach to produce VLP vaccines. The VLPs were characterised by Western immunoblot, enzyme immunoassay, electron and atomic force microscopy, and in vitro and in vivo immunogenicity studies. Microscopy techniques showed proteins self-assemble to form VLPs authentic to native viruses. The inclusion of the glycolipid adjuvant, α-galactosylceramide (α-GalCer) in the vaccine formulation led to high levels of natural killer T (NKT) cell stimulation in vitro, and strong antibody and memory CD8+ T cell responses in vivo, demonstrated with SARS-CoV-2, hepatitis C virus (HCV) and DEN VLPs. This study shows our unique vaccine formulation presents a promising, and much needed, new vaccine platform in the fight against infections caused by enveloped RNA viruses.
RESUMEN
Our understanding of the immune responses that follow SARS-CoV-2 infection and vaccination has progressed considerably since the COVID-19 pandemic was first declared on the 11th of March in 2020. Recovery from infection is associated with the development of protective immune responses, although over time these become less effective against new emerging SARS-CoV-2 variants. Consequently, reinfection with SARS-CoV-2 variants is not infrequent and has contributed to the ongoing pandemic. COVID-19 vaccines have had a tremendous impact on reducing infection and particularly the number of deaths associated with SARS-CoV-2 infection. However, waning of vaccine induced immunity plus the emergence of new variants has necessitated the use of boosters to maintain the benefits of vaccination in reducing COVID-19 associated deaths. Boosting is also beneficial for individuals who have recovered from COVID-19 and developed natural immunity, also enhancing responses immune responses to SARS-CoV-2 variants. This review summarizes our understanding of the immune responses that follow SARS-CoV-2 infection and vaccination, the risks of reinfection with emerging variants and the very important protective role vaccine boosting plays in both vaccinated and previously infected individuals.
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COVID-19 , Vacunas Virales , COVID-19/prevención & control , Vacunas contra la COVID-19 , Humanos , Inmunidad , Pandemias , ARN Viral , Reinfección/prevención & control , SARS-CoV-2RESUMEN
Dengue virus (DENV) and West Nile virus (WNV) are arthropod-transmitted flaviviruses that cause systemic vascular leakage and encephalitis syndromes, respectively, in humans. However, the viral factors contributing to these specific clinical disorders are not completely understood. Flavivirus nonstructural protein 1 (NS1) is required for replication, expressed on the cell surface, and secreted as a soluble glycoprotein, reaching high levels in the blood of infected individuals. Extracellular DENV NS1 and WNV NS1 interact with host proteins and cells, have immune evasion functions, and promote endothelial dysfunction in a tissue-specific manner. To characterize how differences in DENV NS1 and WNV NS1 might function in pathogenesis, we generated WNV NS1 variants with substitutions corresponding to residues found in DENV NS1. We discovered that the substitution NS1-P101K led to reduced WNV infectivity in the brain and attenuated lethality in infected mice, although the virus replicated efficiently in cell culture and peripheral organs and bound at wild-type levels to brain endothelial cells and complement components. The P101K substitution resulted in reduced NS1 antigenemia in mice, and this was associated with reduced WNV spread to the brain. Because exogenous administration of NS1 protein rescued WNV brain infectivity in mice, we conclude that circulating WNV NS1 facilitates viral dissemination into the central nervous system and impacts disease outcomes. IMPORTANCE Flavivirus NS1 serves as an essential scaffolding molecule during virus replication but also is expressed on the cell surface and is secreted as a soluble glycoprotein that circulates in the blood of infected individuals. Although extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, it has been challenging to study specific effects of NS1 on pathogenesis without disrupting its key role in virus replication. Here, we assessed WNV NS1 variants that do not affect virus replication and evaluated their effects on pathogenesis in mice. Our characterization of WNV NS1-P101K suggests that the levels of NS1 in the circulation facilitate WNV dissemination to the brain and affect disease outcomes. Our findings facilitate understanding of the role of NS1 during flavivirus infection and support antiviral strategies for targeting circulating forms of NS1.
