RESUMEN
Parainfluenza viruses enter host cells by fusing the viral and target cell membranes via concerted action of their two envelope glycoproteins: the hemagglutinin-neuraminidase (HN) and the fusion protein (F). Receptor-bound HN triggers F to undergo conformational changes that render it fusion-competent. To address the role of receptor engagement and to elucidate how HN and F interact during the fusion process, we used bimolecular fluorescence complementation to follow the dynamics of human parainfluenza virus type 3 (HPIV3) HN/F pairs in living cells. We show that HN and F associate before receptor engagement. HN drives the formation of HN-F clusters at the site of fusion, and alterations in HN-F interaction determine the fusogenicity of the glycoprotein pair. An interactive site, at the HN dimer interface modulates HN fusion activation property, which is critical for infection of the natural host. This first evidence for the sequence of initial events that lead to viral entry may indicate a new paradigm for understanding Paramyxovirus infection.
Asunto(s)
Hemaglutininas Virales/metabolismo , Neuraminidasa/metabolismo , Virus de la Parainfluenza 3 Humana/fisiología , Proteínas Virales de Fusión/metabolismo , Internalización del Virus , Células HEK293 , Hemaglutininas Virales/química , Hemaglutininas Virales/genética , Humanos , Modelos Moleculares , Imagen Molecular , Mutación , Neuraminidasa/química , Neuraminidasa/genética , Virus de la Parainfluenza 3 Humana/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Receptores de Superficie Celular/metabolismo , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
The hemagglutinin (HA)-neuraminidase protein (HN) of paramyxoviruses carries out three discrete activities, each of which affects the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. Binding of HN to its sialic acid receptor on a target cell triggers its activation of the fusion protein (F), which then inserts into the target cell and mediates the membrane fusion that initiates infection. We provide new evidence for a fourth function of HN: stabilization of the F protein in its pretriggered state before activation. Influenza virus hemagglutinin protein (uncleaved HA) was used as a nonspecific binding protein to tether F-expressing cells to target cells, and heat was used to activate F, indicating that the prefusion state of F can be triggered to initiate structural rearrangement and fusion by temperature. HN expression along with uncleaved HA and F enhances the F activation if HN is permitted to engage the receptor. However, if HN is prevented from engaging the receptor by the use of a small compound, temperature-induced F activation is curtailed. The results indicate that HN helps stabilize the prefusion state of F, and analysis of a stalk domain mutant HN reveals that the stalk domain of HN mediates the F-stabilization effect.
Asunto(s)
Proteína HN/metabolismo , Virus de la Parainfluenza 1 Humana/fisiología , Proteínas Virales de Fusión/metabolismo , Internalización del Virus , Línea Celular , Citometría de Flujo , Humanos , Microscopía Fluorescente , Estabilidad Proteica , Receptores de Superficie Celular/metabolismo , Temperatura , beta-GalactosidasaRESUMEN
Paramyxoviruses, including the childhood pathogen human parainfluenza virus type 3, enter host cells by fusion of the viral and target cell membranes. This fusion results from the concerted action of its two envelope glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). The receptor-bound HN triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We proposed that, if the fusion process could be activated prematurely before the virion reaches the target host cell, infection could be prevented. We identified a small molecule that inhibits paramyxovirus entry into target cells and prevents infection. We show here that this compound works by an interaction with HN that results in F-activation prior to receptor binding. The fusion process is thereby prematurely activated, preventing fusion of the viral membrane with target cells and precluding viral entry. This first evidence that activation of a paramyxovirus F can be specifically induced before the virus contacts its target cell suggests a new strategy with broad implications for the design of antiviral agents.
Asunto(s)
Proteína HN/química , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/metabolismo , Adsorción , Animales , Antivirales/farmacología , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Células Epiteliales/citología , Humanos , Fusión de Membrana , Modelos Químicos , Neuraminidasa/metabolismo , Conformación Proteica , Proteínas Virales/químicaRESUMEN
In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses.
