RESUMEN
Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) and Knob-associated Histidine-rich Protein (KAHRP) are directly linked to malaria pathology. PfEMP1 and KAHRP cluster on protrusions (knobs) on the P. falciparum-infected erythrocyte surface and enable pathogenic cytoadherence of infected erythrocytes to the host microvasculature, leading to restricted blood flow, oxygen deprivation and damage of tissues. Here we characterize the interactions of PfEMP1 and KAHRP with host erythrocyte spectrin using biophysical, structural and computational approaches. These interactions assist knob formation and, thus, promote cytoadherence. We show that the folded core of the PfEMP1 cytosolic domain interacts broadly with erythrocyte spectrin but shows weak, residue-specific preference for domain 17 of α spectrin, which is proximal to the erythrocyte cytoskeletal junction. In contrast, a protein sequence repeat region in KAHRP preferentially associates with domains 10-14 of ß spectrin, proximal to the spectrin-ankyrin complex. Structural models of PfEMP1 and KAHRP with spectrin combined with previous microscopy and protein interaction data suggest a model for knob architecture.
Asunto(s)
Eritrocitos/parasitología , Interacciones Huésped-Parásitos/fisiología , Malaria Falciparum/metabolismo , Péptidos/metabolismo , Proteínas Protozoarias/metabolismo , Espectrina/metabolismo , Cristalografía por Rayos X , Humanos , Simulación del Acoplamiento Molecular , Resonancia Magnética Nuclear Biomolecular , Péptidos/química , Plasmodium falciparum , Proteínas Protozoarias/química , Espectrina/químicaRESUMEN
Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus.
Asunto(s)
Gangliosidosis GM2/metabolismo , Orthoreovirus de los Mamíferos/metabolismo , Receptores Virales/metabolismo , Infecciones por Reoviridae/metabolismo , Proteínas Virales/metabolismo , Animales , Cricetinae , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Embrión de Mamíferos/virología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Gangliosidosis GM2/genética , Células L , Ratones , Mutación , Orthoreovirus de los Mamíferos/genética , Estructura Terciaria de Proteína , Receptores Virales/genética , Infecciones por Reoviridae/genética , Infecciones por Reoviridae/patología , Proteínas Virales/genéticaRESUMEN
BACKGROUND: SARS-CoV-2-neutralizing antibodies (nABs) showed great promise in the early phases of the COVID-19 pandemic. The emergence of resistant strains, however, quickly rendered the majority of clinically approved nABs ineffective. This underscored the imperative to develop nAB cocktails targeting non-overlapping epitopes. METHODS: Undertaking a nAB discovery program, we employed a classical workflow, while integrating artificial intelligence (AI)-based prediction to select non-competing nABs very early in the pipeline. We identified and in vivo validated (in female Syrian hamsters) two highly potent nABs. FINDINGS: Despite the promising results, in depth cryo-EM structural analysis demonstrated that the AI-based prediction employed with the intention to ensure non-overlapping epitopes was inaccurate. The two nABs in fact bound to the same receptor-binding epitope in a remarkably similar manner. INTERPRETATION: Our findings indicate that, even in the Alphafold era, AI-based predictions of paratope-epitope interactions are rough and experimental validation of epitopes remains an essential cornerstone of a successful nAB lead selection. FUNDING: Full list of funders is provided at the end of the manuscript.
Asunto(s)
COVID-19 , SARS-CoV-2 , Cricetinae , Animales , Humanos , Femenino , Epítopos , Pandemias , Inteligencia Artificial , Anticuerpos Antivirales , Anticuerpos Neutralizantes , MesocricetusRESUMEN
Reovirus attachment protein σ1 is an elongated trimer with head-and-tail morphology that engages cell-surface carbohydrate and junctional adhesion molecule A (JAM-A). The σ1 protein is comprised of three domains partitioned by two flexible linkers termed interdomain regions (IDRs). To determine the importance of σ1 length and flexibility at different stages of reovirus infection, we generated viruses with mutant σ1 molecules of altered length and flexibility and tested these viruses for the capacity to bind the cell surface, internalize, uncoat, induce protein synthesis, assemble, and replicate. We reduced the length of the α-helical σ1 tail to engineer mutants L1 and L2 and deleted midpoint and head-proximal σ1 IDRs to generate ΔIDR1 and ΔIDR2 mutant viruses, respectively. Decreasing length or flexibility of σ1 resulted in delayed reovirus infection and reduced viral titers. L1, L2, and ΔIDR1 viruses but not ΔIDR2 virus displayed reduced cell attachment, but altering σ1 length or flexibility did not diminish the efficiency of virion internalization. Replication of ΔIDR2 virus was hindered at a postdisassembly step. Differences between wild-type and σ1 mutant viruses were not attributable to alterations in σ1 folding, as determined by experiments assessing engagement of cell-surface carbohydrate and JAM-A by the length and IDR mutant viruses. However, ΔIDR1 virus harbored substantially less σ1 on the outer capsid. Taken together, these data suggest that σ1 length is required for reovirus binding to cells. In contrast, IDR1 is required for stable σ1 encapsidation, and IDR2 is required for a postuncoating replication step. Thus, the structural architecture of σ1 is required for efficient reovirus infection of host cells.
