RESUMO
The SARS-CoV-2 Omicron variant with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the spike (S) from Omicron reveals amino acid substitutions forging interactions that stably maintain an active conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of the viral fusion step. Alterations in local conformation, charge, and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Structure of the Omicron S bound with human ACE2, together with the analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members, as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies, sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.
Assuntos
Evasão da Resposta Imune/fisiologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Antivirais/imunologia , Sítios de Ligação , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Microscopia Crioeletrônica , Humanos , Mutagênese Sítio-Dirigida , Testes de Neutralização , Ligação Proteica , Domínios Proteicos/imunologia , Estrutura Quaternária de Proteína , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Ressonância de Plasmônio de Superfície , Ligação ViralRESUMO
Cell-penetrating peptides (CPPs) are short protein segments that can transport cargos into cells. Although CPPs are widely studied as potential drug delivery tools, their role in normal cell physiology is poorly understood. Early during infection, the L2 capsid protein of human papillomaviruses binds retromer, a cytoplasmic trafficking factor required for delivery of the incoming non-enveloped virus into the retrograde transport pathway. Here, we show that the C terminus of HPV L2 proteins contains a conserved cationic CPP that drives passage of a segment of the L2 protein through the endosomal membrane into the cytoplasm, where it binds retromer, thereby sorting the virus into the retrograde pathway for transport to the trans-Golgi network. These experiments define the cell-autonomous biological role of a CPP in its natural context and reveal how a luminal viral protein engages an essential cytoplasmic entry factor.
Assuntos
Proteínas do Capsídeo/metabolismo , Peptídeos Penetradores de Células/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Endossomos/metabolismo , Complexo de Golgi/virologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiologia , Humanos , Mutagênese , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Ligação Viral , Internalização do VírusRESUMO
Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with epithelial-cell cancers and B cell lymphomas. An effective EBV vaccine is not available. We found that antibodies to the EBV glycoprotein gH/gL complex were the principal components in human plasma that neutralized infection of epithelial cells and that antibodies to gH/gL and gp42 contributed to B cell neutralization. Immunization of mice and nonhuman primates with nanoparticle vaccines that displayed components of the viral-fusion machinery EBV gH/gL or gH/gL/gp42 elicited antibodies that potently neutralized both epithelial-cell and B cell infection. Immune serum from nonhuman primates inhibited EBV-glycoprotein-mediated fusion of epithelial cells and B cells and targeted an epitope critical for virus-cell fusion. Therefore, unlike the leading EBV gp350 vaccine candidate, which only protects B cells from infection, these EBV nanoparticle vaccines elicit antibodies that inhibit the virus-fusion apparatus and provide cell-type-independent protection from virus infection.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Células Epiteliais/imunologia , Infecções por Vírus Epstein-Barr/prevenção & controle , Herpesvirus Humano 4/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Linfócitos B/virologia , Células CHO , Fusão Celular , Linhagem Celular Tumoral , Cricetulus , Células Epiteliais/virologia , Infecções por Vírus Epstein-Barr/imunologia , Feminino , Células HEK293 , Células HeLa , Humanos , Soros Imunes/administração & dosagem , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Ligação ViralRESUMO
The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site containing multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.
