RESUMEN
Cleavage of the flavivirus premembrane (prM) structural protein during maturation can be inefficient. The contribution of partially mature flavivirus virions that retain uncleaved prM to pathogenesis during primary infection is unknown. To investigate this question, we characterized the functional properties of newly-generated dengue virus (DENV) prM-reactive monoclonal antibodies (mAbs) in vitro and using a mouse model of DENV disease. Anti-prM mAbs neutralized DENV infection in a virion maturation state-dependent manner. Alanine scanning mutagenesis and cryoelectron microscopy of anti-prM mAbs in complex with immature DENV defined two modes of attachment to a single antigenic site. In vivo, passive transfer of intact anti-prM mAbs resulted in an antibody-dependent enhancement of disease. However, protection against DENV-induced lethality was observed when the transferred mAbs were genetically modified to inhibit their ability to interact with Fcγ receptors. These data establish that in addition to mature forms of the virus, partially mature infectious prM+ virions can also contribute to pathogenesis during primary DENV infections.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Virus del Dengue , Dengue , Microscopía por Crioelectrón , Proteínas del Envoltorio Viral/metabolismo , Virión/metabolismo , Animales , RatonesRESUMEN
Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus in the Flaviviridae family. In contrast to most other insect-transmitted flaviviruses, ZIKV also can be transmitted sexually and from mother to fetus in humans. During recent outbreaks, ZIKV infections have been linked to microcephaly, congenital disease, and Guillain-Barré syndrome. Neutralizing antibodies have potential as therapeutic agents. We report here a 4-Å-resolution cryo-electron microscopy structure of the ZIKV virion in complex with Fab fragments of the potently neutralizing human monoclonal antibody ZIKV-195. The footprint of the ZIKV-195 Fab fragment expands across two adjacent envelope (E) protein protomers. ZIKV neutralization by this antibody is presumably accomplished by cross-linking the E proteins, which likely prevents formation of E protein trimers required for fusion of the viral and cellular membranes. A single dose of ZIKV-195 administered 5 days after virus inoculation showed marked protection against lethality in a stringent mouse model of infection.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Microscopía por Crioelectrón/métodos , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Vacunación/métodos , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
African swine fever virus, a double-stranded DNA virus that infects pigs, is the only known member of the Asfarviridae family. Nevertheless, during our isolation and sequencing of the complete genome of faustovirus, followed by the description of kaumoebavirus, carried out over the past 2 years, we observed the emergence of previously unknown related viruses within this group of viruses. Here we describe the isolation of pacmanvirus, a fourth member in this group, which is capable of infecting Acanthamoeba castellanii Pacmanvirus A23 has a linear compact genome of 395,405 bp, with a 33.62% G+C content. The pacmanvirus genome harbors 465 genes, with a high coding density. An analysis of reciprocal best hits shows that 31 genes are conserved between African swine fever virus, pacmanvirus, faustovirus, and kaumoebavirus. Moreover, the major capsid protein locus of pacmanvirus appears to be different from those of kaumoebavirus and faustovirus. Overall, comparative and genomic analyses reveal the emergence of a new group or cluster of viruses encompassing African swine fever virus, faustovirus, pacmanvirus, and kaumoebavirus.IMPORTANCE Pacmanvirus is a newly discovered icosahedral double-stranded DNA virus that was isolated from an environmental sample by amoeba coculture. We describe herein its structure and replicative cycle, along with genomic analysis and genomic comparisons with previously known viruses. This virus represents the third virus, after faustovirus and kaumoebavirus, that is most closely related to classical representatives of the Asfarviridae family. These results highlight the emergence of previously unknown double-stranded DNA viruses which delineate and extend the diversity of a group around the asfarvirus members.