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Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/virología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/inmunología , Virus del Dengue/metabolismo , Células Endoteliales , Femenino , Flavivirus/patogenicidad , Evasión Inmune , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas no Estructurales Virales/análisis , Proteínas no Estructurales Virales/sangre , Proteínas no Estructurales Virales/genética , Replicación Viral/genética , Replicación Viral/fisiología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/efectos de los fármacos , Virus del Nilo Occidental/inmunologíaRESUMEN
There are no licensed therapeutics or vaccines available against Zika virus (ZIKV) to counteract its potential for congenital disease. Antibody-based countermeasures targeting the ZIKV envelope protein have been hampered by concerns for cross-reactive responses that induce antibody-dependent enhancement (ADE) of heterologous flavivirus infection. Nonstructural protein 1 (NS1) is a membrane-associated and secreted glycoprotein that functions in flavivirus replication and immune evasion but is absent from the virion. Although some studies suggest that antibodies against ZIKV NS1 are protective, their activity during congenital infection is unknown. Here we develop mouse and human anti-NS1 monoclonal antibodies that protect against ZIKV in both non-pregnant and pregnant mice. Avidity of antibody binding to cell-surface NS1 along with Fc effector functions engagement correlate with protection in vivo. Protective mAbs map to exposed epitopes in the wing domain and loop face of the ß-platform. Anti-NS1 antibodies provide an alternative strategy for protection against congenital ZIKV infection without causing ADE.
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Anticuerpos Antivirales/administración & dosificación , Complicaciones Infecciosas del Embarazo/prevención & control , Proteínas no Estructurales Virales/inmunología , Infección por el Virus Zika/prevención & control , Virus Zika/inmunología , Animales , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos , Acrecentamiento Dependiente de Anticuerpo , Reacciones Cruzadas , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Embarazo , Complicaciones Infecciosas del Embarazo/inmunología , Complicaciones Infecciosas del Embarazo/virología , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Virus Zika/química , Virus Zika/genética , Infección por el Virus Zika/congénito , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virologíaRESUMEN
Chikungunya virus (CHIKV) is an emerging mosquito-borne virus that has caused explosive outbreaks worldwide. Although neutralizing monoclonal antibodies (mAbs) against CHIKV inhibit infection in animals, the contribution of Fc effector functions to protection remains unknown. Here, we evaluated the activity of therapeutic mAbs that had or lacked the ability to engage complement and Fcγ receptors (FcγR). When administered as post-exposure therapy in mice, the Fc effector functions of mAbs promoted virus clearance from infected cells and reduced joint swelling-results that were corroborated in antibody-treated transgenic animals lacking activating FcγR. The control of CHIKV infection by antibody-FcγR engagement was associated with an accelerated influx of monocytes. A series of immune cell depletions revealed that therapeutic mAbs required monocytes for efficient clearance of CHIKV infection. Overall, our study suggests that in mice, FcγR expression on monocytes is required for optimal therapeutic activity of antibodies against CHIKV and likely other related viruses.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Artritis Experimental/terapia , Virus Chikungunya/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Factores Inmunológicos/uso terapéutico , Monocitos/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos Neutralizantes/uso terapéutico , Artritis Experimental/virología , Fiebre Chikungunya/virología , Activación de Complemento/inmunología , Complemento C1q/inmunología , Modelos Animales de Enfermedad , Epítopos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor de Interferón alfa y beta/genéticaRESUMEN
Japanese encephalitis virus (JEV) remains a leading cause of viral encephalitis worldwide. Although JEV-specific antibodies have been described, an assessment of their ability to neutralize multiple genotypes of JEV has been limited. Here, we describe the development of a panel of mouse and human neutralizing monoclonal antibodies (MAbs) that inhibit infection in cell culture of four different JEV genotypes tested. Mechanism-of-action studies showed that many of these MAbs inhibited infection at a postattachment step, including blockade of virus fusion. Mapping studies using site-directed mutagenesis and hydrogen-deuterium exchange with mass spectrometry revealed that the lateral ridge on domain III of the envelope protein was a primary recognition epitope for our panel of strongly neutralizing MAbs. Therapeutic studies in mice demonstrated protection against lethality caused by genotype I and III strains when MAbs were administered as a single dose even 5 days after infection. This information may inform the development of vaccines and therapeutic antibodies as emerging strains and genotypic shifts become more prevalent.IMPORTANCE Although Japanese encephalitis virus (JEV) is a vaccine-preventable cause of viral encephalitis, the inactivated and live attenuated platforms available are derived from strains belonging to a single genotype (GIII) due to its historical prevalence in areas of JEV epidemics. Related to this, studies with vaccines and antibodies have focused on assessing the in vitro and in vivo protective responses to homologous or heterologous GIII strains. An epidemiological shift in JEV genotype distribution warrants the induction of broadly neutralizing antibody responses that inhibit infection of multiple JEV genotypes. Here, we generated a panel of mouse and human neutralizing monoclonal antibodies and evaluated their inhibitory activity, epitope location, and capacity for protection against multiple JEV genotypes in mice.