Assuntos
Betacoronavirus/química , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/química , Animais , Betacoronavirus/fisiologia , COVID-19 , Linhagem Celular , Chlorocebus aethiops , Furina/química , Furina/genética , Furina/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/virologia , Pandemias , SARS-CoV-2 , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Ligação ViralRESUMO
Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and is associated with 200,000 new cases of cancer and 140,000 deaths annually. Subunit vaccines against this pathogen have focused on the gp350 glycoprotein and remain unsuccessful. We isolated human antibodies recognizing the EBV fusion machinery (gH/gL and gB) from rare memory B cells. One anti-gH/gL antibody, AMMO1, potently neutralized infection of B cells and epithelial cells, the two major cell types targeted by EBV. We determined a cryo-electron microscopy reconstruction of the gH/gL-gp42-AMMO1 complex and demonstrated that AMMO1 bound to a discontinuous epitope formed by both gH and gL at the Domain-I/Domain-II interface. Integrating structural, biochemical, and infectivity data, we propose that AMMO1 inhibits fusion of the viral and cellular membranes. This work identifies a crucial epitope that may aid in the design of next-generation subunit vaccines against this major public health burden.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas do Envelope Viral/imunologia , Células 3T3 , Animais , Linfócitos B/virologia , Células CHO , Linhagem Celular , Cricetulus , Microscopia Crioeletrônica , Células Epiteliais/virologia , Epitopos de Linfócito B/imunologia , Células HEK293 , Humanos , Camundongos , Ligação ViralRESUMO
Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans1. VEEV is a biodefence concern because of its potential for aerosol spread and the current lack of sufficient countermeasures. The host factors that are required for VEEV entry and infection remain poorly characterized. Here, using a genome-wide CRISPR-Cas9-based screen, we identify low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3)-a highly conserved yet poorly characterized member of the scavenger receptor superfamily-as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation with LDLRAD3. LDLRAD3 binds directly to VEEV particles and enhances virus attachment and internalization into host cells. Genetic studies indicate that domain 1 of LDLRAD3 (LDLRAD3(D1)) is necessary and sufficient to support infection by VEEV, and both anti-LDLRAD3 antibodies and an LDLRAD3(D1)-Fc fusion protein block VEEV infection in cell culture. The pathogenesis of VEEV infection is abrogated in mice with deletions in Ldlrad3, and administration of LDLRAD3(D1)-Fc abolishes disease caused by several subtypes of VEEV, including highly virulent strains. The development of a decoy-receptor fusion protein suggests a strategy for the prevention of severe VEEV infection and associated disease in humans.
Assuntos
Vírus da Encefalite Equina Venezuelana/metabolismo , Receptores de LDL/metabolismo , Receptores Virais/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/prevenção & controle , Encefalomielite Equina Venezuelana/virologia , Feminino , Teste de Complementação Genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Receptores de LDL/deficiência , Receptores de LDL/genética , Receptores Virais/genética , Ligação Viral , Internalização do VírusRESUMO
Mammalian orthoreoviruses (reoviruses) serve as potential triggers of celiac disease and have oncolytic properties, making these viruses potential cancer therapeutics. Primary attachment of reovirus to host cells is mainly mediated by the trimeric viral protein, σ1, which engages cell-surface glycans, followed by high-affinity binding to junctional adhesion molecule-A (JAM-A). This multistep process is thought to be accompanied by major conformational changes in σ1, but direct evidence is lacking. By combining biophysical, molecular, and simulation approaches, we define how viral capsid protein mechanics influence virus-binding capacity and infectivity. Single-virus force spectroscopy experiments corroborated by in silico simulations show that GM2 increases the affinity of σ1 for JAM-A by providing a more stable contact interface. We demonstrate that conformational changes in σ1 that lead to an extended rigid conformation also significantly increase avidity for JAM-A. Although its associated lower flexibility impairs multivalent cell attachment, our findings suggest that diminished σ1 flexibility enhances infectivity, indicating that fine-tuning of σ1 conformational changes is required to successfully initiate infection. Understanding properties underlying the nanomechanics of viral attachment proteins offers perspectives in the development of antiviral drugs and improved oncolytic vectors.
Assuntos
Orthoreovirus , Reoviridae , Animais , Proteínas do Capsídeo/química , Reoviridae/metabolismo , Orthoreovirus/metabolismo , Proteínas Virais/metabolismo , Ligação Viral , Anticorpos Antivirais , Mamíferos/metabolismoRESUMO
Nogo-66 receptor 1 (NgR1) binds a variety of structurally dissimilar ligands in the adult central nervous system to inhibit axon extension. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron outgrowth, making NgR1 an important therapeutic target for diverse neurological conditions such as spinal crush injuries and Alzheimer's disease. Human NgR1 serves as a receptor for mammalian orthoreovirus (reovirus), but the mechanism of virus-receptor engagement is unknown. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that central NgR1 surfaces form a bridge between two copies of viral capsid protein σ3, establishing that σ3 serves as a receptor ligand for reovirus. This unusual binding interface produces high-avidity interactions between virus and receptor to prime early entry steps. These studies refine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.