Asunto(s)
Acanthamoeba castellanii/virología , Virus ADN/clasificación , Virus ADN/aislamiento & purificación , ADN Viral/química , ADN Viral/genética , Acanthamoeba castellanii/ultraestructura , Composición de Base , Análisis por Conglomerados , Virus ADN/genética , Genes Virales , Microscopía Electrónica de Transmisión , Filogenia , Sintenía , Virión/ultraestructuraRESUMEN
Nuclear replication of cytomegalovirus relies on elaborate mechanisms of nucleocytoplasmic egress of viral particles. Thus, the role of two essential and conserved viral nuclear egress proteins, pUL50 and pUL53, is pivotal. pUL50 and pUL53 heterodimerize and form a core nuclear egress complex (NEC), which is anchored to the inner nuclear membrane and provides a scaffold for the assembly of a multimeric viral-cellular NEC. Here, we report the crystal structure of the pUL50-pUL53 heterodimer (amino acids 1-175 and 50-292, respectively) at 2.44 Å resolution. Both proteins adopt a globular fold with mixed α and ß secondary structure elements. pUL53-specific features include a zinc-binding site and a hook-like N-terminal extension, the latter representing a hallmark element of the pUL50-pUL53 interaction. The hook-like extension (amino acids 59-87) embraces pUL50 and contributes 1510 Å(2) to the total interface area (1880 Å(2)). The pUL50 structure overall resembles the recently published NMR structure of the murine cytomegalovirus homolog pM50 but reveals a considerable repositioning of the very C-terminal α-helix of pUL50 upon pUL53 binding. pUL53 shows structural resemblance with the GHKL domain of bacterial sensory histidine kinases. A close examination of the crystal structure indicates partial assembly of pUL50-pUL53 heterodimers to hexameric ring-like structures possibly providing additional scaffolding opportunities for NEC. In combination, the structural information on pUL50-pUL53 considerably improves our understanding of the mechanism of HCMV nuclear egress. It may also accelerate the validation of the NEC as a unique target for developing a novel type of antiviral drug and improved options of broad-spectrum antiherpesviral therapy.
Asunto(s)
Citomegalovirus/fisiología , Interacciones Huésped-Patógeno , Proteínas Virales/química , Liberación del Virus , Antivirales/química , Antivirales/farmacología , Sitios de Unión , Cristalografía por Rayos X , Citomegalovirus/efectos de los fármacos , Diseño de Fármacos , Humanos , Membrana Nuclear/virología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Virales/metabolismo , Virión/efectos de los fármacos , Virión/fisiología , Replicación ViralRESUMEN
PML nuclear bodies (PML-NBs) are enigmatic structures of the cell nucleus that act as key mediators of intrinsic immunity against viral pathogens. PML itself is a member of the E3-ligase TRIM family of proteins that regulates a variety of innate immune signaling pathways. Consequently, viruses have evolved effector proteins to modify PML-NBs; however, little is known concerning structure-function relationships of viral antagonists. The herpesvirus human cytomegalovirus (HCMV) expresses the abundant immediate-early protein IE1 that colocalizes with PML-NBs and induces their dispersal, which correlates with the antagonization of NB-mediated intrinsic immunity. Here, we delineate the molecular basis for this antagonization by presenting the first crystal structure for the evolutionary conserved primate cytomegalovirus IE1 proteins. We show that IE1 consists of a globular core (IE1CORE) flanked by intrinsically disordered regions. The 2.3 Å crystal structure of IE1CORE displays an all α-helical, femur-shaped fold, which lacks overall fold similarity with known protein structures, but shares secondary structure features recently observed in the coiled-coil domain of TRIM proteins. Yeast two-hybrid and coimmunoprecipitation experiments demonstrate that IE1CORE binds efficiently to the TRIM family member PML, and is able to induce PML deSUMOylation. Intriguingly, this results in the release of NB-associated proteins into the nucleoplasm, but not of PML itself. Importantly, we show that PML deSUMOylation by IE1CORE is sufficient to antagonize PML-NB-instituted intrinsic immunity. Moreover, co-immunoprecipitation experiments demonstrate that IE1CORE binds via the coiled-coil domain to PML and also interacts with TRIM5α We propose that IE1CORE sequesters PML and possibly other TRIM family members via structural mimicry using an extended binding surface formed by the coiled-coil region. This mode of interaction might render the antagonizing activity less susceptible to mutational escape.