Asunto(s)
Colesterol/uso terapéutico , Infecciones por Henipavirus/prevención & control , Virus Nipah/fisiología , Paramyxovirinae/fisiología , Proteínas Virales de Fusión/antagonistas & inhibidores , Internalización del Virus , Secuencias de Aminoácidos/efectos de los fármacos , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Chlorocebus aethiops , Colesterol/química , Colesterol/farmacología , Regulación hacia Abajo , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/terapia , Humanos , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Terapia Molecular Dirigida , Virus Nipah/efectos de los fármacos , Virus Nipah/inmunología , Virus Nipah/patogenicidad , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/uso terapéutico , Células Vero , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/metabolismo , Proteínas Virales de Fusión/fisiologíaRESUMEN
The fusion of enveloped viruses with the host cell is driven by specialized fusion proteins to initiate infection. The "class I" fusion proteins harbor two regions, typically two heptad repeat (HR) domains, which are central to the complex conformational changes leading to fusion: the first heptad repeat (HRN) is adjacent to the fusion peptide, while the second (HRC) immediately precedes the transmembrane domain. Peptides derived from the HR regions can inhibit fusion, and one HR peptide, T20 (enfuvirtide), is in clinical use for HIV-1. For paramyxoviruses, the activities of two membrane proteins, the receptor-binding protein (hemagglutinin-neuraminidase [HN] or G) and the fusion protein (F), initiate viral entry. The binding of HN or G to its receptor on a target cell triggers the activation of F, which then inserts into the target cell and mediates the membrane fusion that initiates infection. We have shown that for paramyxoviruses, the inhibitory efficacy of HR peptides is inversely proportional to the rate of F activation. For HIV-1, the antiviral potency of an HRC-derived peptide can be dramatically increased by targeting it to the membrane microdomains where fusion occurs, via the addition of a cholesterol group. We report here that for three paramyxoviruses-human parainfluenza virus type 3 (HPIV3), a major cause of lower respiratory tract diseases in infants, and the emerging zoonotic viruses Hendra virus (HeV) and Nipah virus (NiV), which cause lethal central nervous system diseases-the addition of cholesterol to a paramyxovirus HRC-derived peptide increased antiviral potency by 2 log units. Our data suggest that this enhanced activity is indeed the result of the targeting of the peptide to the plasma membrane, where fusion occurs. The cholesterol-tagged peptides on the cell surface create a protective antiviral shield, target the F protein directly at its site of action, and expand the potential utility of inhibitory peptides for paramyxoviruses.
Asunto(s)
Antivirales/farmacología , Virus Hendra/fisiología , Virus Nipah/fisiología , Virus de la Parainfluenza 3 Humana/fisiología , Internalización del Virus/efectos de los fármacos , Animales , Antivirales/química , Línea Celular , Chlorocebus aethiops , Virus Hendra/efectos de los fármacos , Humanos , Virus Nipah/efectos de los fármacos , Virus de la Parainfluenza 3 Humana/efectos de los fármacosRESUMEN
Three discrete activities of the paramyxovirus hemagglutinin-neuraminidase (HN) protein, receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein, each affect the promotion of viral fusion and entry. For human parainfluenza virus type 3 (HPIV3), the effects of specific mutations that alter these functions of the receptor-binding protein have been well characterized using cultured monolayer cells, which have identified steps that are potentially relevant to pathogenesis. In the present study, proposed mechanisms that are relevant to pathogenesis were tested in natural host cell cultures, a model of the human airway epithelium (HAE) in which primary HAE cells are cultured at an air-liquid interface and retain functional properties. Infection of HAE cells with wild-type HPIV3 and variant viruses closely reflects that seen in an animal model, the cotton rat, suggesting that HAE cells provide an ideal system for assessing the interplay of host cell and viral factors in pathogenesis and for screening for inhibitory molecules that would be effective in vivo. Both HN's receptor avidity and the function and timing of F activation by HN require a critical balance for the establishment of ongoing infection in the HAE, and these HN functions independently modulate the production of active virions. Alterations in HN's F-triggering function lead to the release of noninfectious viral particles and a failure of the virus to spread. The finding that the dysregulation of F triggering prohibits successful infection in HAE cells suggests that antiviral strategies targeted to HN's F-triggering activity may have promise in vivo.