Assuntos
Orthoreovirus , Reoviridae , Animais , Humanos , Receptor Nogo 1/metabolismo , Ligação Viral , Proteínas Virais/metabolismo , Ligantes , Reoviridae/metabolismo , Orthoreovirus/metabolismo , Receptores Virais/metabolismo , Mamíferos/metabolismoRESUMO
RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. Although viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when the entry was bypassed, suggesting that the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in murine BV2 cells and infected mice, with reduced viral titers. These results suggest a fitness tradeoff, where increased fitness in a non-native host cell reduces fitness in a natural host environment. Overall, this work suggests that MNV tropism is determined by the presence of not only the viral receptor but also post-entry factors. IMPORTANCE: Viruses infect specific species and cell types, which is dictated by the expression of host factors required for viral entry as well as downstream replication steps. Murine norovirus (MNV) infects mouse cells, but not human cells. However, human cells expressing the murine CD300lf receptor support MNV replication, suggesting that receptor expression is a major determinant of MNV tropism. To determine whether other factors influence MNV tropism, we selected for variants with enhanced replication in human cells. We identified mutations that enhance MNV replication in human cells and demonstrated that these mutations enhance infection at a post-entry replication step. Therefore, MNV infection of human cells is restricted at both entry and post-entry stages. These results shed new light on factors that influence viral tropism and host range.
Assuntos
Norovirus , Tropismo Viral , Internalização do Vírus , Animais , Humanos , Camundongos , Infecções por Caliciviridae/virologia , Genoma Viral , Células HeLa , Especificidade de Hospedeiro , Mutação , Norovirus/genética , Norovirus/fisiologia , Receptores Virais/metabolismo , Receptores Virais/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Ligação Viral , Replicação ViralRESUMO
Many functions of viral attachment proteins are established, but less is known about the biological importance of viral attachment protein encapsidation efficiency. The mammalian orthoreovirus (reovirus) σ1 attachment protein forms filamentous trimers that incorporate into pentamers of the λ2 capsid protein. Reovirus strains vary in the efficiency of σ1 encapsidation onto progeny virions, which influences viral stability during entry into cells and the efficacy of tumor cell lysis. While the role of σ1 encapsidation has been evaluated in studies using cultured cells, the contribution of attachment protein encapsidation efficiency to viral infection in animals is less clear. Polymorphisms in reovirus σ1 at residues 22 and 249 have been implicated in viral dissemination in mice and susceptibility to proteolysis in the murine intestine, respectively. To determine whether these residues contribute to σ1 encapsidation efficiency, we engineered σ1 mutant viruses with single- and double-residue substitutions at sites 22 and 249. We found that substitutions at these sites alter the encapsidation of σ1 and that reoviruses encapsidating higher amounts of σ1 bind cells more avidly and have a modest replication advantage in a cell-type-specific manner relative to low σ1-encapsidating reoviruses. Furthermore, we found that a high σ1-encapsidating reovirus replicates and disseminates more efficiently in mice relative to a low σ1-encapsidating reovirus. These findings provide evidence of a relationship between viral attachment protein encapsidation efficiency and viral replication in cell culture and animal hosts. IMPORTANCE: Viral attachment proteins can serve multiple functions during viral replication, including attachment to host cells, cell entry and disassembly, and modulation of host immune responses. The relationship between viral attachment protein encapsidation efficiency and viral replication in cells and animals is poorly understood. We engineered and characterized a panel of reoviruses that differ in the capacity to encapsidate the σ1 attachment protein. We found that strains encapsidating σ1 with higher efficiency bind cells more avidly and replicate and spread more efficiently in mice relative to those encapsidating σ1 with lower efficiency. These results highlight a function for σ1 attachment protein capsid abundance in viral replication in cells and animals, which may inform future use of reovirus as an oncolytic therapeutic.
