RESUMEN
Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines.
Asunto(s)
Proteína Quinasa C-theta/metabolismo , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Sitios de Unión , Línea Celular , Humanos , Concentración 50 Inhibidora , Interleucina-2/metabolismo , Ratones , Ratones Noqueados , Simulación del Acoplamiento Molecular , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Estructura Terciaria de Proteína , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacología , Relación Estructura-Actividad , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70 percent of infected patients, and there is evidence of human-to-human transmission. Endothelial syncytia, comprised of multinucleated giant-endothelial cells, are frequently found in NiV infections, and are mediated by the fusion (F) and attachment (G) envelope glycoproteins. Identification of the receptor for this virus will shed light on the pathobiology of NiV infection, and spur the rational development of effective therapeutics. Here we report that ephrinB2, the membrane-bound ligand for the EphB class of receptor tyrosine kinases (RTKs), specifically binds to the attachment (G) glycoprotein of NiV. Soluble Fc-fusion proteins of ephrinB2, but not ephrinB1, effectively block NiV fusion and entry into permissive cell types. Moreover, transfection of ephrinB2 into non-permissive cells renders them permissive for NiV fusion and entry. EphrinB2 is expressed on endothelial cells and neurons, which is consistent with the known cellular tropism for NiV. Significantly, we find that NiV-envelope-mediated infection of microvascular endothelial cells and primary cortical rat neurons is inhibited by soluble ephrinB2, but not by the related ephrinB1 protein. Cumulatively, our data show that ephrinB2 is a functional receptor for NiV.
Asunto(s)
Efrina-B2/metabolismo , Virus Nipah/metabolismo , Virus Nipah/patogenicidad , Receptores Virales/metabolismo , Animales , Línea Celular , Efrina-B2/genética , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Fusión de Membrana , Peso Molecular , Virus Nipah/fisiología , Unión Proteica , Estructura Terciaria de Proteína , Conejos , Ratas , Ratas Sprague-Dawley , Receptores Virales/genética , Solubilidad , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismoRESUMEN
EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1-A5 and ephrinB1-B3), only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (K(off) = 1.94 x 10(-3) s(-1) versus 1.06 x 10(-4) s(-1) for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu-Trp residues in the solvent exposed G-H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr-Met residues in the homologous positions in ephrinB1 with Leu-Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G-H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity.
Asunto(s)
Efrina-B3/química , Efrina-B3/metabolismo , Virus Nipah/metabolismo , Receptores Virales/metabolismo , Animales , Sitios de Unión , Células CHO , Cricetinae , Cricetulus , Efrina-B2/genética , Efrina-B2/metabolismo , Efrina-B3/genética , Infecciones por Henipavirus/virología , Humanos , Leucina , Virus Nipah/fisiología , TriptófanoRESUMEN
Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB-inducing kinase (NIK)-dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin ß receptor (LTßR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)-dependent nuclear translocation of p52, but not of the TNF-α receptor-mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTßR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.
Asunto(s)
Antineoplásicos/farmacología , Quinasas Ciclina-Dependientes/metabolismo , Regulación Neoplásica de la Expresión Génica , FN-kappa B/metabolismo , Osteosarcoma/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Ciclinas/metabolismo , Perfilación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Indoles/farmacología , Receptor beta de Linfotoxina/antagonistas & inhibidores , Receptor beta de Linfotoxina/genética , Receptor beta de Linfotoxina/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , Subunidad p52 de NF-kappa B/genética , Subunidad p52 de NF-kappa B/metabolismo , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/patología , Propionatos/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteoma , Transducción de Señal , Receptor de TWEAK/antagonistas & inhibidores , Receptor de TWEAK/genética , Receptor de TWEAK/metabolismo , Células Tumorales Cultivadas , Quinasa de Factor Nuclear kappa BRESUMEN
Germinal-center kinase-like kinase (GLK, Map4k3), a GCK-I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation-loop swapped dimer with more than 20 amino acids of ordered density at the carboxy-terminus. This C-terminal PEST region binds intermolecularly to the hydrophobic groove of the N-terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an "active" kinase, including the salt bridge between the C-helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P-loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.
