RESUMO
Nipah virus infection, one of the top priority diseases recognized by the World Health Organization, underscores the urgent need to develop effective countermeasures against potential epidemics and pandemics. Here, we identify a fully human single-domain antibody that targets a highly conserved cryptic epitope situated at the dimeric interface of the Nipah virus G protein (receptor binding protein, RBP), as elucidated through structures by high-resolution cryo-electron microscopy (cryo-EM). This unique binding mode disrupts the tetramerization of the G protein, consequently obstructing the activation of the F protein and inhibiting viral membrane fusion. Furthermore, our investigations reveal that this compact antibody displays enhanced permeability across the blood-brain barrier (BBB) and demonstrates superior efficacy in eliminating pseudovirus within the brain in a murine model of Nipah virus infection, particularly compared to the well-characterized antibody m102.4 in an IgG1 format. Consequently, this single-domain antibody holds promise as a therapeutic candidate to prevent Nipah virus infections and has potential implications for vaccine development.
Assuntos
Anticorpos Antivirais , Microscopia Crioeletrônica , Epitopos , Infecções por Henipavirus , Vírus Nipah , Anticorpos de Domínio Único , Vírus Nipah/imunologia , Humanos , Animais , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/prevenção & controle , Infecções por Henipavirus/virologia , Epitopos/imunologia , Camundongos , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Anticorpos Antivirais/imunologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/química , Feminino , Células HEK293RESUMO
The Hendra and Nipah viruses (HNVs) are highly pathogenic pathogens without approved interventions for human use. In addition, the interaction pattern between the attachment (G) and fusion (F) glycoproteins required for virus entry remains unclear. Here, we isolate a panel of Macaca-derived G-specific antibodies that cross-neutralize HNVs via multiple mechanisms. The most potent antibody, 1E5, confers adequate protection against the Nipah virus challenge in female hamsters. Crystallography demonstrates that 1E5 has a highly similar binding pattern to the receptor. In cryo-electron microscopy studies, the tendency of 1E5 to bind to the upper or lower heads results in two distinct quaternary structures of G. Furthermore, we identify the extended outer loop ß1S2-ß1S3 of G and two pockets on the apical region of fusion (F) glycoprotein as the essential sites for G-F interactions. This work highlights promising drug candidates against HNVs and contributes deeper insights into the viruses.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Microscopia Crioeletrônica , Infecções por Henipavirus , Proteínas Virais de Fusão , Animais , Anticorpos Neutralizantes/imunologia , Feminino , Anticorpos Antivirais/imunologia , Infecções por Henipavirus/virologia , Infecções por Henipavirus/imunologia , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/química , Humanos , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/química , Vírus Nipah/imunologia , Internalização do Vírus/efeitos dos fármacos , Henipavirus/imunologia , Cricetinae , Reações Cruzadas/imunologia , Vírus Hendra/imunologia , Macaca , Mesocricetus , Cristalografia por Raios XRESUMO
Hendra virus (HeV) is a zoonotic enveloped member of the family Paramyoxviridae. To successfully infect a host cell, HeV utilizes two surface glycoproteins: the attachment (G) protein to bind, and the trimeric fusion (F) protein to merge the viral envelope with the membrane of the host cell. The transmembrane (TM) region of HeV F has been shown to have roles in F protein stability and the overall trimeric association of F. Previously, alanine scanning mutagenesis has been performed on the C-terminal end of the protein, revealing the importance of ß-branched residues in this region. Additionally, residues S490 and Y498 have been demonstrated to be important for F protein endocytosis, needed for the proteolytic processing of F required for fusion. To complete the analysis of the HeV F TM, we performed alanine scanning mutagenesis to explore the residues in the N-terminus of this region (residues 487-506). In addition to confirming the critical roles for S490 and Y498, we demonstrate that mutations at residues M491 and L492 alter F protein function, suggesting a role for these residues in the fusion process.
Assuntos
Vírus Hendra/genética , Infecções por Henipavirus/virologia , Fusão de Membrana , Proteínas Virais de Fusão/metabolismo , Alanina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Membrana Celular/metabolismo , Chlorocebus aethiops , Endocitose , Endossomos/metabolismo , Genes Reporter , Vírus Hendra/fisiologia , Humanos , Mutagênese Sítio-Dirigida , Domínios Proteicos , Estabilidade Proteica , Células Vero , Proteínas Virais de Fusão/genéticaRESUMO
Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we demonstrate quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct C-terminal stalk lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3- to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.