Asunto(s)
Proteínas Portadoras/metabolismo , Citomegalovirus/química , Citomegalovirus/metabolismo , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/metabolismo , Cuerpos de Inclusión Intranucleares/metabolismo , Factores de Restricción Antivirales , Proteínas Portadoras/genética , Línea Celular , Cristalografía por Rayos X , Citomegalovirus/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/virología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
Protection of the endothelium is provided by circulating sphingosine-1-phosphate (S1P), which maintains vascular integrity. We show that HDL-associated S1P is bound specifically to both human and murine apolipoprotein M (apoM). Thus, isolated human ApoM(+) HDL contained S1P, whereas ApoM(-) HDL did not. Moreover, HDL in Apom(-/-) mice contains no S1P, whereas HDL in transgenic mice overexpressing human apoM has an increased S1P content. The 1.7-Å structure of the S1P-human apoM complex reveals that S1P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM(+) HDL induced S1P(1) receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoM(-) HDL did not. Importantly, lack of S1P in the HDL fraction of Apom(-/-) mice decreased basal endothelial barrier function in lung tissue. Our results demonstrate that apoM, by delivering S1P to the S1P(1) receptor on endothelial cells, is a vasculoprotective constituent of HDL.
Asunto(s)
Apolipoproteínas/metabolismo , Endotelio Vascular/metabolismo , Lipoproteínas HDL/metabolismo , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Apolipoproteínas/química , Apolipoproteínas/genética , Apolipoproteínas M , Western Blotting , Células Cultivadas , Cristalografía por Rayos X , Endocitosis , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Activación Enzimática , Células HEK293 , Humanos , Lipocalinas/química , Lipocalinas/genética , Lipocalinas/metabolismo , Lipoproteínas HDL/química , Lisofosfolípidos/química , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/química , Esfingosina/metabolismoRESUMEN
Herpesviral capsids are assembled in the host cell nucleus and are subsequently translocated to the cytoplasm. During this process it has been demonstrated that the human cytomegalovirus proteins pUL50 and pUL53 interact and form, together with other viral and cellular proteins, the nuclear egress complex at the nuclear envelope. In this study we provide evidence that specific residues of a conserved N-terminal region of pUL50 determine its intranuclear interaction with pUL53. In silico evaluation and biophysical analyses suggested that the conserved region forms a regular secondary structure adopting a globular fold. Importantly, site-directed replacement of individual amino acids by alanine indicated a strong functional influence of specific residues inside this globular domain. In particular, mutation of the widely conserved residues Glu-56 or Tyr-57 led to a loss of interaction with pUL53. Consistent with the loss of binding properties, mutants E56A and Y57A showed a defective function in the recruitment of pUL53 to the nuclear envelope in expression plasmid-transfected and human cytomegalovirus-infected cells. In addition, in silico analysis suggested that residues 3-20 form an amphipathic α-helix that appears to be conserved among Herpesviridae. Point mutants revealed a structural role of this N-terminal α-helix for pUL50 stability rather than a direct role in the binding of pUL53. In contrast, the central part of the globular domain including Glu-56 and Tyr-57 is directly responsible for the functional interaction with pUL53 and thus determines formation of the basic nuclear egress complex.
Asunto(s)
Aminoácidos/metabolismo , Citomegalovirus/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Sitios de Unión/genética , Western Blotting , Núcleo Celular/virología , Secuencia Conservada/genética , Citomegalovirus/genética , Ácido Glutámico/química , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopía Confocal , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Tirosina/química , Tirosina/genética , Tirosina/metabolismo , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The molecular mechanisms of hepatitis C virus (HCV) persistence and pathogenesis are poorly understood. The design of an effective HCV vaccine is challenging despite a robust humoral immune response against closely related strains of HCV. This is primarily because of the huge genetic diversity of HCV and the molecular evolution of various virus escape mechanisms. These mechanisms are steered by the presence of a high mutational rate in HCV, structural plasticity of the immunodominant regions on the virion surface of diverse HCV genotypes, and constant amino acid substitutions on key structural components of HCV envelope glycoproteins. Here, we review the molecular basis of neutralizing antibody (nAb)-mediated immune response against diverse HCV variants, HCV-steered humoral immune evasion strategies and explore the essential structural elements to consider for designing a universal HCV vaccine. Structural perspectives on key escape pathways mediated by a point mutation within the epitope, allosteric modulation of the epitope by distant mutations and glycan shift on envelope glycoproteins will be highlighted (abstract graphic).