Asunto(s)
Proteínas de la Cápside/metabolismo , Regulación Viral de la Expresión Génica , Reoviridae/metabolismo , Animales , Cápside/metabolismo , Carbohidratos/química , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Eliminación de Gen , Genoma Viral , Células HeLa , Humanos , Ratones , Mutación , Unión Proteica , Pliegue de Proteína , Receptores de Superficie Celular/metabolismo , Replicación ViralRESUMEN
Mammalian reoviruses display serotype-specific patterns of tropism and disease in the murine central nervous system (CNS) attributable to polymorphisms in viral attachment protein σ1. While all reovirus serotypes use junctional adhesion molecule-A as a cellular receptor, they differ in their utilization of carbohydrate coreceptors. This observation raises the possibility that carbohydrate binding by σ1 influences reovirus pathology in the CNS. In this study, we sought to define the function of carbohydrate binding in reovirus neuropathogenesis. Newborn mice were inoculated intramuscularly with wild-type strain type 3 Dearing (T3D) and T3D-σ1R202W, a point mutant T3D derivative that does not bind sialic acid (SA). Infected mice were monitored for survival, and viral loads at the sites of primary and secondary replication were quantified. Fewer mice inoculated with the wild-type virus survived in comparison to those inoculated with the mutant virus. The wild-type virus also produced higher titers in the spinal cord and brain at late times postinoculation but lower titers in the liver in comparison to those produced by the mutant virus. In addition, the wild-type virus was more virulent and produced higher titers in the brain than the mutant following intracranial inoculation. These animal infectivity studies suggest that T3D-σ1R202W harbors a defect in neural growth. Concordantly, compared with the wild-type virus, the mutant virus displayed a decreased capacity to infect and replicate in primary cultures of cortical neurons, a property dependent on cell surface SA. These results suggest that SA binding enhances the kinetics of reovirus replication in neural tissues and highlight a functional role for sialylated glycans as reovirus coreceptors in the CNS.
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Sistema Nervioso Central/virología , Orthoreovirus Mamífero 3/patogenicidad , Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/metabolismo , Receptores Virales/metabolismo , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Inmunohistoquímica , Orthoreovirus Mamífero 3/aislamiento & purificación , Orthoreovirus Mamífero 3/metabolismo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Carga Viral , Virulencia , Replicación ViralRESUMEN
Many viruses attach to target cells by binding to cell-surface glycans. To gain a better understanding of strategies used by viruses to engage carbohydrate receptors, we determined the crystal structures of reovirus attachment protein σ1 in complex with α-2,3-sialyllactose, α-2,6-sialyllactose, and α-2,8-di-siallylactose. All three oligosaccharides terminate in sialic acid, which serves as a receptor for the reovirus serotype studied here. The overall structure of σ1 resembles an elongated, filamentous trimer. It contains a globular head featuring a compact ß-barrel, and a fibrous extension formed by seven repeating units of a triple ß-spiral that is interrupted near its midpoint by a short α-helical coiled coil. The carbohydrate-binding site is located between ß-spiral repeats two and three, distal from the head. In all three complexes, the terminal sialic acid forms almost all of the contacts with σ1 in an identical manner, while the remaining components of the oligosaccharides make little or no contacts. We used this structural information to guide mutagenesis studies to identify residues in σ1 that functionally engage sialic acid by assessing hemagglutination capacity and growth in murine erythroleukemia cells, which require sialic acid binding for productive infection. Our studies using σ1 mutant viruses reveal that residues 198, 202, 203, 204, and 205 are required for functional binding to sialic acid by reovirus. These findings provide insight into mechanisms of reovirus attachment to cell-surface glycans and contribute to an understanding of carbohydrate binding by viruses. They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to endow other trimeric proteins with carbohydrate-binding properties.
Asunto(s)
Proteínas de la Cápside/química , Ácido N-Acetilneuramínico/química , Oligosacáridos/química , Orthoreovirus de los Mamíferos/química , Sustitución de Aminoácidos , Proteínas de la Cápside/genética , Cristalografía por Rayos X , Mutación Missense , Ácido N-Acetilneuramínico/genética , Oligosacáridos/genética , Orthoreovirus de los Mamíferos/genética , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Adenoviruses (Ads) are important human pathogens and valuable gene delivery vehicles. We report here the crystal structure of the species B Ad11 knob complexed with the Ad11-binding region of its receptor CD46. The conformation of bound CD46 differs profoundly from its unbound state, with the bent surface structure straightened into an elongated rod. This mechanism of interaction is likely to be conserved among many pathogens that target CD46 or related molecules.