Assuntos
Henipavirus/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Células Gigantes/metabolismo , Glicoproteínas/genética , Células HEK293 , Henipavirus/genética , Infecções por Henipavirus/metabolismo , Infecções por Henipavirus/virologia , Humanos , Fusão de Membrana/fisiologia , Receptores Virais/metabolismo , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/fisiologia , Ligação Viral , Internalização do VírusRESUMO
Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic, bat-borne paramyxoviruses in the genus Henipavirus that cause severe and often fatal acute respiratory and/or neurologic diseases in humans and livestock. There are currently no approved antiviral therapeutics or vaccines for use in humans to treat or prevent NiV or HeV infection. To facilitate development of henipavirus antivirals, a high-throughput screening (HTS) platform was developed based on a well-characterized recombinant version of the nonpathogenic Henipavirus, Cedar virus (rCedV). Using reverse genetics, a rCedV encoding firefly luciferase (rCedV-Luc) was rescued and its utility evaluated for high-throughput antiviral compound screening. The luciferase reporter gene signal kinetics of rCedV-Luc in different human cell lines was characterized and validated as an authentic real-time measure of viral growth. The rCedV-Luc platform was optimized as an HTS assay that demonstrated high sensitivity with robust Z' scores, excellent signal-to-background ratios and coefficients of variation. Eight candidate compounds that inhibited rCedV replication were identified for additional validation and demonstrated that 4 compounds inhibited authentic NiV-Bangladesh replication. Further evaluation of 2 of the 4 validated compounds in a 9-point dose response titration demonstrated potent antiviral activity against NiV-Bangladesh and HeV, with minimal cytotoxicity. This rCedV reporter can serve as a surrogate yet authentic BSL-2 henipavirus platform that will dramatically accelerate drug candidate identification in the development of anti-henipavirus therapies.
Assuntos
Antivirais/farmacologia , Infecções por Henipavirus/tratamento farmacológico , Henipavirus/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Proteínas do Envelope Viral/metabolismo , Linhagem Celular , Genes Reporter , Henipavirus/fisiologia , Infecções por Henipavirus/virologia , Humanos , Luciferases/genética , Luciferases/metabolismo , Recombinação Genética , Proteínas do Envelope Viral/genética , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Mojiang virus (MojV) is the first henipavirus identified in a rodent and known only by sequence data, whereas all other henipaviruses have been isolated from bats (Hendra virus, Nipah virus, Cedar virus) or discovered by sequence data from material of bat origin (Ghana virus). Ephrin-B2 and -B3 are entry receptors for Hendra and Nipah viruses, but Cedar virus can utilize human ephrin-B1, -B2, -A2 and -A5 and mouse ephrin-A1. However, the entry receptor for MojV remains unknown, and its species tropism is not well characterized. Here, we utilized recombinant full-length and soluble forms of the MojV fusion (F) and attachment (G) glycoproteins in membrane fusion and receptor tropism studies. MojV F and G were functionally competent and mediated cell-cell fusion in primate and rattine cells, albeit with low levels and slow fusion kinetics. Although a relative instability of the pre-fusion conformation of a soluble form of MojV F was observed, MojV F displayed significantly greater fusion activity when heterotypically paired with Ghana virus G. An exhaustive investigation of A- and B-class ephrins indicated that none serve as a primary receptor for MojV. The MojV cell fusion phenotype is therefore likely the result of receptor restriction rather than functional defects in recombinant MojV F and G glycoproteins.