Asunto(s)
Mutación Missense , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Sustitución de Aminoácidos , Cristalografía por Rayos X , Humanos , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Estructura Secundaria de ProteínaRESUMEN
MLKL is a pore forming pseudokinase involved in the final stage of necroptosis, a form of programmed cell death. Its phosphorylation by RIPK3 is necessary for triggering necroptosis but not for triggering apoptosis, which makes it a unique target for pharmacological inhibition to block necroptotic cell death. This mechanism has been described as playing a role in disease progression in neurodegenerative and inflammatory diseases. A type II kinase inhibitor (cpd 1) has been described that reportedly binds to the MLKL pseudokinase domain and prevents necroptosis. Here we describe five compounds that bind to the MLKL ATP-binding site, however the four MLKL-selective compounds have no activity in rescuing cells from necroptosis. We use kinase selectivity panels, crystallography and a new conformationally sensitive method of measuring protein conformational changes (SHG) to confirm that the one previously reported compound that can rescue cells (cpd 1) is a non-selective type II inhibitor that also inhibits the upstream kinase RIPK1. Although this compound can shift the GFE motif of the activation loop to an "out" position, the accessibility of the key residue Ser358 in the MLKL activation loop is not affected by compound binding to the MLKL active site. Our studies indicate that an ATP-pocket inhibitor of the MLKL pseudokinase domain does not have any impact on the necroptosis pathway, which is contrary to a previously reported study.
Asunto(s)
Adenosina Trifosfato/metabolismo , Muerte Celular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Células Jurkat , Modelos Moleculares , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Proteínas Quinasas/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
Andes virus (ANDV), a member of the Hantavirus genus in the family Bunyaviridae, causes an acute disease characteristic of New-World hantaviruses called hantavirus pulmonary syndrome (HPS). HPS is a highly pathogenic disease with a case-fatality rate of 40%. ANDV is the only hantavirus reported to spread directly from human-to-human. The aim of the present study was to develop a quantitative and high-throughput pseudovirion assay to study ANDV infection and neutralization in biosafety level 2 facilities (BSL-2). This pseudovirion assay is based on incorporation of ANDV glycoproteins onto replication-defective vesicular stomatitis virus (VSV) cores in which the gene for the surface G protein has been replaced by that encoding Renilla luciferase. Infection by the pseudovirions can be quantified by luciferase activity of infected cell lysates. ANDV pseudovirions were neutralized by ANDV-specific antisera, and there was good concordance between specificity and neutralization titers of ANDV hamster sera as determined by our pseudovirion assay and a commonly used plaque reduction neutralization titer (PRNT) assay. In addition, the pseudovirions were used to evaluate the requirements for ANDV entry, like pH dependency and the role of beta3 integrin, the reported receptor for other pathogenic hantaviruses, on entry.
Asunto(s)
Vectores Genéticos , Pruebas de Neutralización/métodos , Orthohantavirus/fisiología , Vesiculovirus/genética , Virología/métodos , Internalización del Virus , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Línea Celular , Chlorocebus aethiops , Cricetinae , Genes Reporteros , Orthohantavirus/genética , Humanos , Luciferasas de Renilla/genética , Luciferasas de Renilla/metabolismo , Glicoproteínas de Membrana/genética , Conejos , Estadística como Asunto , Ensayo de Placa Viral , Proteínas Estructurales Virales/genéticaRESUMEN
Methionine oxidation commonly occurs in the Fc fragment of therapeutic monoclonal antibodies; however, its impact on antibody function has not been addressed. Using surface plasmon resonance and cell binding assays, we examined the impact of methionine oxidation on the binding of two humanized IgG1 antibodies to Fc gamma receptors (Fc gammaR) and to the neonatal Fc receptor (Fc Rn). A panel of Fc gammaRs, including Fc gammaRI, Fc gammaRIIa-131H, Fc gammaRIIa-131R, Fc gammaRIIb/c, Fc gammaRIII ALF, Fc gammaRIII ALV, and Fc gammaRIIIb was evaluated. The binding of oxidized IgG1 molecules to individual receptors remained the same with the exception of Fc gammaRIIa where a subtle decrease in binding to the 131H allele was observed. In contrast, but in agreement with recently reported structural changes associated with Met oxidation, binding to Fc Rn was significantly affected. An increase in K(D) values at pH 6.0 was observed with increasing degree of oxidation, reaching several-fold greater value in highly oxidized samples. To our knowledge this is the first report demonstrating that chemical degradations in the constant region of monoclonal antibodies can impact their function and it highlights the importance of avoiding oxidation in therapeutic antibodies.