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Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Antivirales/administración & dosificación , Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Japonesa/prevención & control , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Virus de la Encefalitis Japonesa (Especie)/clasificación , Virus de la Encefalitis Japonesa (Especie)/genética , Epítopos/inmunología , Genotipo , Humanos , Ratones , Modelos Biológicos , Resultado del Tratamiento , Células Vero , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRß signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion.
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Neoplasias Encefálicas/inmunología , Puntos de Control del Ciclo Celular , Glioblastoma/inmunología , Células Asesinas Naturales/inmunología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Neoplasias Encefálicas/patología , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Femenino , Glioblastoma/patología , Humanos , Inmunidad Innata , Interferón gamma/metabolismo , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor 2 Gatillante de la Citotoxidad Natural/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.
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Infecciones por Alphavirus/inmunología , Alphavirus/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Epítopos , Proteínas del Envoltorio Viral/inmunología , Alphavirus/clasificación , Alphavirus/metabolismo , Infecciones por Alphavirus/prevención & control , Infecciones por Alphavirus/terapia , Secuencia de Aminoácidos , Animales , Virus Chikungunya/química , Virus Chikungunya/inmunología , Microscopía por Crioelectrón , Glicoproteínas/química , Glicoproteínas/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Proteínas del Envoltorio Viral/química , Vacunas Virales/inmunología , Internalización del VirusRESUMEN
We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1) infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII) of DENV-1 envelope (E) protein to 2.45 Å resolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a â¼20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Virus del Dengue , Dengue , Inmunoglobulina G , Proteínas del Envoltorio Viral , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/farmacología , Afinidad de Anticuerpos , Dengue/tratamiento farmacológico , Dengue/inmunología , Virus del Dengue/química , Virus del Dengue/genética , Virus del Dengue/inmunología , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Ratones , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Virión/química , Virión/genética , Virión/inmunologíaRESUMEN
The Flavivirus nonstructural protein 1 (NS1) is a conserved, membrane-associated and secreted glycoprotein with replication and immune evasion functions. Secreted NS1 is a hexameric, barrel-shaped lipoprotein that can bind back to the plasma membrane of cells. Antibodies targeting cell surface-associated NS1 can be protective in vivo in a manner dependent on Fc effector functions. We describe here the crystal structure of a C-terminal fragment (residues 172-352) of West Nile (WNV) and Dengue virus NS1 proteins at 1.85 and 2.7 Å resolution, respectively. NS1(172-352) assembles as a unique rod-shaped dimer composed of a 16-stranded ß-platform flanked on one face by protruding connecting loops. We also determined the 3.0 Å resolution structure of WNV NS1(172-352) with the protective 22NS1 antibody Fab, which engages the loop-face of the rod. The head-to-head NS1(172-352) dimer we observe in crystal lattices is supported by multiangle light and small-angle X-ray scattering studies. We used the available cryo-electron microscopy reconstruction to develop a pseudoatomic model of the NS1 hexamer. The model was constructed with the NS1(172-352) dimeric rod aligned with the long axis of the barrel, and with the loop-face oriented away from the core. Difference densities suggest that the N-terminal region of NS1 forms globular lobes that mediate lateral contacts between dimers in the hexamer. Our model also suggests that the N-terminal lobe forms the surface of the central cavity where lipid binding may occur.
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Anticuerpos Antivirales/química , Virus del Dengue/genética , Modelos Moleculares , Conformación Proteica , Proteínas no Estructurales Virales/química , Virus del Nilo Occidental/genética , Secuencia de Aminoácidos , Anticuerpos Antivirales/inmunología , Microscopía por Crioelectrón , Cristalización , Virus del Dengue/inmunología , Dimerización , Regulación Viral de la Expresión Génica/inmunología , Datos de Secuencia Molecular , Alineación de Secuencia , Proteínas no Estructurales Virales/inmunología , Virus del Nilo Occidental/inmunologíaRESUMEN
Flavivirus-infected cells secrete a structurally heterogeneous population of viruses because of an inefficient virion maturation process. Flaviviruses assemble as noninfectious, immature virions composed of trimers of envelope (E) and precursor membrane (prM) protein heterodimers. Cleavage of prM is a required process during virion maturation, although this often remains incomplete for infectious virus particles. Previous work demonstrated that the efficiency of virion maturation could impact antibody neutralization through changes in the accessibility of otherwise cryptic epitopes on the virion. In this study, we show that the neutralization potency of monoclonal antibody (MAb) E33 is sensitive to the maturation state of West Nile virus (WNV), despite its recognition of an accessible epitope, the domain III lateral ridge (DIII-LR). Comprehensive epitope mapping studies with 166 E protein DIII-LR variants revealed that the functional footprint of MAb E33 on the E protein differs subtly from that of the well-characterized DIII-LR MAb E16. Remarkably, aromatic substitutions at E protein residue 306 ablated the maturation state sensitivity of E33 IgG, and the neutralization efficacy of E33 Fab fragments was not affected by changes in the virion maturation state. We propose that E33 IgG binding on mature virions orients the Fc region in a manner that impacts subsequent antibody binding to nearby sites. This Fc-mediated steric constraint is a novel mechanism by which the maturation state of a virion modulates the efficacy of the humoral immune response to flavivirus infection.