Assuntos
Glicoproteínas/metabolismo , Infecções por Henipavirus/virologia , Henipavirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Células A549 , Animais , Células CHO , Chlorocebus aethiops , Cricetulus , Células HEK293 , Células HeLa , Humanos , Fusão de Membrana , Células Vero , Internalização do VírusRESUMO
Nipah virus (NiV) is a zoonotic bat henipavirus in the family Paramyxoviridae NiV is deadly to humans, infecting host cells by direct fusion of the viral and host cell plasma membranes. This membrane fusion process is coordinated by the receptor-binding attachment (G) and fusion (F) glycoproteins. Upon G-receptor binding, F fuses membranes via a cascade that sequentially involves F-triggering, fusion pore formation, and viral or genome entry into cells. Using NiV as an important paramyxoviral model, we identified two novel regions in F that modulate the membrane fusion cascade. For paramyxoviruses and other viral families with class I fusion proteins, the heptad repeat 1 (HR1) and HR2 regions in the fusion protein prefusion conformation bind to form a six-helix bundle in the postfusion conformation. Here, structural comparisons between the F prefusion and postfusion conformations revealed that a short loop region (N1) undergoes dramatic spatial reorganization and a short alpha helix (N4) undergoes secondary structural changes. The roles of the N1 and N4 regions during the membrane fusion cascade, however, remain unknown for henipaviruses and paramyxoviruses. By performing alanine scanning mutagenesis and various functional analyses, we report that specific residues within these regions alter various steps in the membrane fusion cascade. While the N1 region affects early F-triggering, the N4 region affects F-triggering, F thermostability, and extensive fusion pore expansion during syncytium formation, also uncovering a link between F-G interactions and F-triggering. These novel mechanistic roles expand our understanding of henipaviral and paramyxoviral F-triggering, viral entry, and cell-cell fusion (syncytia), a pathognomonic feature of paramyxoviral infections.IMPORTANCE Henipaviruses infect bats, agriculturally important animals, and humans, with high mortality rates approaching â¼75% in humans. Known human outbreaks have been concentrated in Southeast Asia and Australia. Furthermore, about 20 new henipaviral species have been recently discovered in bats, with geographical spans in Asia, Africa, and South America. The development of antiviral therapeutics requires a thorough understanding of the mechanism of viral entry into host cells. In this study, we discovered novel roles of two regions within the fusion protein of the deadly henipavirus NiV. Such roles were in allowing viral entry into host cells and cell-cell fusion, a pathological hallmark of this and other paramyxoviruses. These novel roles were in the previously undescribed N1 and N4 regions within the fusion protein, modulating early and late steps of these important processes of viral infection and henipaviral disease. Notably, this knowledge may apply to other henipaviruses and more broadly to other paramyxoviruses.
Assuntos
Infecções por Henipavirus/virologia , Fusão de Membrana , Vírus Nipah/fisiologia , Proteínas Virais de Fusão/química , Internalização do Vírus , Animais , Chlorocebus aethiops , Células HEK293 , Humanos , Conformação Proteica , Conformação Proteica em alfa-Hélice , Células VeroRESUMO
Although enveloped viruses canonically mediate particle entry through virus-cell fusion, certain viruses can spread by cell-cell fusion, brought about by receptor engagement and triggering of membrane-bound, viral-encoded fusion proteins on the surface of cells. The formation of pathogenic syncytia or multinucleated cells is seen in vivo, but their contribution to viral pathogenesis is poorly understood. For the negative-strand paramyxoviruses respiratory syncytial virus (RSV) and Nipah virus (NiV), cell-cell spread is highly efficient because their oligomeric fusion protein complexes are active at neutral pH. The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been reported to induce syncytia formation in infected cells, with the spike protein initiating cell-cell fusion. Whilst it is well established that fusion protein-specific antibodies can block particle attachment and/or entry into the cell (canonical virus neutralization), their capacity to inhibit cell-cell fusion and the consequences of this neutralization for the control of infection are not well characterized, in part because of the lack of specific tools to assay and quantify this activity. Using an adapted bimolecular fluorescence complementation assay, based on a split GFP-Renilla luciferase reporter, we have established a micro-fusion inhibition test (mFIT) that allows the identification and quantification of these neutralizing antibodies. This assay has been optimized for high-throughput use and its applicability has been demonstrated by screening monoclonal antibody (mAb)-mediated inhibition of RSV and NiV fusion and, separately, the development of fusion-inhibitory antibodies following NiV vaccine immunization in pigs. In light of the recent emergence of coronavirus disease 2019 (COVID-19), a similar assay was developed for SARS-CoV-2 and used to screen mAbs and convalescent patient plasma for fusion-inhibitory antibodies. Using mFITs to assess antibody responses following natural infection or vaccination is favourable, as this assay can be performed entirely at low biocontainment, without the need for live virus. In addition, the repertoire of antibodies that inhibit cell-cell fusion may be different to those that inhibit particle entry, shedding light on the mechanisms underpinning antibody-mediated neutralization of viral spread.