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulina G/metabolismo , Metionina/metabolismo , Metionina/fisiología , Receptores Fc/metabolismo , Receptores de IgG/metabolismo , Anticuerpos Monoclonales/efectos de los fármacos , Anticuerpos Monoclonales/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Peróxido de Hidrógeno/farmacología , Oxidación-Reducción/efectos de los fármacos , Unión Proteica/efectos de los fármacosRESUMEN
The structural glycoproteins of Crimean-Congo hemorrhagic fever virus (CCHFV; genus Nairovirus, family Bunyaviridae) are derived through endoproteolytic cleavage of a 1,684-amino-acid M RNA segment-encoded polyprotein. This polyprotein is cotranslationally cleaved into the PreGN and PreGC precursors, which are then cleaved by SKI-1 and a SKI-1-like protease to generate the N termini of GN and GC, respectively. However, the resulting polypeptide defined by the N termini of GN and GC is predicted to be larger (58 kDa) than mature GN (37 kDa). By analogy to the topologically similar M segment-encoded polyproteins of viruses in the Orthobunyavirus genus, the C-terminal region of PreGN that contains four predicted transmembrane domains may also contain a nonstructural protein, NSM. To characterize potential PreGN C-terminal cleavage events, a panel of epitope-tagged PreGN truncation and internal deletion mutants was developed. These constructs allowed for the identification of a C-terminal endoproteolytic cleavage within, or very proximal to, the second predicted transmembrane domain following the GN ectodomain and the subsequent generation of a C-terminal fragment. Pulse-chase experiments showed that PreGN C-terminal cleavage occurred shortly after synthesis of the precursor and prior to generation of the GN glycoprotein. The resulting fragment trafficked to the Golgi compartment, the site of virus assembly. Development of an antiserum specific to the second cytoplasmic loop of PreGN allowed detection of cell-associated NSM proteins derived from transient expression of the complete CCHFV M segment and also in the context of virus infection.
Asunto(s)
Virus de la Fiebre Hemorrágica de Crimea-Congo/metabolismo , Fiebre Hemorrágica de Crimea/virología , Animales , Transporte Biológico , Chlorocebus aethiops , Cricetinae , Epítopos/química , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Plásmidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , ARN Viral/química , Células VeroRESUMEN
Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5- or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5- and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to small-molecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function, screening for new inhibitors of gp120 core interactions with chemokine receptors, and designing novel immunogens for vaccines.
Asunto(s)
Farmacorresistencia Viral , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Internalización del Virus , Replicación Viral , Fármacos Anti-VIH/farmacología , Bencilaminas , Antagonistas de los Receptores CCR5 , Antígenos CD4/genética , Antígenos CD4/metabolismo , Línea Celular , Ciclamas , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/genética , VIH-2/genética , Compuestos Heterocíclicos/farmacología , Humanos , Oligopéptidos/farmacología , Estructura Secundaria de Proteína/genética , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Eliminación de Secuencia/genética , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by beta-galactosidase alpha-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.
Asunto(s)
Alphavirus/genética , Vectores Genéticos , Glicoproteínas/metabolismo , Fusión de Membrana , Replicón , Virus de la Fiebre del Valle del Rift/patogenicidad , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Glicoproteínas/genética , Humanos , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/genética , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusion-inhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F's interactions with NiV-G, providing support for the attachment protein "displacement" model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this "shielding" role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.
