RESUMO
We report the first cryoEM structure of the Hendra henipavirus nucleoprotein in complex with RNA, at 3.5 Å resolution, derived from single particle analysis of a double homotetradecameric RNA-bound N protein ring assembly exhibiting D14 symmetry. The structure of the HeV N protein adopts the common bi-lobed paramyxoviral N protein fold; the N-terminal and C-terminal globular domains are bisected by an RNA binding cleft containing six RNA nucleotides and are flanked by the N-terminal and C-terminal arms, respectively. In common with other paramyxoviral nucleocapsids, the lateral interface between adjacent Ni and Ni+1 protomers involves electrostatic and hydrophobic interactions mediated primarily through the N-terminal arm and globular domains with minor contribution from the C-terminal arm. However, the HeV N multimeric assembly uniquely identifies an additional protomer-protomer contact between the Ni+1 N-terminus and Ni-1 C-terminal arm linker. The model presented here broadens the understanding of RNA-bound paramyxoviral nucleocapsid architectures and provides a platform for further insight into the molecular biology of HeV, as well as the development of antiviral interventions.
Assuntos
Microscopia Crioeletrônica , Vírus Hendra , Nucleocapsídeo , Nucleoproteínas , Vírus Hendra/química , Nucleoproteínas/química , Nucleoproteínas/ultraestrutura , Nucleoproteínas/metabolismo , Nucleocapsídeo/química , Nucleocapsídeo/ultraestrutura , Nucleocapsídeo/metabolismo , Modelos Moleculares , RNA Viral/química , RNA Viral/metabolismo , RNA Viral/genética , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/ultraestrutura , Proteínas do Nucleocapsídeo/metabolismoRESUMO
Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes frequent outbreaks of severe neurologic and respiratory disease in humans with high case fatality rates. The 2 glycoproteins displayed on the surface of the virus, NiV-G and NiV-F, mediate host-cell attachment and membrane fusion, respectively, and are targets of the host antibody response. Here, we provide a molecular basis for neutralization of NiV through antibody-mediated targeting of NiV-F. Structural characterization of a neutralizing antibody (nAb) in complex with trimeric prefusion NiV-F reveals an epitope at the membrane-distal domain III (DIII) of the molecule, a region that undergoes substantial refolding during host-cell entry. The epitope of this monoclonal antibody (mAb66) is primarily protein-specific and we observe that glycosylation at the periphery of the interface likely does not inhibit mAb66 binding to NiV-F. Further characterization reveals that a Hendra virus-F-specific nAb (mAb36) and many antibodies in an antihenipavirus-F polyclonal antibody mixture (pAb835) also target this region of the molecule. Integrated with previously reported paramyxovirus F-nAb structures, these data support a model whereby the membrane-distal region of the F protein is targeted by the antibody-mediated immune response across henipaviruses. Notably, our domain-specific sequence analysis reveals no evidence of selective pressure at this region of the molecule, suggestive that functional constraints prevent immune-driven sequence variation. Combined, our data reveal the membrane-distal region of NiV-F as a site of vulnerability on the NiV surface.