Assuntos
Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , COVID-19/diagnóstico , Infecções por Henipavirus/diagnóstico , Ensaios de Triagem em Larga Escala , Infecções por Vírus Respiratório Sincicial/diagnóstico , Proteínas Virais de Fusão/antagonistas & inibidores , Animais , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/metabolismo , COVID-19/imunologia , COVID-19/virologia , Fusão Celular , Convalescença , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/virologia , Humanos , Soros Imunes/química , Luciferases/genética , Luciferases/metabolismo , Modelos Moleculares , Vírus Nipah/imunologia , Vírus Nipah/patogenicidade , Conformação Proteica , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/imunologia , Vírus Sincicial Respiratório Humano/patogenicidade , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Suínos , Inibidores de Proteínas Virais de Fusão/química , Inibidores de Proteínas Virais de Fusão/metabolismo , Inibidores de Proteínas Virais de Fusão/farmacologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologiaRESUMO
Nipah Virus (NiV) has been designated as a priority disease with an urgent need for therapeutic development by World Health Organization. The monoclonal antibody m102.4 binds to the immunodominant NiV receptor-binding glycoprotein (GP), and potently neutralizes NiV, indicating its potential as a therapeutic agent. Although the co-crystal structure of m102.3, an m102.4 derivative, in complex with the GP of the related Hendra Virus (HeV) has been solved, the structural interaction between m102.4 and NiV is uncharacterized. Herein, we used structure-guided alanine-scanning mutagenesis to map the functional epitope and paratope residues that govern the antigen-antibody interaction. Our results revealed that the binding of m102.4 is mediated predominantly by two residues in the HCDR3 region, which is unusually small for an antibody-antigen interaction. We performed computational docking to generate a structural model of m102.4-NiV interaction. Our model indicates that m102.4 targets the common hydrophobic central cavity and a hydrophilic rim on the GP, as observed for the m102.3-HeV co-crystal, albeit with Fv orientation differences. In summary, our study provides insight into the m102.4-NiV interaction, demonstrating that structure-guided alanine-scanning and computational modeling can serve as the starting point for additional antibody reengineering (e.g. affinity maturation) to generate potential therapeutic candidates.
Assuntos
Alanina/genética , Anticorpos Monoclonais/metabolismo , Simulação por Computador , Glicoproteínas/metabolismo , Infecções por Henipavirus/virologia , Vírus Nipah/metabolismo , Proteínas do Envelope Viral/metabolismo , Alanina/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Epitopos/imunologia , Glicoproteínas/química , Glicoproteínas/genética , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Vírus Nipah/imunologia , Vírus Nipah/isolamento & purificação , Elementos Estruturais de Proteínas/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr Virus (EBV) establish life-long infections and are associated with malignancies. Striking geographic variation in incidence and the fact that virus alone is insufficient to cause disease, suggests other co-factors are involved. Here we present epidemiological analysis and genome-wide association study (GWAS) in 4365 individuals from an African population cohort, to assess the influence of host genetic and non-genetic factors on virus antibody responses. EBV/KSHV co-infection (OR = 5.71(1.58-7.12)), HIV positivity (OR = 2.22(1.32-3.73)) and living in a more rural area (OR = 1.38(1.01-1.89)) are strongly associated with immunogenicity. GWAS reveals associations with KSHV antibody response in the HLA-B/C region (p = 6.64 × 10-09). For EBV, associations are identified for VCA (rs71542439, p = 1.15 × 10-12). Human leucocyte antigen (HLA) and trans-ancestry fine-mapping substantiate that distinct variants in HLA-DQA1 (p = 5.24 × 10-44) are driving associations for EBNA-1 in Africa. This study highlights complex interactions between KSHV and EBV, in addition to distinct genetic architectures resulting in important differences in pathogenesis and transmission.