Assuntos
Proteínas do Capsídeo , Infecções por Reoviridae , Replicação Viral , Animais , Camundongos , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Infecções por Reoviridae/virologia , Infecções por Reoviridae/metabolismo , Ligação Viral , Polimorfismo Genético , Orthoreovirus de Mamíferos/genética , Orthoreovirus de Mamíferos/fisiologia , Orthoreovirus de Mamíferos/metabolismo , Montagem de Vírus , Linhagem Celular , Capsídeo/metabolismo , HumanosRESUMO
The role of Culex mosquitoes in the transmission of Japanese encephalitis virus (JEV) is crucial, yet the mechanisms of JEV infection in these vectors remain unclear. Previous research has indicated that various host factors participate in JEV infection. Herein, we present evidence that mosquito sialic acids enhance JEV infection both in vivo and in vitro. By treating mosquitoes and C6/36 cells with neuraminidase or lectin, the function of sialic acids is effectively blocked, resulting in significant inhibition of JEV infection. Furthermore, knockdown of the sialic acid biosynthesis genes in Culex mosquitoes also leads to a reduction in JEV infection. Moreover, our research revealed that sialic acids play a role in the attachment of JEV to mosquito cells, but not in its internalization. To further explore the mechanisms underlying the promotion of JEV attachment by sialic acids, we conducted immunoprecipitation experiments to confirm the direct binding of sialic acids to the last α-helix in JEV envelope protein domain III. Overall, our study contributes to a molecular comprehension of the interaction between mosquitoes and JEV and offers potential strategies for preventing the dissemination of flavivirus in natural environments.IMPORTANCEIn this study, we aimed to investigate the impact of glycoconjugate sialic acids on mosquito infection with Japanese encephalitis virus (JEV). Our findings demonstrate that sialic acids play a crucial role in enhancing JEV infection by facilitating the attachment of the virus to the cell membrane. Furthermore, our investigation revealed that sialic acids directly bind to the final α-helix in the JEV envelope protein domain III, thereby accelerating virus adsorption. Collectively, our results highlight the significance of mosquito sialic acids in JEV infection within vectors, contributing to a better understanding of the interaction between mosquitoes and JEV.
Assuntos
Culex , Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa , Ácidos Siálicos , Ligação Viral , Animais , Camundongos , Linhagem Celular , Culex/virologia , Culex/metabolismo , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Vírus da Encefalite Japonesa (Espécie)/metabolismo , Encefalite Japonesa/virologia , Encefalite Japonesa/metabolismo , Mosquitos Vetores/virologia , Neuraminidase/metabolismo , Neuraminidase/genética , Ácidos Siálicos/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/genética , Internalização do VírusRESUMO
Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galß1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates.IMPORTANCEAll cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease.
Assuntos
Metapneumovirus , Infecções por Paramyxoviridae , Polissacarídeos , Receptores Virais , Proteínas Virais de Fusão , Ligação Viral , Humanos , Linhagem Celular , Metapneumovirus/metabolismo , Metapneumovirus/fisiologia , Infecções por Paramyxoviridae/virologia , Infecções por Paramyxoviridae/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Receptores Virais/química , Receptores Virais/metabolismo , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Interações entre Hospedeiro e Microrganismos , Proteoglicanas de Heparan Sulfato/metabolismoRESUMO
Hexosylceramides (HexCer) are implicated in the infection process of various pathogens. However, the molecular and cellular functions of HexCer in infectious cycles are poorly understood. Investigating the enveloped virus Uukuniemi (UUKV), a bunyavirus of the Phenuiviridae family, we performed a lipidomic analysis with mass spectrometry and determined the lipidome of both infected cells and derived virions. We found that UUKV alters the processing of HexCer to glycosphingolipids (GSL) in infected cells. The infection resulted in the overexpression of glucosylceramide (GlcCer) synthase (UGCG) and the specific accumulation of GlcCer and its subsequent incorporation into viral progeny. UUKV and several pathogenic bunyaviruses relied on GlcCer in the viral envelope for binding to various host cell types. Overall, our results indicate that GlcCer is a structural determinant of virions crucial for bunyavirus infectivity. This study also highlights the importance of glycolipids on virions in facilitating interactions with host cell receptors and infectious entry of enveloped viruses.
Assuntos
Orthobunyavirus , Glucosilceramidas , Ligação Viral , Lipidômica , Espectrometria de MassasRESUMO
Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and selectively utilizes the decay-accelerating factor (DAF; CD55) to infect cells. However, the differential receptor usage mechanism for CVB remains elusive. This study identified VP3-234 residues (234Q/N/V/D/E) as critical population selection determinants during CVB3 virus evolution, contributing to diverse binding affinities to CD55. Cryoelectron microscopy (cryo-EM) structures of CD55-binding/nonbinding isolates and their complexes with CD55 or CAR were obtained under both neutral and acidic conditions, and the molecular mechanism of VP3-234 residues determining CD55 affinity/specificity for naturally occurring CVB3 strains was elucidated. Structural and biochemical studies in vitro revealed the dynamic entry process of CVB3 and the function of the uncoating receptor CAR with different pH preferences. This work provides detailed insight into the molecular mechanism of CVB infection and contributes to an in-depth understanding of enterovirus attachment receptor usage.