Asunto(s)
Proteína HN/metabolismo , Virus de la Parainfluenza 3 Humana/patogenicidad , Infecciones por Paramyxoviridae/virología , Proteínas Virales de Fusión/metabolismo , Animales , Línea Celular , Femenino , Regulación Viral de la Expresión Génica , Humanos , Pulmón/patología , Pulmón/virología , Ratas , Ratas Endogámicas , Receptores Virales/metabolismoRESUMEN
The structures of canine parvovirus (CPV) and feline parvovirus (FPV) complexed with antibody fragments from eight different neutralizing monoclonal antibodies were determined by cryo-electron microscopy (cryoEM) reconstruction to resolutions varying from 8.5 to 18 A. The crystal structure of one of the Fab molecules and the sequence of the variable domain for each of the Fab molecules have been determined. The structures of Fab fragments not determined crystallographically were predicted by homology modeling according to the amino acid sequence. Fitting of the Fab and virus structures into the cryoEM densities identified the footprints of each antibody on the viral surface. As anticipated from earlier analyses, the Fab binding sites are directed to two epitopes, A and B. The A site is on an exposed part of the surface near an icosahedral threefold axis, whereas the B site is about equidistant from the surrounding five-, three-, and twofold axes. One antibody directed to the A site binds CPV but not FPV. Two of the antibodies directed to the B site neutralize the virus as Fab fragments. The differences in antibody properties have been linked to the amino acids within the antibody footprints, the position of the binding site relative to the icosahedral symmetry elements, and the orientation of the Fab structure relative to the surface of the virus. Most of the exposed surface area was antigenic, although each of the antibodies had a common area of overlap that coincided with the positions of the previously mapped escape mutations.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Cápside/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Parvovirus/química , Parvovirus/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/ultraestructura , Especificidad de Anticuerpos , Antígenos/química , Antígenos/inmunología , Cápside/química , Cápside/ultraestructura , Biología Computacional , Secuencia Conservada , Microscopía por Crioelectrón , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Parvovirus/ultraestructura , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
Human paramyxoviruses include global causes of lower respiratory disease like the parainfluenza viruses, as well as agents of lethal encephalitis like Nipah virus. Infection is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. Paramyxovirus viral fusion proteins (F) insert into the target cell membrane, and form a transient intermediate that pulls the viral and cell membranes together as two heptad-repeat regions refold to form a six-helix bundle structure that can be specifically targeted by fusion-inhibitory peptides. Antiviral potency can be improved by sequence modification and lipid conjugation, and by adding linkers between the protein and lipid components. We exploit the uniquely broad spectrum antiviral activity of a parainfluenza F-derived peptide sequence that inhibits both parainfluenza and Nipah viruses, to investigate the influence of peptide orientation and intervening linker length on the peptides' interaction with transitional states of F, solubility, membrane insertion kinetics, and protease sensitivity. We assessed the impact of these features on biodistribution and antiviral efficacy in vitro and in vivo. The engineering approach based on biophysical parameters resulted in a peptide that is a highly effective inhibitor of both paramyxoviruses and a set of criteria to be used for engineering broad spectrum antivirals for emerging paramyxoviruses.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Paramyxoviridae/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Proteínas Virales de Fusión/antagonistas & inhibidores , Proteínas Virales de Fusión/química , Secuencia de Aminoácidos , Animales , Antivirales/farmacocinética , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Cricetinae , Estructura Molecular , Péptidos/farmacocinética , Unión Proteica , Ratas , Solubilidad , Ensayo de Placa ViralRESUMEN
Recapitulation of lung development from human pluripotent stem cells (hPSCs) in three dimensions (3D) would allow deeper insight into human development, as well as the development of innovative strategies for disease modelling, drug discovery and regenerative medicine. We report here the generation from hPSCs of lung bud organoids (LBOs) that contain mesoderm and pulmonary endoderm and develop into branching airway and early alveolar structures after xenotransplantation and in Matrigel 3D culture. Expression analysis and structural features indicated that the branching structures reached the second trimester of human gestation. Infection in vitro with respiratory syncytial virus, which causes small airway obstruction and bronchiolitis in infants, led to swelling, detachment and shedding of infected cells into the organoid lumens, similar to what has been observed in human lungs. Introduction of mutation in HPS1, which causes an early-onset form of intractable pulmonary fibrosis, led to accumulation of extracellular matrix and mesenchymal cells, suggesting the potential use of this model to recapitulate fibrotic lung disease in vitro. LBOs therefore recapitulate lung development and may provide a useful tool to model lung disease.