Assuntos
Anticorpos Antivirais/biossíntese , Resistência à Doença/genética , Infecções por Vírus Epstein-Barr/genética , Infecções por Henipavirus/genética , Interações Hospedeiro-Patógeno/genética , Sarcoma de Kaposi/genética , Adolescente , Adulto , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Coinfecção , Infecções por Vírus Epstein-Barr/epidemiologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Feminino , Expressão Gênica , Estudo de Associação Genômica Ampla , HIV/genética , HIV/imunologia , HIV/patogenicidade , Cadeias alfa de HLA-DQ/genética , Cadeias alfa de HLA-DQ/imunologia , Infecções por Henipavirus/epidemiologia , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/virologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/patogenicidade , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/imunologia , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , População Rural , Sarcoma de Kaposi/epidemiologia , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/virologia , Uganda/epidemiologia , População UrbanaRESUMO
Medically important paramyxoviruses, such as measles, mumps, parainfluenza, Nipah, and Hendra viruses, infect host cells by directing fusion of the viral and cellular plasma membranes. Upon infection, paramyxoviruses cause a second type of membrane fusion, cell-cell fusion (syncytium formation), which is linked to pathogenicity. Host cell receptor binding causes conformational changes in the attachment glycoprotein (HN, H, or G) that trigger a conformational cascade in the fusion (F) glycoprotein that mediates membrane fusion. F, a class I fusion protein, contains the archetypal heptad repeat regions 1 (HR1) and 2 (HR2). It is well established that binding of HR1 and HR2 is key to fusing viral and cellular membranes. In this study, we uncovered a novel fusion-modulatory role of a third structurally conserved helical region (HR3) in F. Based on its location within the F structure, and structural differences between its prefusion and postfusion conformations, we hypothesized that the HR3 modulates triggering of the F conformational cascade (still requiring G). We used the deadly Nipah virus (NiV) as an important paramyxoviral model to perform alanine scan mutagenesis and a series of multidisciplinary structural/functional analyses that dissect the various states of the membrane fusion cascade. Remarkably, we found that specific residues within the HR3 modulate not only early F-triggering but also late extensive fusion pore expansion steps in the membrane fusion cascade. Our results characterize these novel fusion-modulatory roles of the F HR3, improving our understanding of the membrane fusion process for NiV and likely for the related Henipavirus genus and possibly Paramyxoviridae family members.IMPORTANCE The Paramyxoviridae family includes important human and animal pathogens, such as measles, mumps, and parainfluenza viruses and the deadly henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviruses infect the respiratory tract and the central nervous system (CNS) and can be highly infectious. Most paramyxoviruses have a limited host range. However, the biosafety level 4 NiV and HeV are highly pathogenic and have a wide mammalian host range. Nipah viral infections result in acute respiratory syndrome and severe encephalitis in humans, leading to 40 to 100% mortality rates. The lack of licensed vaccines or therapeutic approaches against NiV and other important paramyxoviruses underscores the need to understand viral entry mechanisms. In this study, we uncovered a novel role of a third helical region (HR3) of the NiV fusion glycoprotein in the membrane fusion process that leads to viral entry. This discovery sets HR3 as a new candidate target for antiviral strategies for NiV and likely for related viruses.
Assuntos
Fusão de Membrana/fisiologia , Vírus Nipah/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Animais , Chlorocebus aethiops , Encefalite/virologia , Células HEK293 , Infecções por Henipavirus/virologia , Especificidade de Hospedeiro , Humanos , Modelos Moleculares , Vírus Nipah/genética , Paramyxovirinae , Conformação Proteica , Domínios Proteicos , Alinhamento de Sequência , Homologia Estrutural de Proteína , Células Vero , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/genética , Internalização do VírusRESUMO
Licensed vaccines or therapeutics are rarely available for pathogens with epidemic or pandemic potential. Developing interventions for specific pathogens and defining generalizable approaches for related pathogens is a global priority and inherent to the UN Sustainable Development Goals. Nipah virus (NiV) poses a significant epidemic threat, and zoonotic transmission from bats-to-humans with high fatality rates occurs almost annually. Human-to-human transmission of NiV has been documented in recent outbreaks leading public health officials and government agencies to declare an urgent need for effective vaccines and therapeutics. Here, we evaluate NiV vaccine antigen design options including the fusion glycoprotein (F) and the major attachment glycoprotein (G). A stabilized prefusion F (pre-F), multimeric G constructs, and chimeric proteins containing both pre-F and G were developed as protein subunit candidate vaccines. The proteins were evaluated for antigenicity and structural integrity using kinetic binding assays, electron microscopy, and other biophysical properties. Immunogenicity of the vaccine antigens was evaluated in mice. The stabilized pre-F trimer and hexameric G immunogens both induced serum neutralizing activity in mice, while the post-F trimer immunogen did not elicit neutralizing activity. The pre-F trimer covalently linked to three G monomers (pre-F/G) induced potent neutralizing antibody activity, elicited responses to the greatest diversity of antigenic sites, and is the lead candidate for clinical development. The specific stabilizing mutations and immunogen designs utilized for NiV were successfully applied to other henipaviruses, supporting the concept of identifying generalizable solutions for prototype pathogens as an approach to pandemic preparedness.