Asunto(s)
Fusión de Membrana/fisiología , Virus Nipah/fisiología , Polisacáridos/fisiología , Proteínas del Envoltorio Viral/fisiología , Proteínas Virales de Fusión/fisiología , Animales , Línea Celular , Perros , Glicosilación , Células HeLa , Infecciones por Henipavirus/prevención & control , Infecciones por Henipavirus/virología , Humanos , Fusión de Membrana/genética , Pruebas de Neutralización , Virus Nipah/genética , Virus Nipah/patogenicidad , Polisacáridos/genética , Polisacáridos/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales de Fusión/biosíntesis , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismoRESUMEN
Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease with high rates of mortality in humans. The CCHFV M RNA segment encodes the virus glycoproteins G(N) and G(C). To understand the processing and intracellular localization of the CCHFV glycoproteins as well as their neutralization and protection determinants, we produced and characterized monoclonal antibodies (MAbs) specific for both G(N) and G(C). Using these MAbs, we found that G(N) predominantly colocalized with a Golgi marker when expressed alone or with G(C), while G(C) was transported to the Golgi apparatus only in the presence of G(N). Both proteins remained endo-beta-N-acetylglucosaminidase H sensitive, indicating that the CCHFV glycoproteins are most likely targeted to the cis Golgi apparatus. Golgi targeting information partly resides within the G(N) ectodomain, because a soluble version of G(N) lacking its transmembrane and cytoplasmic domains also localized to the Golgi apparatus. Coexpression of soluble versions of G(N) and G(C) also resulted in localization of soluble G(C) to the Golgi apparatus, indicating that the ectodomains of these proteins are sufficient for the interactions needed for Golgi targeting. Finally, the mucin-like and P35 domains, located at the N terminus of the G(N) precursor protein and removed posttranslationally by endoproteolysis, were required for Golgi targeting of G(N) when it was expressed alone but were dispensable when G(C) was coexpressed. In neutralization assays on SW-13 cells, MAbs to G(C), but not to G(N), prevented CCHFV infection. However, only a subset of G(C) MAbs protected mice in passive-immunization experiments, while some nonneutralizing G(N) MAbs efficiently protected animals from a lethal CCHFV challenge. Thus, neutralization of CCHFV likely depends not only on the properties of the antibody, but on host cell factors as well. In addition, nonneutralizing antibody-dependent mechanisms, such as antibody-dependent cell-mediated cytotoxicity, may be involved in the in vivo protection seen with the MAbs to G(C).
Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Aparato de Golgi/metabolismo , Virus de la Fiebre Hemorrágica de Crimea-Congo/inmunología , Virus de la Fiebre Hemorrágica de Crimea-Congo/metabolismo , Fiebre Hemorrágica de Crimea/metabolismo , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Modelos Animales de Enfermedad , Fiebre Hemorrágica de Crimea/prevención & control , Humanos , Inmunización Pasiva , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/farmacología , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , SolubilidadRESUMEN
Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease in humans with high rates of mortality. The virus has a tripartite genome composed of a small (S), a medium (M) and a large (L) RNA segment; the M segment encodes the two viral glycoproteins, G(N) and G(C). Whilst relatively few full-length M segment sequences are available, it is apparent that both G(N) and G(C) may exhibit significant sequence diversity. It is unknown whether considerable antigenic differences exist between divergent CCHFV strains, or whether there are conserved neutralizing epitopes. The M segments derived from viral isolates of a human case of CCHF in South Africa (SPU 41/84), an infected tick (Hyalomma marginatum) in South Africa (SPU 128/81), a human case in Congo (UG 3010), an infected individual in Uzbekistan (U2-2-002) and an infected tick (Hyalomma asiaticum) in China (Hy13) were sequenced fully, and the glycoproteins were expressed. These novel sequences showed high variability in the N-terminal region of G(N) and more modest differences in the remainder of G(N) and in G(C). Phylogenetic analyses placed these newly identified strains in three of the four previously described M segment groups. Studies with a panel of mAbs specific to G(N) and G(C) indicated that there were significant antigenic differences between the M segment groups, although several neutralizing epitopes in both G(N) and G(C) were conserved among all strains examined. Thus, the genetic diversity exhibited by CCHFV strains results in significant antigenic differences that will need to be taken into consideration for vaccine development.
Asunto(s)
Antígenos Virales/inmunología , Epítopos/inmunología , Glicoproteínas/inmunología , Virus de la Fiebre Hemorrágica de Crimea-Congo/inmunología , Proteínas Virales/inmunología , Animales , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Mapeo Epitopo , Expresión Génica , Glicoproteínas/genética , Virus de la Fiebre Hemorrágica de Crimea-Congo/genética , Virus de la Fiebre Hemorrágica de Crimea-Congo/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Pruebas de Neutralización , Filogenia , Polimorfismo Genético , ARN Viral/genética , Análisis de Secuencia de ADN , Homología de Secuencia , Proteínas Virales/genéticaRESUMEN
Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3' untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3' terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3' elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3'UTR (ORF2+3+3'UTR). In contrast, expression of VP1 from constructs that lacked the 3'UTR (ORF2+3), ORF3 (ORF2+3'UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3'UTR revealed that the 3'UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.