Asunto(s)
Adenovirus Humanos/metabolismo , Receptores Virales/química , Proteínas Virales/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , Receptores Virales/metabolismo , Proteínas Virales/metabolismoRESUMEN
Adenoviruses (Ads) are icosahedral, nonenveloped viruses with a double-stranded DNA genome. The 51 known Ad serotypes exhibit profound variations in cell tropism and disease types. The number of observed Ad infections is steadily increasing, sometimes leading to fatal outcomes even in healthy individuals. Species B Ads can cause kidney infections, hemorrhagic cystitis, and severe respiratory infections, and most of them use the membrane cofactor protein CD46 as a cellular receptor. The crystal structure of the human Ad type 11 (Ad11) knob complexed with CD46 is known; however, the determinants of CD46 binding in related species B Ads remain unclear. We report here a structural and functional analysis of the Ad11 knob, as well as the Ad7 and Ad14 knobs, which are closely related in sequence to the Ad11 knob but have altered CD46-binding properties. The comparison of the structures of the three knobs, which we determined at very high resolution, provides a platform for understanding these differences and allows us to propose a mechanism for productive high-affinity engagement of CD46. At the center of this mechanism is an Ad knob arginine that needs to switch its orientation in order to engage CD46 with high affinity. Quantum chemical calculations showed that the CD46-binding affinity of Ad11 is significantly higher than that of Ad7. Thus, while Ad7 and Ad14 also bind CD46, the affinity and kinetics of these interactions suggest that these Ads are unlikely to use CD46 productively. The proposed mechanism is likely to determine the receptor usage of all CD46-binding Ads.
Asunto(s)
Adenoviridae/química , Adenoviridae/fisiología , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Proteína Cofactora de Membrana/metabolismo , Acoplamiento Viral , Cristalografía por Rayos X , Humanos , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Amyloid fibrils are best known as a product of human and animal protein misfolding disorders, where amyloid formation is associated with cytotoxicity and disease. It is now evident that for some proteins, the amyloid state constitutes the native structure and serves a functional role. These functional amyloids are proving widespread in bacteria and fungi, fulfilling diverse functions as structural components in biofilms or spore coats, as toxins and surface-active fibers, as epigenetic material, peptide reservoirs or adhesins mediating binding to and internalization into host cells. In this review, we will focus on the role of functional amyloids in bacterial pathogenesis. The role of functional amyloids as virulence factor is diverse but mostly indirect. Nevertheless, functional amyloid pathways deserve consideration for the acute and long-term effects of the infectious disease process and may form valid antimicrobial targets.
Asunto(s)
Amiloide/metabolismo , Bacterias/metabolismo , Bacterias/patogenicidad , Infecciones Bacterianas/microbiología , Proteínas Bacterianas/metabolismo , Amiloide/química , Amiloide/ultraestructura , Amiloidosis/etiología , Amiloidosis/metabolismo , Animales , Antígenos/inmunología , Antígenos/metabolismo , Bacterias/genética , Bacterias/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biopelículas , Humanos , Multimerización de Proteína , Toxinas Biológicas/inmunología , Toxinas Biológicas/metabolismo , VirulenciaRESUMEN
Viral infections are initiated by attachment of the virus to host cell surface receptors, including sialic acid-containing glycans. It is now possible to rapidly identify specific glycan receptors using glycan array screening, to define atomic-level structures of virus-glycan complexes and to alter the glycan-binding site to determine the function of glycan engagement in viral disease. This Review highlights general principles of virus-glycan interactions and provides specific examples of sialic acid binding by viruses with stalk-like attachment proteins, including influenza virus, reovirus, adenovirus and rotavirus. Understanding virus-glycan interactions is essential to combating viral infections and designing improved viral vectors for therapeutic applications.