Assuntos
Antígenos Virais/imunologia , Infecções por Henipavirus/prevenção & controle , Imunogenicidade da Vacina , Vírus Nipah/química , Vírus Nipah/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Células HEK293 , Infecções por Henipavirus/virologia , Humanos , Imunização/métodos , Camundongos , Camundongos Endogâmicos C57BL , Transfecção , Proteínas Virais de Fusão/imunologia , Internalização do VírusRESUMO
BACKGROUND: Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses that cause severe disease in both animals and humans. There are no approved vaccines or treatments for use in humans; however, therapeutic treatment of both NiV and HeV infection in ferrets and non-human primates with a cross-reactive, neutralizing human monoclonal antibody (mAb), m102.4, targeting the G glycoprotein has been demonstrated. In a previous study, we isolated, characterized, and humanized a cross-reactive, neutralizing anti-F mAb (h5B3.1). The mAb h5B3.1 blocks the required F conformational change needed to facilitate membrane fusion and virus infection, and the epitope recognized by h5B3.1 has been structurally defined; however, the efficacy of h5B3.1 in vivo is unknown. METHODS: The post-infection antiviral activity of h5B3.1 was evaluated in vivo by administration in ferrets after NiV and HeV virus challenge. RESULTS: All subjects that received h5B3.1 from 1 to several days after infection with a high-dose, oral-nasal virus challenge were protected from disease, whereas all controls died. CONCLUSIONS: This is the first successful post-exposure antibody therapy for NiV and HeV using a humanized cross-reactive mAb targeting the F glycoprotein, and the findings suggest that a combination therapy targeting both F and G should be evaluated as a therapy for NiV/HeV infection.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Vírus Hendra , Infecções por Henipavirus/terapia , Vírus Nipah , Proteínas Virais de Fusão/imunologia , Animais , Reações Cruzadas , Furões , Infecções por Henipavirus/prevenção & controle , Infecções por Henipavirus/virologia , HumanosRESUMO
Interferon (IFN) type I plays a critical role in the protection of mice from lethal Nipah virus (NiV) infection, but mechanisms responsible for IFN-I induction remain unknown. In the current study, we demonstrated the critical role of the mitochondrial antiviral signaling protein signaling pathway in IFN-I production and NiV replication in murine embryonic fibroblasts in vitro, and the redundant but essential roles of both mitochondrial antiviral signaling protein and myeloid differentiation primary response 88 adaptors, but not toll/interleukin-1 receptor/resistance [TIR] domain-containing adaptor-inducing IFN-ß (TRIF), in the control of NiV infection in mice. These results reveal potential novel targets for antiviral intervention and help in understanding NiV immunopathogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/virologia , Fator 88 de Diferenciação Mieloide/metabolismo , Vírus Nipah , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Regulação da Expressão Gênica/imunologia , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
The emergent zoonotic henipaviruses, Hendra, and Nipah are responsible for frequent and fatal disease outbreaks in domestic animals and humans. Specificity of henipavirus attachment glycoproteins (G) for highly species-conserved ephrin ligands underpins their broad host range and is associated with systemic and neurological disease pathologies. Here, we demonstrate that Cedar virus (CedV)-a related henipavirus that is ostensibly nonpathogenic-possesses an idiosyncratic entry receptor repertoire that includes the common henipaviral receptor, ephrin-B2, but, distinct from pathogenic henipaviruses, does not include ephrin-B3. Uniquely among known henipaviruses, CedV can use ephrin-B1 for cellular entry. Structural analyses of CedV-G reveal a key region of molecular specificity that directs ephrin-B1 utilization, while preserving a universal mode of ephrin-B2 recognition. The structural and functional insights presented uncover diversity within the known henipavirus receptor repertoire and suggest that only modest structural changes may be required to modulate receptor specificities within this group of lethal human pathogens.