Assuntos
Antígenos CD55/metabolismo , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/virologia , Enterovirus Humano B/fisiologia , Interações Hospedeiro-Patógeno , Receptores Virais/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Enterovirus Humano B/ultraestrutura , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/química , Relação Estrutura-Atividade , Ligação ViralRESUMO
Emergence of new viral agents is driven by evolution of interactions between viral proteins and host targets. For instance, increased infectivity of SARS-CoV-2 compared to SARS-CoV-1 arose in part through rapid evolution along the interface between the spike protein and its human receptor ACE2, leading to increased binding affinity. To facilitate broader exploration of how pathogen-host interactions might impact transmission and virulence in the ongoing COVID-19 pandemic, we performed state-of-the-art interface prediction followed by molecular docking to construct a three-dimensional structural interactome between SARS-CoV-2 and human. We additionally carried out downstream meta-analyses to investigate enrichment of sequence divergence between SARS-CoV-1 and SARS-CoV-2 or human population variants along viral-human protein-interaction interfaces, predict changes in binding affinity by these mutations/variants and further prioritize drug repurposing candidates predicted to competitively bind human targets. We believe this resource ( http://3D-SARS2.yulab.org ) will aid in development and testing of informed hypotheses for SARS-CoV-2 etiology and treatments.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/virologia , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Ligação Viral , Evolução Biológica , COVID-19/imunologia , Variação Genética , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/fisiologiaRESUMO
African swine fever (ASF) is an acute and hemorrhagic infectious disease caused by African swine fever virus (ASFV), which is listed as an animal epidemic disease that must be reported by The World Organization for Animal Health and that causes serious economic losses to China and even the whole world. Currently, the entry mechanism of ASFV is not fully understood. Especially in the early stages of virus entry, the host factors required for ASFV entry have not yet been identified and characterized. In this study, we demonstrated that ASFV externalized phosphatidylserine (PS) on the envelope functioned as viral apoptotic mimicry, which interacts with AXL, a tyrosine kinase receptor, to mediate ASFV entry into porcine alveolar macrophages (PAMs). We found that AXL was the most pronounced phosphatidylserine receptor (PSR) affecting ASFV entry in PAMs by RNA interference screening. Knockout AXL gene expression remarkably decreased ASFV internalization and replication in MA104 cells. Furthermore, the antibody against AXL extracellular domains effectively inhibited the ASFV entry. Consistent with these results, the deletion of the intracellular kinase domain of AXL and the treatment of the AXL inhibitor, R428, significantly inhibited the internalization of ASFV. Mechanistically, AXL facilitated the internalization of ASFV virions via macropinocytosis. Collectively, we provide evidence that AXL is a coreceptor for ASFV entry into PAMs, which expands our knowledge of ASFV entry and provides a theoretical basis for identifying new antiviral targets. IMPORTANCE African swine fever (ASF) is a highly contagious infectious disease caused by the ASF virus (ASFV), with a mortality rate of up to 100%. ASFV has caused huge economic losses to pig farming worldwide. Specific cellular surface receptors are considered crucial determinants of ASFV tropism. However, the host factors required for ASFV entry have not yet been identified, and the molecular mechanism of its entry remains unclear. Here, we found that ASFV utilized phosphatidylserine (PS) on the surface of virions to masquerade as apoptotic mimicry and facilitated virus entry by interacting with host factor AXL. We found that knockout of AXL remarkably decreased ASFV internalization and replication. The antibody against AXL extracellular domains and AXL inhibitor R428 significantly inhibited the internalization of ASFV via macropinocytosis. The current work deepens our understanding of ASFV entry and provides clues for the development of antiviral drugs to control ASFV infection.