Asunto(s)
Técnicas de Cultivo de Célula , Pulmón/patología , Organoides/patología , Células Madre Pluripotentes/patología , Fibrosis Pulmonar/patología , Infecciones por Virus Sincitial Respiratorio/patología , Ingeniería de Tejidos/métodos , Animales , Diferenciación Celular , Células Cultivadas , Femenino , Predisposición Genética a la Enfermedad , Humanos , Pulmón/metabolismo , Pulmón/virología , Trasplante de Pulmón , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos NOD , Mutación , Organogénesis , Organoides/metabolismo , Organoides/trasplante , Organoides/virología , Fenotipo , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/trasplante , Células Madre Pluripotentes/virología , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Factores de TiempoRESUMEN
UNLABELLED: Respiratory paramyxoviruses, including the highly prevalent human parainfluenza viruses, cause the majority of childhood croup, bronchiolitis, and pneumonia, yet there are currently no vaccines or effective treatments. Paramyxovirus research has relied on the study of laboratory-adapted strains of virus in immortalized cultured cell lines. We show that findings made in such systems about the receptor interaction and viral fusion requirements for entry and fitness-mediated by the receptor binding protein and the fusion protein-can be drastically different from the requirements for infection in vivo. Here we carried out whole-genome sequencing and genomic analysis of circulating human parainfluenza virus field strains to define functional and structural properties of proteins of circulating strains and to identify the genetic basis for properties that confer fitness in the field. The analysis of clinical strains suggests that the receptor binding-fusion molecule pairs of circulating viruses maintain a balance of properties that result in an inverse correlation between fusion in cultured cells and growth in vivo. Future analysis of entry mechanisms and inhibitory strategies for paramyxoviruses will benefit from considering the properties of viruses that are fit to infect humans, since a focus on viruses that have adapted to laboratory work provides a distinctly different picture of the requirements for the entry step of infection. IMPORTANCE: Mechanistic information about viral infection-information that impacts antiviral and vaccine development-is generally derived from viral strains grown under laboratory conditions in immortalized cells. This study uses whole-genome sequencing of clinical strains of human parainfluenza virus 3-a globally important respiratory paramyxovirus-in cell systems that mimic the natural human host and in animal models. By examining the differences between clinical isolates and laboratory-adapted strains, the sequence differences are correlated to mechanistic differences in viral entry. For this ubiquitous and pathogenic respiratory virus to infect the human lung, modulation of the processes of receptor engagement and fusion activation occur in a manner quite different from that carried out by the entry glycoprotein-expressing pair of laboratory strains. These marked contrasts in the viral properties necessary for infection in cultured immortalized cells and in natural host tissues and animals will influence future basic and clinical studies.