Assuntos
Efrina-B1/metabolismo , Infecções por Henipavirus/metabolismo , Henipavirus/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Animais , Quirópteros/virologia , Chlorocebus aethiops , Efrina-B1/genética , Efrina-B2/genética , Efrina-B2/metabolismo , Células HEK293 , Henipavirus/isolamento & purificação , Infecções por Henipavirus/virologia , Humanos , Ligantes , Ligação Proteica , Estrutura Secundária de Proteína , Receptores Virais/metabolismo , Transfecção , Células VeroRESUMO
Nipah virus (NiV) is a pathogenic paramyxovirus and zoononis with very high human fatality rates. Previous protein over-expression studies have shown that various mutations to the common N-terminal STAT1-binding motif of the NiV P, V, and W proteins affected the STAT1-binding ability of these proteins thus interfering with he JAK/STAT pathway and reducing their ability to inhibit type-I IFN signaling, but due to differing techniques it was unclear which amino acids were most important in this interaction or what impact this had on pathogenesis in vivo. We compared all previously described mutations in parallel and found the amino acid mutation Y116E demonstrated the greatest reduction in binding to STAT1 and the greatest reduction in interferon antagonism. A similar reduction in binding and activity was seen for a deletion of twenty amino acids constituting the described STAT1-binding domain. To investigate the contribution of this STAT1-binding motif in NiV-mediated disease, we produced rNiVs with complete deletion of the STAT1-binding motif or the Y116E mutation for ferret challenge studies (rNiVM-STAT1blind). Despite the reduced IFN inhibitory function, ferrets challenged with these rNiVM-STAT1blind mutants had a lethal, albeit altered, NiV-mediated disease course. These data, together with our previously published data, suggest that the major role of NiV P, V, and W in NiV-mediated disease in the ferret model are likely to be in the inhibition of viral recognition/innate immune signaling induction with a minor role for inhibition of IFN signaling.
Assuntos
Infecções por Henipavirus/patologia , Infecções por Henipavirus/virologia , Vírus Nipah/fisiologia , Fosfoproteínas/metabolismo , Fator de Transcrição STAT1/antagonistas & inibidores , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Modelos Animais de Doenças , Progressão da Doença , Feminino , Furões , Infecções por Henipavirus/metabolismo , Fosfoproteínas/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Proteínas Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness with fatality rates of 50-100%. No vaccines or licensed therapeutics currently exist to protect humans against NiV or HeV. HNVs enter host cells by fusing the viral and cellular membranes via the concerted action of the attachment (G) and fusion (F) glycoproteins, the main targets of the humoral immune response. Here, we describe the isolation and humanization of a potent monoclonal antibody cross-neutralizing NiV and HeV. Cryo-electron microscopy, triggering and fusion studies show the antibody binds to a prefusion-specific quaternary epitope, conserved in NiV F and HeV F glycoproteins, and prevents membrane fusion and viral entry. This work supports the importance of the HNV prefusion F conformation for eliciting a robust immune response and paves the way for using this antibody for prophylaxis and post-exposure therapy with NiV- and HeV-infected individuals.
Assuntos
Anticorpos Neutralizantes/farmacologia , Antivirais/farmacologia , Vírus Hendra/efeitos dos fármacos , Infecções por Henipavirus/tratamento farmacológico , Vírus Nipah/efeitos dos fármacos , Proteínas Virais de Fusão/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Células HEK293 , Vírus Hendra/metabolismo , Infecções por Henipavirus/metabolismo , Infecções por Henipavirus/virologia , Humanos , Modelos Moleculares , Vírus Nipah/metabolismo , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
Cedar virus (CedV) is a bat-borne henipavirus related to Nipah virus (NiV) and Hendra virus (HeV), zoonotic agents of fatal human disease. CedV receptor-binding protein (G) shares only â¼30% sequence identity with those of NiV and HeV, although they can all use ephrin-B2 as an entry receptor. We demonstrate that CedV also enters cells through additional B- and A-class ephrins (ephrin-B1, ephrin-A2, and ephrin-A5) and report the crystal structure of the CedV G ectodomain alone and in complex with ephrin-B1 or ephrin-B2. The CedV G receptor-binding site is structurally distinct from other henipaviruses, underlying its capability to accommodate additional ephrin receptors. We also show that CedV can enter cells through mouse ephrin-A1 but not human ephrin-A1, which differ by 1 residue in the key contact region. This is evidence of species specific ephrin receptor usage by a henipavirus, and implicates additional ephrin receptors in potential zoonotic transmission.