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Anticuerpos Antivirales/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Mapeo Epitopo , Células HEK293 , Humanos , Pruebas de Neutralización , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/química , Virus del Nilo Occidental/genéticaRESUMEN
Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar(-/-) ) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans.
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Infecciones por Alphavirus/prevención & control , Infecciones por Alphavirus/terapia , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Receptor de Interferón alfa y beta/genética , Proteínas Estructurales Virales/inmunología , Células 3T3 , Aedes , Infecciones por Alphavirus/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Línea Celular , Fiebre Chikungunya , Virus Chikungunya/inmunología , Chlorocebus aethiops , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Vero , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
COPI mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) and within the Golgi stack, sorting transmembrane proteins bearing C-terminal KKxx or KxKxx motifs. The structure of KxKxx motifs bound to the N-terminal WD-repeat domain of ß'-COP identifies electrostatic contacts between the motif and complementary patches at the center of the ß'-COP propeller. An absolute requirement of a two-residue spacing between the terminal carboxylate group and first lysine residue results from interactions of carbonyl groups in the motif backbone with basic side chains of ß'-COP. Similar interactions are proposed to mediate binding of KKxx motifs by the homologous α-COP domain. Mutation of key interacting residues in either domain or in their cognate motifs abolishes in vitro binding and results in mistrafficking of dilysine-containing cargo in yeast without compromising cell viability. Flexibility between ß'-COP WD-repeat domains and the location of cargo binding have implications for COPI coat assembly.
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Proteína Coat de Complejo I/metabolismo , Proteína Coatómero/metabolismo , Dipéptidos/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Proteína Coat de Complejo I/química , Proteína Coat de Complejo I/genética , Proteína Coatómero/química , Proteína Coatómero/genética , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Modelos Moleculares , Unión Proteica , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/síntesis química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC' loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC' loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Virus del Dengue/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos , Dengue/inmunología , Dengue/prevención & control , Virus del Dengue/genética , Mapeo Epitopo , Epítopos/genética , Epítopos/inmunología , Humanos , Estructura Terciaria de ProteínaRESUMEN
Flavivirus NS1 is a nonstructural glycoprotein that is expressed on the cell surface and secreted into the extracellular space. Despite its transit through the secretory pathway, NS1 is an essential gene linked to early viral RNA replication. How this occurs has remained a mystery given the disparate localization of NS1 and the viral RNA replication complex, as the latter is present on the cytosolic face of the endoplasmic reticulum (ER). We recently identified an N-terminal di-amino acid motif in NS1 that modulates protein targeting and affected viral replication. Exchange of two amino acids at positions 10 and 11 from dengue virus (DENV) into West Nile virus (WNV) NS1 (RQ10NK) changed its relative surface expression and secretion and attenuated infectivity. However, the phenotype of WNV containing NS1 RQ10NK was unstable, as within two passages heterogeneous plaque variants were observed. Here, using a mutant WNV encoding the NS1 RQ10NK mutation, we identified a suppressor mutation (F86C) in NS4B, a virally encoded transmembrane protein with loops on both the luminal and cytoplasmic sides of the ER membrane. Introduction of NS4B F86C specifically rescued RNA replication of mutant WNV but did not affect the wild-type virus. Mass spectrometry and coimmunoprecipitation studies established a novel physical interaction between NS1 and NS4B, suggesting a mechanism for how luminal NS1 conveys signals to the cytoplasm to regulate RNA replication.
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Mapeo de Interacción de Proteínas , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Virus del Nilo Occidental/fisiología , Sustitución de Aminoácidos , Animales , Línea Celular , Cricetinae , Inmunoprecipitación , Espectrometría de Masas , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Unión Proteica , Supresión GenéticaRESUMEN
Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential.