Asunto(s)
Sistema Respiratorio/virología , Respirovirus/fisiología , Internalización del Virus , Animales , Genoma Viral , Humanos , Respirovirus/aislamiento & purificación , Respirovirus/patogenicidad , Respirovirus/ultraestructura , Infecciones por Respirovirus/virología , Análisis de Secuencia de ADN , Sigmodontinae , VirulenciaRESUMEN
UNLABELLED: Paramyxoviruses, including the human pathogen measles virus (MV), enter host cells by fusing their viral envelope with the target cell membrane. This fusion process is driven by the concerted actions of the two viral envelope glycoproteins, the receptor binding protein (hemagglutinin [H]) and the fusion (F) protein. H attaches to specific proteinaceous receptors on host cells; once the receptor engages, H activates F to directly mediate lipid bilayer fusion during entry. In a recent MV outbreak in South Africa, several HIV-positive people died of MV central nervous system (CNS) infection. We analyzed the virus sequences from these patients and found that specific intrahost evolution of the F protein had occurred and resulted in viruses that are "CNS adapted." A mutation in F of the CNS-adapted virus (a leucine-to-tryptophan change present at position 454) allows it to promote fusion with less dependence on engagement of H by the two known wild-type (wt) MV cellular receptors. This F protein is activated independently of H or the receptor and has reduced thermal stability and increased fusion activity compared to those of the corresponding wt F. These functional effects are the result of the single L454W mutation in F. We hypothesize that in the absence of effective cellular immunity, such as HIV infection, MV variants bearing altered fusion machinery that enabled efficient spread in the CNS underwent positive selection. IMPORTANCE: Measles virus has become a concern in the United States and Europe due to recent outbreaks and continues to be a significant global problem. While live immunization is available, there are no effective therapies or prophylactics to combat measles infection in unprotected people. Additionally, vaccination does not adequately protect immunocompromised people, who are vulnerable to the more severe CNS manifestations of disease. We found that strains isolated from patients with measles virus infection of the CNS have fusion properties different from those of strains previously isolated from patients without CNS involvement. Specifically, the viral entry machinery is more active and the virus can spread, even in the absence of H. Our findings are consistent with an intrahost evolution of the fusion machinery that leads to neuropathogenic MV variants.
Asunto(s)
Enfermedades del Sistema Nervioso Central/virología , Infecciones por VIH/complicaciones , Virus del Sarampión/fisiología , Sarampión/virología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/virología , Enfermedades del Sistema Nervioso Central/etiología , Enfermedades del Sistema Nervioso Central/metabolismo , Hemaglutininas/genética , Hemaglutininas/metabolismo , Humanos , Sarampión/etiología , Sarampión/metabolismo , Virus del Sarampión/genética , Virus del Sarampión/aislamiento & purificación , Mutación Missense , Receptores Virales/metabolismo , Internalización del VirusRESUMEN
The Paramyxoviridae family includes many viruses that are pathogenic in humans, including parainfluenza viruses, measles virus, respiratory syncytial virus, and the emerging zoonotic Henipaviruses. No effective treatments are currently available for these viruses, and there is a need for efficient antiviral therapies. Paramyxoviruses enter the target cell by binding to a cell surface receptor and then fusing the viral envelope with the target cell membrane, allowing the release of the viral genome into the cytoplasm. Blockage of these crucial steps prevents infection and disease. Binding and fusion are driven by two virus-encoded glycoproteins, the receptor-binding protein and the fusion protein, that together form the viral "fusion machinery." The development of efficient antiviral drugs requires a deeper understanding of the mechanism of action of the Paramyxoviridae fusion machinery, which is still controversial. Here, we review recent structural and functional data on these proteins and the current understanding of the mechanism of the paramyxovirus cell entry process.
Asunto(s)
Paramyxoviridae/fisiología , Internalización del Virus , Animales , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Paramyxoviridae/clasificación , Unión Proteica , Receptores de Superficie Celular/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
UNLABELLED: Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain's HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN's altered ability to activate F and reveal properties that are critical for infection in vivo. IMPORTANCE: Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by specific interactions between the receptor binding molecule (hemagglutinin-neuraminidase [HN]) and the fusion protein (F). Here we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion and directly impact infection. The crystallographic and biochemical data point to a structural explanation for the HN's altered ability to activate F for fusion and reveal properties that are critical for infection by this important lung virus in vivo.