Asunto(s)
Hepacivirus/inmunología , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/virología , Evasión Inmune , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Epítopos , Variación Genética , Hepacivirus/química , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Inmunidad Humoral , Epítopos Inmunodominantes , Mutación , Conformación Proteica , Dominios Proteicos , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR. We investigated the origin of this activity reversal by limited proteolysis, CD spectroscopy and X-ray crystallography. We show that the TetR mutant Leu17Gly switches its function via a disorder-order mechanism that differs completely from the allosteric mechanism of wild-type TetR. Our study emphasizes how single point mutations can engender unexpected leaps in protein function thus enabling the appearance of new functionalities in proteins without the need for promiscuous intermediates.
Asunto(s)
Mutación Puntual , Proteínas Represoras/química , Proteínas Represoras/genética , Sustitución de Aminoácidos , Cristalografía por Rayos X , ADN/metabolismo , Modelos Moleculares , Movimiento (Física) , Regiones Operadoras Genéticas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Represoras/metabolismo , Tetraciclinas/químicaRESUMEN
Flaviviruses are emerging arthropod-borne RNA viruses, causing a broad spectrum of life-threatening disease symptoms such as encephalitis and hemorrhagic fever. Successful vaccines exist against yellow fever virus, Japanese encephalitis virus and tick-borne encephalitis virus. However, vaccine development against other flaviviruses like dengue virus is not straightforward. This is partly because of the high sequence conservation and immunological cross-reactivity among flavivirus envelope glycoproteins leading to antibody mediated enhancement of disease. A comprehensive analyses of the structural landscape of humoral immune response against flaviviruses is crucial for antigen design. Here, we compare the available structural data of several flavivirus antibody complexes with a major focus on Zika virus and dengue virus and discuss the mapped epitopes, the stoichiometry of antibody binding and mechanisms of neutralization.
Asunto(s)
Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas/inmunología , Flavivirus/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/clasificación , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/clasificación , Anticuerpos Antivirales/uso terapéutico , Dengue/prevención & control , Virus del Dengue/inmunología , Flavivirus/clasificación , Infecciones por Flavivirus/terapia , Humanos , Fiebre Amarilla , Virus de la Fiebre Amarilla/inmunología , Virus Zika/inmunología , Infección por el Virus ZikaRESUMEN
The flavivirus genus encompasses more than 75 unique viruses, including dengue virus which accounts for almost 390 million global infections annually. Flavivirus infection can result in a myriad of symptoms ranging from mild rash and flu-like symptoms, to severe encephalitis and even hemorrhagic fever. Efforts to combat the impact of these viruses have been hindered due to limited antiviral drug and vaccine development. However, the advancement of knowledge in the structural biology of flaviviruses over the last 25 years has produced unique perspectives for the identification of potential therapeutic targets. With particular emphasis on the assembly and maturation stages of the flavivirus life cycle, it is the goal of this review to comparatively analyze the structural similarities between flaviviruses to provide avenues for new research and innovation.
Asunto(s)
Cápside/metabolismo , Infecciones por Flavivirus/virología , Flavivirus/química , Flavivirus/fisiología , Ensamble de Virus , Libros , Cápside/química , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Flavivirus/clasificación , Flavivirus/genética , Infecciones por Flavivirus/fisiopatología , Humanos , Liberación del VirusRESUMEN
Zika virus (ZIKV), a mosquito-borne human flavivirus that causes microcephaly and other neurological disorders, has been a recent focus for the development of flavivirus vaccines and therapeutics. We report here a 4.0 Å resolution structure of the mature ZIKV in complex with ADI-30056, a ZIKV-specific human monoclonal antibody (hMAb) isolated from a ZIKV infected donor with a prior dengue virus infection. The structure shows that the hMAb interactions span across the E protein dimers on the virus surface, inhibiting conformational changes required for the formation of infectious fusogenic trimers similar to the hMAb, ZIKV-117. Structure-based functional analysis, and structure and sequence comparisons, identified ZIKV residues essential for neutralization and crucial for the evolution of highly potent E protein crosslinking Abs in ZIKV. Thus, this epitope, ZIKV's "Achilles heel", defined by the contacts between ZIKV and ADI-30056, could be a suitable target for the design of therapeutic antibodies.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Coinfección , Reacciones Cruzadas/inmunología , Infecciones por Flavivirus/inmunología , Flavivirus/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Dengue/inmunología , Virus del Dengue/inmunología , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Infecciones por Flavivirus/virología , Humanos , Imagenología Tridimensional , Modelos Moleculares , Pruebas de Neutralización , Conformación Proteica , Células Vero , Virus Zika/ultraestructura , Infección por el Virus Zika/virologíaRESUMEN
AmtR, a member of the TetR family of transcription regulators, is a global regulator of nitrogen control in Corynebacterium glutamicum. Unlike other TetR-family members, which are regulated by small-molecule effectors, AmtR is regulated by a protein called GlnK. It has been shown that a GlnK trimer has to become adenylylated prior to formation of a complex with AmtR. The physiological function of AmtR has been very well studied, but structural characterization of the mechanistic aspects of AmtR-regulated transcription has yet to be accomplished. AmtR has successfully been crystallized in space group P2(1)2(1)2, with six molecules in the asymmetric unit and unit-cell parameters a = 153.34, b = 163.10, c = 51.93 angstrom . Preliminary phases were obtained using Se-SAD.