Asunto(s)
Regiones no Traducidas 3'/genética , Proteínas de la Cápside/metabolismo , Regulación Viral de la Expresión Génica , Virus Norwalk/metabolismo , ARN Mensajero/genética , Animales , Baculoviridae/genética , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Línea Celular , Células Cultivadas , Humanos , Virus Norwalk/genética , ARN Mensajero/metabolismo , SpodopteraRESUMEN
Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. Rotavirus VLPs composed of simian SA11 strain VP2 and VP6 proteins (homologous 2/6-VLPs) were produced by cloning the rotavirus simian SA11 genes 2 and 6 into a single baculovirus transfer vector (pAcAB4). The overall yield of homologous 2/6-VLPs produced with the dual recombinant baculovirus was at least 30-fold higher than that of VLPs composed of bovine RF strain VP2 and simian SA11 strain VP6 (heterologous 2/6-VLPs), produced with single recombinant baculoviruses. Adult mice were immunized intramuscularly twice with various doses of homologous or heterologous 2/6-VLPs in QS-21, orally with or without cholera toxin (CT), or intranasally with mutant Escherichia coli heat-labile enterotoxin (LT-R192G). Both homologous and heterologous 2/6-VLPs were immunogenic and induced protection from challenge, with those administered parenterally or intranasally affording the highest mean protection from challenge. The 2/6-VLPs did not induce serum neutralizing antibody (N-Ab) responses, but these VLPs primed for a broad heterotypic N-Ab response, which was elicited after rotavirus challenge. Heterotypic N-Ab responses were not observed in 2/6-VLP vaccinated mice that were > or =94% protected from challenge. After challenge, control mice immunized with adjuvant alone developed only homotypic serum N-Ab responses. Similar results were obtained after challenge of rabbits immunized parenterally or intranasally with heterologous 2/6-VLPs. These results suggest that 2/6-VLPs prime the immune system to enhance the production of heterotypic N-Ab responses, but the induction of heterotypic N-Abs requires that virus replication occurs after challenge. The use of 2/6-VLPs expressed from a single recombinant baculovirus simplifies production and would reduce the cost of a VLP-based vaccine.
Asunto(s)
Baculoviridae/genética , Vectores Genéticos/genética , Vacunas contra Rotavirus/inmunología , Rotavirus/inmunología , Adyuvantes Inmunológicos/farmacología , Administración Intranasal , Administración Oral , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/biosíntesis , Toxina del Cólera/farmacología , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Heces/virología , Femenino , Inyecciones Intramusculares , Ratones , Pruebas de Neutralización , Conejos , Rotavirus/genética , Rotavirus/aislamiento & purificación , Infecciones por Rotavirus/prevención & control , Vacunas contra Rotavirus/administración & dosificación , Vacunas contra Rotavirus/genética , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
Norwalk virus (NV) is the prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant NV virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. The X-ray structure of the rNV VLPs has revealed that the capsid protein folds into two principal domains: a shell (S) domain and a protruding (P) domain (B. V. V. Prasad, M. E. Hardy, T. Dokland, J. Bella, M. G. Rossmann, and M. K. Estes, Science 286:287-290, 1999). To investigate the structural requirements for the assembly of rNV VLPs, we performed mutational analyses of the capsid protein. We examined the ability of 10 deletion mutants of the capsid protein to assemble into VLPs in insect cell cultures. Deletion of the N-terminal 20 residues, suggested by the X-ray structure to be involved in a switching mechanism during assembly, did not affect the ability of the mutant capsid protein to self-assemble into 38-nm VLPs with a T=3 icosahedral symmetry. Further deletions in the N-terminal region affected particle assembly. Deletions in the C-terminal regions of the P domain, involved in the interactions between the P and S domains, did not block the assembly process, but they affected the size and stability of the particles. Mutants carrying three internal deletion mutations in the P domain, involved in maintaining dimeric interactions, produced significantly larger 45-nm particles, albeit in low yields. The complete removal of the protruding domain resulted in the formation of smooth particles with a diameter that is slightly smaller than the 30-nm diameter expected from the rNV structure. These studies indicate that the shell domain of the NV capsid protein contains everything required to initiate the assembly of the capsid, whereas the entire protruding domain contributes to the increased stability of the capsid by adding intermolecular contacts between the dimeric subunits and may control the size of the capsid.