Asunto(s)
Proteína HN/metabolismo , Virus de la Parainfluenza 3 Humana/crecimiento & desarrollo , Virus de la Parainfluenza 3 Humana/metabolismo , Infecciones por Respirovirus/virología , Proteínas Virales de Fusión/metabolismo , Proteínas Virales/metabolismo , Animales , Cristalografía por Rayos X , Dimerización , Femenino , Proteína HN/química , Proteína HN/genética , Humanos , Virus de la Parainfluenza 3 Humana/enzimología , Virus de la Parainfluenza 3 Humana/genética , Unión Proteica , Ratas , Sigmodontinae , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
UNLABELLED: Paramyxoviruses, a family of RNA enveloped viruses that includes human parainfluenza virus type 3 (HPIV3), cause the majority of childhood croup, bronchiolitis, and pneumonia worldwide. Infection starts with host cell receptor binding and fusion of the viral envelope with the cell membrane at the cell surface. The fusion process requires interaction of the two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). We have previously shown that viruses with an HN/F pair that is highly fusogenic in monolayers of immortalized cells due to mutations in HN's secondary sialic acid binding site are growth impaired in differentiated human airway epithelium (HAE) cultures and in vivo. Here we have shown that adaptation of HPIV3 to growth in the lung is determined by specific features of HN and F that are different from those required for growth in cultured immortalized cells. An HPIV3 virus bearing a mutated HN (H552Q), which is fit and fusogenic in immortalized cells but unfit for growth in the lung, evolved into a less-fusogenic but viable virus in differentiated human airway epithelium. Stepwise evolution led to a progressive decrease in efficiency of fusion activation by the HN/F pair, with a mutation in F first decreasing the activation of F by HN and a mutation in HN's secondary sialic acid binding site decreasing fusion activation further and producing a stable virus. Adaptation of HPIV3 to successful growth in HAE is determined by specific features of HN and F that lead to a less easily activated fusion mechanism. IMPORTANCE: Human parainfluenza viruses (HPIVs) are paramyxoviruses that cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide, but there are currently no vaccines or antivirals available for treatment. Enveloped viruses must fuse their membrane with the target cell membrane in order to initiate infection. Parainfluenza virus fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. The receptor-binding hemagglutinin-neuraminidase (HN), upon receptor engagement, activates the fusion protein (F) to penetrate the target cell and mediate viral entry. In this study, we show that the precise balance of fusion activation properties of these two glycoproteins during entry is key for infection. In clinically relevant tissues, viruses evolve to acquire a set of fusion features that provide key clues about requirements for infection in human beings.
Asunto(s)
Adaptación Biológica , Células Epiteliales/virología , Proteína HN/genética , Virus de la Parainfluenza 3 Humana/fisiología , Proteínas Virales de Fusión/genética , Internalización del Virus , Animales , Línea Celular , Chlorocebus aethiops , Evolución Molecular , Humanos , Modelos Moleculares , Proteínas Mutantes/genética , Mutación Missense , Virus de la Parainfluenza 3 Humana/genética , Virus de la Parainfluenza 3 Humana/crecimiento & desarrollo , Virus de la Parainfluenza 3 Humana/patogenicidad , Conformación ProteicaRESUMEN
Paramyxoviruses, including the childhood respiratory pathogen human parainfluenza virus type 3 (HPIV3), possess an envelope protein hemagglutinin-neuraminidase (HN) that has receptor-cleaving (neuraminidase), as well as receptor-binding, activity. HN is a type II transmembrane glycoprotein, present on the surface of the virus as a tetramer composed of two dimers. HN is also essential for activating the fusion protein (F) to mediate merger of the viral envelope with the host cell membrane. This initial step of viral entry occurs at the host cell surface at neutral pH. The HN molecule carries out these three different critical activities at specific points in the process of viral entry, and understanding the regulation of these activities is key for the design of strategies that block infection. One bifunctional site (site I) on the HN of HPIV3 possesses both receptor binding and neuraminidase activities, and we recently obtained experimental evidence for a second receptor binding site (site II) on HPIV3 HN. Mutation of HN at specific residues at this site, which is next to the HN dimer interface, confers enhanced fusion properties, without affecting neuraminidase activity or receptor binding at neutral pH. We now demonstrate that mutations at this site II, as well as at site I, confer pH dependence on HN's receptor avidity. These mutations permit pH to modulate the binding and fusion processes of the virus, potentially providing regulation at specific stages of the viral life cycle.