Asunto(s)
Proteínas Bacterianas/química , Corynebacterium glutamicum/química , Proteínas Represoras/química , Cristalización , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Nucleotidiltransferasas/química , Proteínas PII Reguladoras del Nitrógeno/química , Conformación Proteica , Difracción de Rayos XRESUMEN
The putative transcriptional regulator protein YvoA (BSU35030) from Bacillus subtilis was cloned and heterologously expressed in Escherichia coli. The protein was purified by immobilized metal-affinity chromatography and size-exclusion chromatography and subsequently crystallized. A complete native data set was collected to 2.50 A resolution. The crystals belonged to the monoclinic space group C2 and preliminary analysis of the diffraction data indicated the presence of approximately 12 molecules per asymmetric unit.
Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Difracción de Rayos X , Proteínas Bacterianas/metabolismo , Cromatografía en Gel , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Estructura Terciaria de ProteínaRESUMEN
In contrast to twinning by merohedry, the reciprocal lattices of the different domains of non-merohedral twins do not overlap exactly. This leads to three kinds of reflections: reflections with no overlap, reflections with an exact overlap and reflections with a partial overlap of a reflection from a second domain. This complicates the unit-cell determination, indexing, data integration and scaling of X-ray diffraction data. However, with hindsight it is possible to detwin the data because there are reflections that are not affected by the twinning. In this article, the successful solution and refinement of one mineral, one organometallic and two protein non-merohedral twins using a common strategy are described. The unit-cell constants and the orientation matrices were determined by the program CELL_NOW. The data were then integrated with SAINT. TWINABS was used for scaling, empirical absorption corrections and the generation of two different data files, one with detwinned data for structure solution and refinement and a second one for (usually more accurate) structure refinement against total integrated intensities. The structures were solved by experimental phasing using SHELXT for the first two structures and SHELXC/D/E for the two protein structures; all models were refined with SHELXL.
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Cristalización/métodos , Cristalografía por Rayos X/métodos , Insulina Regular Porcina/química , Minerales/química , Modelos MolecularesRESUMEN
Zika virus (ZIKV) is an enveloped, icosahedral flavivirus that has structural and functional similarities to other human flavivirus pathogens such as dengue (DENV), West Nile (WNV) and Japanese encephalitis (JEV) viruses. ZIKV infections have been linked to fetal microcephaly and the paralytic Guillain-Barré syndrome. This review provides a comparative structural analysis of the assembly, maturation and host-cell entry of ZIKV with other flaviviruses, especially DENV. We also discuss the mechanisms of neutralization by antibodies.