Assuntos
Febre Suína Africana , Receptor Tirosina Quinase Axl , Interações entre Hospedeiro e Microrganismos , Internalização do Vírus , Animais , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/genética , Suínos , Receptor Tirosina Quinase Axl/genética , Receptor Tirosina Quinase Axl/metabolismo , Macrófagos Alveolares/virologia , Técnicas de Inativação de Genes , Linhagem Celular , Envelope Viral/metabolismo , Ligação Viral , Domínios ProteicosRESUMO
IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several B cell malignancies and Kaposi's sarcoma. We analyzed the function of K8.1, the major antigenic component of the KSHV virion in the infection of different cells. To do this, we deleted K8.1 from the viral genome. It was found that K8.1 is critical for the infection of certain epithelial cells, e.g., a skin model cell line but not for infection of many other cells. K8.1 was found to mediate attachment of the virus to cells where it plays a role in infection. In contrast, we did not find K8.1 or a related protein from a closely related monkey virus to activate fusion of the viral and cellular membranes, at least not under the conditions tested. These findings suggest that K8.1 functions in a highly cell-specific manner during KSHV entry, playing a crucial role in the attachment of KSHV to, e.g., skin epithelial cells.
Assuntos
Glicoproteínas , Herpesvirus Humano 8 , Queratinócitos , Proteínas Virais , Ligação Viral , Internalização do Vírus , Humanos , Glicoproteínas/deficiência , Glicoproteínas/genética , Glicoproteínas/metabolismo , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Queratinócitos/metabolismo , Queratinócitos/virologia , Sarcoma de Kaposi/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Fusão de Membrana , Pele/citologiaRESUMO
Pseudorabies virus (PRV) is a neurotropic virus causing obvious neurological disorders and reproductive failure in pigs. PRV entry into target cells is a complex multistep process initiated by interacting viral envelope glycoproteins with cellular receptors. In the current study, we found that thrombospondin 3 (THBS3) plays an important role in PRV entry into target cells, indicating that THBS3 is a new PRV coreceptor. To confirm this hypothesis, the knockdown of THBS3 in several permissive cells inhibited PRV primary infection, and overexpression of THBS3 in PK15 cells promoted PRV infection. CRISPR-Cas9 knockout markedly reduced PRV infection in PK15 cells. Antibodies against THBS3 blocked PRV infection in naturally permissive target cells. Moreover, soluble THBS3 protein neutralized the infectivity of PRV. Mechanistically, THBS3 interacted with the PRV gD via its N and C termini to facilitate PRV binding in permissive and nonpermissive cells. Also, in the absence of Nectin-1, THBS3 promoted cell-to-cell fusion mediated by virus glycoproteins. While THBS3 alone could not increase virus entry, overexpression of it in the presence of Nectin-1 promoted virus entry into CHO-K1 cells. Our results have identified THBS3 as a critical player in PRV binding and subsequent membrane fusion and entry. IMPORTANCE Herpesvirus entry occurs through a cascade of virus-cell interactions, and multiple surface glycoproteins play a role in virus binding and entry during the virus invasion process. Early studies showed that attachment to cells by PRV, as well as other alphaherpesviruses, is mediated by interactions between the viral glycoprotein gC and cell membrane proteoglycans carrying heparan sulfate chains (HSPGs). However, gD may also be involved in virus binding in an HSPG-independent manner. To date, the respective cellular receptors are still unknown. In this report, we identified a host molecule, THBS3, involved in gD-mediated PRV binding and subsequent membrane fusion and entry, which increases our understanding of the initial events in alpha herpesvirus infections.