Asunto(s)
Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/metabolismo , Receptores de Superficie Celular/metabolismo , Virosis/virología , Virus/metabolismo , Animales , Sitios de Unión , Interacciones Huésped-Patógeno , Humanos , Receptores Virales/metabolismo , Especificidad de la Especie , Acoplamiento ViralRESUMEN
Intestinal barrier function is regulated by epithelial tight junctions (TJs), structures that control paracellular permeability. Junctional adhesion molecule-A (JAM-A) is a TJ-associated protein that regulates barrier; however, mechanisms linking JAM-A to epithelial permeability are poorly understood. Here we report that JAM-A associates directly with ZO-2 and indirectly with afadin, and this complex, along with PDZ-GEF1, activates the small GTPase Rap2c. Supporting a functional link, small interfering RNA-mediated down-regulation of the foregoing regulatory proteins results in enhanced permeability similar to that observed after JAM-A loss. JAM-A-deficient mice and cultured epithelial cells demonstrate enhanced paracellular permeability to large molecules, revealing a potential role of JAM-A in controlling perijunctional actin cytoskeleton in addition to its previously reported role in regulating claudin proteins and small-molecule permeability. Further experiments suggest that JAM-A does not regulate actin turnover but modulates activity of RhoA and phosphorylation of nonmuscle myosin, both implicated in actomyosin contraction. These results suggest that JAM-A regulates epithelial permeability via association with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and control contraction of the apical cytoskeleton.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Proteína de la Zonula Occludens-2/metabolismo , Proteínas ras/metabolismo , Animales , Proteínas de la Cápside/metabolismo , Moléculas de Adhesión Celular/deficiencia , Línea Celular , Permeabilidad de la Membrana Celular , Polaridad Celular , Citoesqueleto/metabolismo , Regulación hacia Abajo , Endocitosis , Humanos , Ratones , Modelos Biológicos , Peso Molecular , Unión Proteica , Transporte de Proteínas , Receptores de Superficie Celular/deficiencia , Uniones Estrechas/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Sulfolobus turreted icosahedral virus (STIV) infects Sulfolobus species found in the hot springs of Yellowstone National Park. Its 37 open reading frames (ORFs) generally lack sequence similarity to other genes. One exception, however, is ORF B116. While its function is unknown, orthologs are found in three additional crenarchaeal viral families. Due to the central importance of this protein family to crenarchaeal viruses, we have undertaken structural and biochemical studies of B116. The structure reveals a previously unobserved fold consisting of a five-stranded beta-sheet flanked on one side by three alpha helices. Two subunits come together to form a homodimer with a 10-stranded mixed beta-sheet, where the topology of the central strands resembles an unclosed beta-barrel. Highly conserved loops rise above the surface of the saddle-shaped protein and suggest an interaction with the major groove of DNA. The predicted B116-DNA interaction is confirmed by electrophoretic mobility shift assays.
Asunto(s)
Virus de Archaea/química , Proteínas de Unión al ADN/química , Sulfolobus/virología , Proteínas Virales/química , Secuencia de Aminoácidos , Virus de Archaea/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/fisiología , Estructura Secundaria de Proteína , Alineación de Secuencia , Proteínas Virales/genética , Proteínas Virales/metabolismo , Microbiología del AguaRESUMEN
Sulfolobus turreted icosahedral virus (STIV) was the first non-tailed icosahedral virus to be isolated from an archaeal host. Like other archaeal viruses, its 37 open reading frames generally lack sequence similarity to genes with known function. The roles of the gene products in this and other archaeal viruses are thus largely unknown. However, a protein's three-dimensional structure may provide functional and evolutionary insight in cases of minimal sequence similarity. In this vein, the structure of STIV F93 reveals a homodimer with strong similarity to the winged-helix family of DNA-binding proteins. Importantly, an interchain disulfide bond is found at the dimer interface, prompting analysis of the cysteine distribution in the putative intracellular proteins of the viral proteome. The analysis suggests that intracellular disulfide bonds are common in cellular STIV proteins, where they enhance the thermostability of the viral proteome.
Asunto(s)
Virus de Archaea/química , Disulfuros/química , Calor , Sulfolobus/virología , Proteínas Virales/química , Virus de Archaea/genética , Virus de Archaea/metabolismo , Cristalización , Cisteína/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Electroforesis en Gel de Poliacrilamida , Secuencias Hélice-Giro-Hélice , Modelos Moleculares , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Sulfolobus turreted icosahedral virus (STIV) was the first icosahedral virus characterized from an archaeal host. It infects Sulfolobus species that thrive in the acidic hot springs (pH 2.9 to 3.9 and 72 to 92 degrees C) of Yellowstone National Park. The overall capsid architecture and the structure of its major capsid protein are very similar to those of the bacteriophage PRD1 and eukaryotic viruses Paramecium bursaria Chlorella virus 1 and adenovirus, suggesting a viral lineage that predates the three domains of life. The 17,663-base-pair, circular, double-stranded DNA genome contains 36 potential open reading frames, whose sequences generally show little similarity to other genes in the sequence databases. However, functional and evolutionary information may be suggested by a protein's three-dimensional structure. To this end, we have undertaken structural studies of the STIV proteome. Here we report our work on A197, the product of an STIV open reading frame. The structure of A197 reveals a GT-A fold that is common to many members of the glycosyltransferase superfamily. A197 possesses a canonical DXD motif and a putative catalytic base that are hallmarks of this family of enzymes, strongly suggesting a glycosyltransferase activity for A197. Potential roles for the putative glycosyltransferase activity of A197 and their evolutionary implications are discussed.