Asunto(s)
Proteína HN/metabolismo , Virus de la Parainfluenza 3 Humana/fisiología , Acoplamiento Viral , Internalización del Virus , Sustitución de Aminoácidos/genética , Sitios de Unión/efectos de los fármacos , Línea Celular , Dimerización , Proteína HN/química , Proteína HN/genética , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación Missense , Virus de la Parainfluenza 3 Humana/genéticaRESUMEN
Antibody binding and neutralization are major host defenses against viruses, yet the mechanisms are often not well understood. Eight monoclonal antibodies and their Fab fragments were tested for neutralization of canine parvovirus and feline panleukopenia virus. All IgGs neutralized >85% of virus infectivity. Two Fabs neutralized when present at 5 nM, while the others gave little or no neutralization even at 20-100 nM. The antibodies bind two antigenic sites on the capsids which overlap the binding site of the host transferrin receptor (TfR). There was no specific correlation between Fab binding affinity and neutralization. All Fabs reduced capsid binding of virus to purified feline TfR in vitro, but the highly neutralizing Fabs were more efficient competitors. All partially prevented binding and uptake of capsids by feline TfR on cells. The virus appears adapted to allow some infectivity in the presence of at least low levels of antibodies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Virus de la Leucemia Felina/inmunología , Animales , Afinidad de Anticuerpos , Antígenos Virales/inmunología , Unión Competitiva/inmunología , Cápside/inmunología , Línea Celular , Epítopos , Inmunoglobulina G/inmunología , Pruebas de Neutralización , Parvovirus Canino/inmunología , Unión Proteica/inmunología , Receptores de Transferrina/inmunologíaRESUMEN
The most extensive structural information on viruses relates to apparently icosahedral virions and is based on X-ray crystallography and on cryo-electron microscopy (cryo-EM) single-particle reconstructions. Both techniques lean heavily on imposing icosahedral symmetry, thereby obscuring any deviation from the assumed symmetry. However, tailed bacteriophages have icosahedral or prolate icosahedral heads that have one obvious unique vertex where the genome can enter for DNA packaging and exit when infecting a host cell. The presence of the tail allows cryo-EM reconstructions in which the special vertex is used to orient the head in a unique manner. Some very large dsDNA icosahedral viruses also develop special vertices thought to be required for infecting host cells. Similarly, preliminary cryo-EM data for the small ssDNA canine parvovirus complexed with receptor suggests that these viruses, previously considered to be accurately icosahedral, might have some asymmetric properties that generate one preferred receptor-binding site on the viral surface. Comparisons are made between rhinoviruses that bind receptor molecules uniformly to all 60 equivalent binding sites, canine parvovirus, which appears to have a preferred receptor-binding site, and bacteriophage T4, which gains major biological advantages on account of its unique vertex and tail organelle.
Asunto(s)
Bacteriófagos/química , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X/métodos , Virus/química , Virus/ultraestructura , Bacteriófagos/ultraestructura , Sitios de Unión , Cápside/química , Cristalización , ADN/química , Empaquetamiento del ADN , Modelos Moleculares , Conformación Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Virión/química , Ensamble de VirusRESUMEN
Although many viruses are icosahedral when they initially bind to one or more receptor molecules on the cell surface, such an interaction is asymmetric, probably causing a breakdown in the symmetry and conformation of the original infecting virion in preparation for membrane penetration and release of the viral genome. Cryoelectron microscopy and biochemical analyses show that transferrin receptor, the cellular receptor for canine parvovirus, can bind to only one or a few of the 60 icosahedrally equivalent sites on the virion, indicating that either canine parvovirus has inherent asymmetry or binding of receptor induces asymmetry. The asymmetry of receptor binding to canine parvovirus is reminiscent of the special portal in tailed bacteriophages and some large, icosahedral viruses. Asymmetric interactions of icosahedral viruses with their hosts might be a more common phenomenon than previously thought and may have been obscured by averaging in previous crystallographic and electron microscopic structure determinations.