Assuntos
Herpesvirus Suídeo 1 , Pseudorraiva , Ligação Viral , Internalização do Vírus , Animais , Cricetinae , Células CHO , Herpesvirus Suídeo 1/metabolismo , Herpesvirus Suídeo 1/patogenicidade , Nectinas/genética , Nectinas/metabolismo , Suínos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Técnicas de Silenciamento de GenesRESUMO
Porcine epidemic diarrhea virus (PEDV) has caused huge economic losses to the global pig industry. The swine enteric coronavirus spike (S) protein recognizes various cell surface molecules to regulate viral infection. In this study, we identified 211 host membrane proteins related to the S1 protein by pulldown combined with liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis. Among these, heat shock protein family A member 5 (HSPA5) was identified through screening as having a specific interaction with the PEDV S protein, and positive regulation of PEDV infection was validated by knockdown and overexpression tests. Further studies verified the role of HSPA5 in viral attachment and internalization. In addition, we found that HSPA5 interacts with S proteins through its nucleotide-binding structural domain (NBD) and that polyclonal antibodies can block viral infection. In detail, HSPA5 was found to be involved in viral trafficking via the endo-/lysosomal pathway. Inhibition of HSPA5 activity during internalization would reduce the subcellular colocalization of PEDV with lysosomes in the endo-/lysosomal pathway. Together, these findings show that HSPA5 is a novel PEDV potential target for the creation of therapeutic drugs. IMPORTANCE PEDV infection causes severe piglet mortality and threatens the global pig industry. However, the complex invasion mechanism of PEDV makes its prevention and control difficult. Here, we determined that HSPA5 is a novel target for PEDV which interacts with its S protein and is involved in viral attachment and internalization, influencing its transport via the endo-/lysosomal pathway. Our work extends knowledge about the relationship between the PEDV S and host proteins and provides a new therapeutic target against PEDV infection.
Assuntos
Infecções por Coronavirus , Chaperona BiP do Retículo Endoplasmático , Vírus da Diarreia Epidêmica Suína , Glicoproteína da Espícula de Coronavírus , Doenças dos Suínos , Internalização do Vírus , Animais , Chlorocebus aethiops , Infecções por Coronavirus/fisiopatologia , Infecções por Coronavirus/virologia , Lisossomos/metabolismo , Lisossomos/virologia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Suínos , Doenças dos Suínos/fisiopatologia , Doenças dos Suínos/virologia , Células Vero , Chaperona BiP do Retículo Endoplasmático/genética , Chaperona BiP do Retículo Endoplasmático/metabolismo , Ligação Viral , Endocitose/genéticaRESUMO
Entry of influenza A viruses (IAVs) into host cells is initiated by binding to sialic acids (Sias), their primary host cell receptor, followed by endocytosis and membrane fusion to release the viral genome into the cytoplasm of the host cell. Host tropism is affected by these entry processes, with a primary factor being receptor specificity. Sias exist in several different chemical forms, including the hydroxylated N-glycolylneuraminic acid (Neu5Gc), which is found in many hosts; however, it has not been clear how modified Sias affect viral binding and entry. Neu5Gc is commonly found in many natural influenza hosts, including pigs and horses, but not in humans or ferrets. Here, we engineered HEK293 cells to express the hydoxylase gene (CMAH) that converts Neu5Ac to Neu5Gc, or knocked out the Sia-CMP transport gene (SLC35A1), resulting in cells that express 95% Neu5Gc or minimal level of Sias, respectively. H3N2 (X-31) showed significantly reduced infectivity in Neu5Gc-rich cells compared to wild-type HEK293 (>95% Neu5Ac). To determine the effects on binding and fusion, we generated supported lipid bilayers (SLBs) derived from the plasma membranes of these cells and carried out single particle microscopy. H3N2 (X-31) exhibited decreased binding to Neu5Gc-containing SLBs, but no significant difference in H3N2 (X-31)'s fusion kinetics to either SLB type, suggesting that reduced receptor binding does not affect subsequent membrane fusion. This finding suggests that for this virus to adapt to host cells rich in Neu5Gc, only receptor affinity changes are required without further adaptation of virus fusion machinery. IMPORTANCE Influenza A virus (IAV) infections continue to threaten human health, causing over 300,000 deaths yearly. IAV infection is initiated by the binding of influenza glycoprotein hemagglutinin (HA) to host cell sialic acids (Sias) and the subsequent viral-host membrane fusion. Generally, human IAVs preferentially bind to the Sia N-acetylneuraminic acid (Neu5Ac). Yet, other mammalian hosts, including pigs, express diverse nonhuman Sias, including N-glycolylneuraminic acid (Neu5Gc). The role of Neu5Gc in human IAV infections in those hosts is not well-understood, and the variant form may play a role in incidents of cross-species transmission and emergence of new epidemic variants. Therefore, it is important to investigate how human IAVs interact with Neu5Ac and Neu5Gc. Here, we use membrane platforms that mimic the host cell surface to examine receptor binding and membrane fusion events of human IAV H3N2. Our findings improve the understanding of viral entry mechanisms that can affect host tropism and virus evolution.