Assuntos
Efrina-B1/metabolismo , Efrina-B2/metabolismo , Efrina-B3/metabolismo , Infecções por Henipavirus/virologia , Henipavirus/fisiologia , Receptores Virais/metabolismo , Proteínas do Envelope Viral/química , Animais , Fusão Celular , Efrina-B1/genética , Efrina-B2/genética , Efrina-B3/genética , Infecções por Henipavirus/genética , Infecções por Henipavirus/metabolismo , Humanos , Camundongos , Mutação , Ligação Proteica , Conformação Proteica , Receptores Virais/genética , Especificidade da Espécie , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Internalização do VírusRESUMO
Nipah virus is an emerging pathogen in the Paramyxoviridae family. Upon transmission of Nipah virus from its natural reservoir, Pteropus spp. fruit bats, to humans, it causes respiratory and neurological disease with a case-fatality rate about 70%. Human-to-human transmission has been observed during Nipah virus outbreaks in Bangladesh and India. A therapeutic treatment for Nipah virus disease is urgently needed. Here, we tested the efficacy of remdesivir (GS-5734), a broad-acting antiviral nucleotide prodrug, against Nipah virus Bangladesh genotype in African green monkeys. Animals were inoculated with a lethal dose of Nipah virus, and a once-daily intravenous remdesivir treatment was initiated 24 hours later and continued for 12 days. Mild respiratory signs were observed in two of four treated animals, whereas all control animals developed severe respiratory disease signs. In contrast to control animals, which all succumbed to the infection, all remsdesivir-treated animals survived the lethal challenge, indicating that remdesivir represents a promising antiviral treatment for Nipah virus infection.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Infecções por Henipavirus/tratamento farmacológico , Infecções por Henipavirus/virologia , Vírus Nipah/efeitos dos fármacos , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/farmacologia , Alanina/uso terapêutico , Animais , Encéfalo/patologia , Encéfalo/virologia , Chlorocebus aethiops , Feminino , Infecções por Henipavirus/sangue , Masculino , Meningoencefalite/tratamento farmacológico , Meningoencefalite/virologia , Testes de Neutralização , Viremia/sangue , Viremia/tratamento farmacológico , Viremia/virologia , Replicação Viral/efeitos dos fármacosRESUMO
Nipah and Hendra viruses (NiV and HeV) exhibit high lethality in humans and are biosafety level 4 (BSL-4) paramyxoviruses in the growing genus Henipavirus The attachment (G) and fusion (F) envelope glycoproteins are both required for viral entry into cells and for cell-cell fusion, which is pathognomonic of henipaviral infections. Here, we compared the fusogenic capacities between homologous and heterologous pairs of NiV and HeV glycoproteins. Importantly, to accurately measure their fusogenic capacities, as these depend on glycoprotein cell surface expression (CSE) levels, we inserted identical extracellular tags to both fusion (FLAG tags) or both attachment (hemagglutinin [HA] tags) glycoproteins. Importantly, these tags were placed in extracellular sites where they did not affect glycoprotein expression or function. NiV and HeV glycoproteins induced comparable levels of homologous HEK293T cell-cell fusion. Surprisingly, however, while the heterologous NiV F/HeV G (NF/HG) combination yielded a hypofusogenic phenotype, the heterologous HeV F/NiV G (HF/NG) combination yielded a hyperfusogenic phenotype. Pseudotyped viral entry levels primarily corroborated the fusogenic phenotypes of the glycoprotein pairs analyzed. Furthermore, we constructed G and F chimeras that allowed us to map the overall regions in G and F that contributed to these hyperfusogenic or hypofusogenic phenotypes. Importantly, the fusogenic phenotypes of the glycoprotein combinations negatively correlated with the avidities of F-G interactions, supporting the F/G dissociation model of henipavirus-induced membrane fusion, even in the context of heterologous glycoprotein pairs.IMPORTANCE The NiV and HeV henipaviruses are BSL-4 pathogens transmitted from bats. NiV and HeV often lead to human death and animal diseases. The formation of multinucleated cells (syncytia) is a hallmark of henipaviral infections and is caused by fusion of cells coordinated by interactions of the viral attachment (G) and fusion (F) glycoproteins. We found via various assays that viral entry and syncytium formation depend on the viral origin of the glycoproteins, with HeV F and NiV G promoting higher membrane fusion levels than their counterparts. This is important knowledge, since both viruses use the same bat vector species and potential coinfections of these or subsequent hosts may alter the outcome of disease.