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Anticuerpos Neutralizantes/inmunología , Virus del Dengue/genética , Virus del Dengue/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Genotipo , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
PACSIN/Syndapin proteins are membrane-active scaffolds that participate in endocytosis. The structure of the Drosophila Syndapin N-terminal EFC domain reveals a crescent shaped antiparallel dimer with a high affinity for phosphoinositides and a unique membrane-inserting prong upon the concave surface. Combined structural, biochemical and reverse genetic approaches in zebrafish define an important role for Syndapin orthologue, Pacsin3, in the early formation of the notochord during embryonic development. In pacsin3-morphant embryos, midline convergence of notochord precursors is defective as axial mesodermal cells fail to polarize, migrate and differentiate properly. The pacsin3 morphant phenotype of a stunted body axis and contorted trunk is rescued by ectopic expression of Drosophila Syndapin, and depends critically on both the prong that protrudes from the surface of the bowed Syndapin EFC domain and the ability of the antiparallel dimer to bind tightly to phosphoinositides. Our data confirm linkage between directional migration, endocytosis and cell specification during embryonic morphogenesis and highlight a key role for Pacsin3 in this coupling in the notochord.
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Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Desarrollo Embrionario , Notocorda/embriología , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Movimiento Celular/efectos de los fármacos , Embrión no Mamífero/anomalías , Embrión no Mamífero/patología , Embrión no Mamífero/ultraestructura , Desarrollo Embrionario/efectos de los fármacos , Células HeLa , Humanos , Liposomas/metabolismo , Datos de Secuencia Molecular , Notocorda/anomalías , Notocorda/efectos de los fármacos , Notocorda/patología , Oligonucleótidos Antisentido/farmacología , Fenotipo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
SNAREs provide the specificity and energy for the fusion of vesicles with their target membrane, but how they are sorted into the appropriate vesicles on post-Golgi trafficking pathways is largely unknown. We demonstrate that the clathrin-mediated endocytosis of the SNARE VAMP7 is directly mediated by Hrb, a clathrin adaptor and ArfGAP. Hrb wraps 20 residues of its unstructured C-terminal tail around the folded VAMP7 longin domain, demonstrating that unstructured regions of clathrin adaptors can select cargo. Disrupting this interaction by mutation of the VAMP7 longin domain or depletion of Hrb causes VAMP7 to accumulate on the cell's surface. However, the SNARE helix of VAMP7 binds back onto its longin domain, outcompeting Hrb for binding to the same groove and suggesting that Hrb-mediated endocytosis of VAMP7 occurs only when VAMP7 is incorporated into a cis-SNARE complex. These results elucidate the mechanism of retrieval of a postfusion SNARE complex in clathrin-coated vesicles.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Proteínas R-SNARE/química , Proteínas R-SNARE/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Endocitosis , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transporte de Proteínas , Técnicas del Sistema de Dos HíbridosRESUMEN
Clathrin-associated sorting proteins (CLASPs) expand the repertoire of endocytic cargo sorted into clathrin-coated vesicles beyond the transmembrane proteins that bind physically to the AP-2 adaptor. LDL and GPCRs are internalized by ARH and beta-arrestin, respectively. We show that these two CLASPs bind selectively to the AP-2 beta2 appendage platform via an alpha-helical [DE](n)X(1-2)FXX[FL]XXXR motif, and that this motif also occurs and is functional in the epsins. In beta-arrestin, this motif maintains the endocytosis-incompetent state by binding back on the folded core of the protein in a beta strand conformation. Triggered via a beta-arrestin/GPCR interaction, the motif must be displaced and must undergo a strand to helix transition to enable the beta2 appendage binding that drives GPCR-beta-arrestin complexes into clathrin coats. Another interaction surface on the beta2 appendage sandwich is identified for proteins such as eps15 and clathrin, suggesting a mechanism by which clathrin displaces eps15 to lattice edges during assembly.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Clatrina/metabolismo , Endocitosis/fisiología , Conformación Proteica , Estructura Terciaria de Proteína , Factor de Transcripción AP-2/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Secuencia de Aminoácidos , Arrestinas/química , Arrestinas/genética , Arrestinas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Células HeLa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Receptores Acoplados a Proteínas G/metabolismo , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Factor de Transcripción AP-2/genética , Factor de Transcripción AP-2/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , beta-ArrestinasRESUMEN
Membrane sorting between secretory and endocytic organelles is predominantly controlled by small carrier vesicles or tubules that have specific protein coats on their cytoplasmic surfaces. Clathrin-clathrin-adaptor coats function in many steps of intracellular transport and are the most extensively studied of all transport-vesicle coats. In recent years, the determination of structures of clathrin assemblies by electron microscopy, of domains of clathrin and of its adaptors has improved our understanding of the molecular mechanisms of clathrin-coated-vesicle assembly and disassembly.