Asunto(s)
Cápside/química , Cápside/metabolismo , Parvovirus Canino/química , Parvovirus Canino/metabolismo , Receptores de Transferrina/química , Receptores de Transferrina/metabolismo , Animales , Sitios de Unión , Gatos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Perros , Virus de la Panleucopenia Felina/química , Virus de la Panleucopenia Felina/metabolismo , Virus de la Panleucopenia Felina/ultraestructura , Humanos , Parvovirus Canino/ultraestructura , Unión Proteica , Receptores de Transferrina/genética , Spodoptera , Virión/química , Virión/metabolismoRESUMEN
The cell infection processes and host ranges of canine parvovirus (CPV) and feline panleukopenia virus (FPV) are controlled by their capsid interactions with the transferrin receptors (TfR) on their host cells. Here, we expressed the ectodomains of wild-type and mutant TfR and tested those for binding to purified viral capsids and showed that different naturally variant strains of the viruses were associated with variant interactions with the receptors which likely reflect the optimization of the viral infection processes in the different hosts. While all viruses bound the feline TfR, reflecting their tissue culture host ranges, a naturally variant mutant of CPV (represented by the CPV type-2b strain) that became the dominant virus worldwide in 1979 showed significantly lower levels of binding to the feline TfR. The canine TfR ectodomain did not bind to a detectable level in the in vitro assays, but this appears to reflect the naturally low affinity of that interaction, as only low levels of binding were seen when the receptor was expressed on mammalian cells; however, that was sufficient to allow endocytosis and infection. The apical domain of the canine TfR controls the specific interaction with CPV capsids, as a canine TfR mutant altering a glycosylation site in that domain bound FPV, CPV-2, and CPV-2b capsids efficiently. Enzymatic removal of the N-linked glycans did not allow FPV binding to the canine TfR, suggesting that the protein sequence difference is itself important. The purified feline TfR inhibited FPV and CPV-2 binding and infection of feline cells but not CPV-2b, indicating that the receptor binding may be able to prevent the attachment to the same receptor on cells.
Asunto(s)
Proteínas de la Cápside/metabolismo , Virus de la Panleucopenia Felina/patogenicidad , Parvovirus Canino/patogenicidad , Receptores de Transferrina/metabolismo , Receptores Virales/metabolismo , Animales , Células CHO , Proteínas de la Cápside/genética , Gatos , Línea Celular , Células Cultivadas , Cricetinae , Perros , Virus de la Panleucopenia Felina/metabolismo , Mariposas Nocturnas , Parvovirus Canino/metabolismo , Receptores de Transferrina/química , Receptores Virales/aislamiento & purificación , Especificidad de la Especie , SpodopteraRESUMEN
The feline and canine transferrin receptors (TfRs) bind canine parvovirus to host cells and mediate rapid capsid uptake and infection. The TfR and its ligand transferrin have well-described pathways of endocytosis and recycling. Here we tested several receptor-dependent steps in infection for their role in virus infection of cells. Deletions of cytoplasmic sequences or mutations of the Tyr-Thr-Arg-Phe internalization motif reduced the rate of receptor uptake from the cell surface, while polar residues introduced into the transmembrane sequence resulted in increased degradation of transferrin. However, the mutant receptors still mediated efficient virus infection. In contrast, replacing the cytoplasmic and transmembrane sequences of the feline TfR with those of the influenza virus neuraminidase (NA) resulted in a receptor that bound and endocytosed the capsid but did not mediate viral infection. This chimeric receptor became localized to detergent-insoluble membrane domains. To test the effect of structural virus receptor interaction on infection, two chimeric receptors were prepared which contained antibody-variable domains that bound the capsid in place of the TfR ectodomain. These chimeric receptors bound CPV capsids and mediated uptake but did not result in cell infection. Adding soluble feline TfR ectodomain to the virus during that uptake did not allow infection.