Asunto(s)
Ensamble de Virus , Internalización del Virus , Infección por el Virus Zika/virología , Virus Zika/química , Virus Zika/fisiología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Microscopía por Crioelectrón , Virus del Dengue/química , Virus del Dengue/fisiología , Virus de la Encefalitis Japonesa (Especie)/química , Virus de la Encefalitis Japonesa (Especie)/fisiología , Femenino , Síndrome de Guillain-Barré/virología , Humanos , Masculino , Ratones , Microcefalia/virología , Modelos Biológicos , Embarazo , Conformación Proteica , Estados Unidos , Virus del Nilo Occidental/química , Virus del Nilo Occidental/fisiologíaRESUMEN
Among the several arthropod-borne human flaviviral diseases, the recent outbreak of Zika virus (ZIKV) has caused devastating birth defects and neurological disorders, challenging the world with another major public health concern. We report here the refined structure of the mature ZIKV at a resolution of 3.1 Å as determined by cryo-electron microscopic single-particle reconstruction. The improvement in the resolution, compared with previous enveloped virus structures, was the result of optimized virus preparation methods and data processing techniques. The glycoprotein interactions and surface properties of ZIKV were compared with other mosquito-borne flavivirus structures. The largest structural differences and sequence variations occur at the glycosylation loop associated with receptor binding. Probable drug binding pockets were identified on the viral surface. These results also provide a structural basis for the design of vaccines against ZIKV.
Asunto(s)
Flavivirus/química , Virus Zika/química , Virus Zika/ultraestructura , Microscopía por Crioelectrón , Diseño de Fármacos , Flavivirus/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Unión Proteica , Imagen Individual de Molécula/métodos , Relación Estructura-Actividad , Estructuras Virales/química , Vacunas Virales/química , Vacunas Virales/farmacología , Virus Zika/metabolismoRESUMEN
AmtR belongs to the TetR family of transcription regulators and is a global nitrogen regulator that is induced under nitrogen-starvation conditions in Corynebacterium glutamicum. AmtR regulates the expression of transporters and enzymes for the assimilation of ammonium and alternative nitrogen sources, for example urea, amino acids etc. The recognition of operator DNA by homodimeric AmtR is not regulated by small-molecule effectors as in other TetR-family members but by a trimeric adenylylated PII-type signal transduction protein named GlnK. The crystal structure of ligand-free AmtR (AmtRorth) has been solved at a resolution of 2.1â Å in space group P21212. Comparison of its quaternary assembly with the previously solved native AmtR structure (PDB entry 5dy1) in a trigonal crystal system (AmtRtri) not only shows how a solvent-content reduction triggers a space-group switch but also suggests a model for how dimeric AmtR might stoichiometrically interact with trimeric adenylylated GlnK.
Asunto(s)
Proteínas Bacterianas/química , Corynebacterium glutamicum/química , Proteínas PII Reguladoras del Nitrógeno/química , Proteínas Represoras/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Corynebacterium glutamicum/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , Proteínas PII Reguladoras del Nitrógeno/genética , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
The ethyl acetate extract of the Streptomyces sp. isolate GW99/1572 exhibited significant biological activity against Gram-positive bacteria and delivered kettapeptin (1), a new hexadepsipeptide antibiotic of the azinothricin type. The structure was elucidated by various 1D and 2D NMR techniques, mass spectrometry and by comparison of the NMR data with those of closely related antibiotics. The absolute configuration of the compound was derived by crystal structure analysis and by comparison with the optical rotation data of related compounds.
Asunto(s)
Antiinfecciosos/química , Depsipéptidos/química , Depsipéptidos/farmacología , Péptidos Cíclicos/química , Streptomyces/química , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/farmacología , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/aislamiento & purificación , Antibióticos Antineoplásicos/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Depsipéptidos/aislamiento & purificación , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Rotación Óptica , Péptidos Cíclicos/aislamiento & purificación , Péptidos Cíclicos/farmacología , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría Infrarroja , Espectrofotometría UltravioletaRESUMEN
Steroidal aryliminium salts were prepared from D-seco-pregnene aldehyde 2b, and their BF3.OEt2-catalyzed reactions were studied. The nature of the substituent R1 in the anilines 3-6 essentially influenced the chemoselectivity. Using unsubstituted 3, 4-methoxy- (4) or 4-bromoaniline (5), different tetrahydroquinoline derivatives 7a-13a via intramolecular hetero Diels-Alder reaction were formed. In the case of 4-nitroaniline (6) the N-arylamino-D-homopregnane (14a) were also obtained. We assume, that an intramolecular Prins reaction led to this type of fluoro-D-homosteroid. The main products represent a new class of tetrahydroquinolino-androstenes.