Assuntos
Anticorpos Neutralizantes , Vírus Hendra , Proteínas Virais de Fusão , Internalização do Vírus , Anticorpos Monoclonais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Linhagem Celular Tumoral , Glicosilação , Células HEK293 , Vírus Hendra/química , Vírus Hendra/imunologia , Vírus Hendra/metabolismo , Vírus Hendra/fisiologia , Humanos , Modelos Moleculares , Ligação Proteica , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/metabolismoRESUMO
Enveloped viruses utilize surface glycoproteins to bind and fuse with a target cell membrane. The zoonotic Hendra virus (HeV), a member of the family Paramyxoviridae, utilizes the attachment protein (G) and the fusion protein (F) to perform these critical functions. Upon triggering, the trimeric F protein undergoes a large, irreversible conformation change to drive membrane fusion. Previously, we have shown that the transmembrane (TM) domain of the F protein, separate from the rest of the protein, is present in a monomer-trimer equilibrium. This TM-TM association contributes to the stability of the prefusion form of the protein, supporting a role for TM-TM interactions in the control of F protein conformational changes. To determine the impact of disrupting TM-TM interactions, constructs expressing the HeV F TM with limited flanking sequences were synthesized. Coexpression of these constructs with HeV F resulted in dramatic reductions in the stability of F protein expression and fusion activity. In contrast, no effects were observed when the HeV F TM constructs were coexpressed with the nonhomologous parainfluenza virus 5 (PIV5) fusion protein, indicating a requirement for specific interactions. To further examine this, a TM peptide homologous to the PIV5 F TM domain was synthesized. Addition of the peptide prior to infection inhibited infection with PIV5 but did not significantly affect infection with human metapneumovirus, a related virus. These results indicate that targeted disruption of TM-TM interactions significantly impact viral fusion protein stability and function, presenting these interactions as a novel target for antiviral development.IMPORTANCE Enveloped viruses require virus-cell membrane fusion to release the viral genome and replicate. The viral fusion protein triggers from the pre- to the postfusion conformation, an essentially irreversible change, to drive membrane fusion. We found that small proteins containing the TM and a limited flanking region homologous to the fusion protein of the zoonotic Hendra virus reduced protein expression and fusion activity. The introduction of exogenous TM peptides may displace a TM domain, disrupting native TM-TM interactions and globally destabilizing the fusion protein. Supporting this hypothesis, we showed that a sequence-specific transmembrane peptide dramatically reduced viral infection in another enveloped virus model, suggesting a broader inhibitory mechanism. Viral fusion protein TM-TM interactions are important for protein function, and disruption of these interactions dramatically reduces protein stability.
Assuntos
Paramyxovirinae/metabolismo , Peptídeos/farmacologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Animais , Sítios de Ligação/efeitos dos fármacos , Chlorocebus aethiops , Vírus Hendra/química , Vírus Hendra/genética , Vírus Hendra/metabolismo , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Vírus da Parainfluenza 5/química , Vírus da Parainfluenza 5/genética , Vírus da Parainfluenza 5/metabolismo , Paramyxovirinae/química , Paramyxovirinae/genética , Conformação Proteica/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Estabilidade Proteica , Células Vero , Proteínas Virais de Fusão/efeitos dos fármacosRESUMO
Henipavirus, including Hendra virus (HeV) and Nipah virus (NiV), is a newly discovered human pathogen genus. The nucleoprotein of Henipavirus contains an α-helical molecular recognition element (α-MoRE) that folds upon binding to the X domain (XD) of the phosphoprotein (P). In order to explore the conformational dynamics of free α-MoREs and the underlying binding-folding mechanism with XD, atomic force field-based and hybrid structure-based MD simulations were carried out. In our empirical force field-based simulations, characteristic structures and helicities of α-MoREs reveal the co-existence of partially structured and disordered conformations, as in the case of the well characterized cognate measles virus (MeV) α-MoRE. In spite of their overall similarity, the two α-MoREs display subtle helicity differences in their C-terminal region, but much different from that of MeV. For the α-MoRE/XD complexes, the results of our hybrid structure-based simulations provide the coupled binding-folding landscapes, and unveil a wide conformational selection mechanism at early binding stages, followed by a final induce-fit mechanism selection process. However, the HeV and NiV complexes have a lower binding barrier compared to that of MeV. Moreover, the HeV α-MoRE/XD complex shows much less coupling effects between binding and folding compared to that from both NiV and MeV. Our analysis revealed that contrary to NiV and MeV, the N- and C-terminal regions of the HeV α-MoRE maintains a low helicity also in the bound form.
Assuntos
Vírus Hendra/química , Simulação de Dinâmica Molecular , Vírus Nipah/química , Nucleoproteínas/química , Proteínas Virais/química , Vírus Hendra/metabolismo , Vírus Nipah/metabolismo , Nucleoproteínas/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Virais/metabolismoRESUMO
Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of ß-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability.
Assuntos
Vírus Hendra/química , Proteínas do Core Viral/química , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Chlorocebus aethiops , Vírus Hendra/genética , Vírus Hendra/metabolismo , Mutação de Sentido Incorreto , Domínios Proteicos , Relação Estrutura-Atividade , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismoRESUMO
Hendra virus (HeV) is an emerging zoonotic paramyxovirus within the genus Henipavirus that has caused severe morbidity and mortality in humans and horses in Australia since 1994. HeV infection of host cells is mediated by the membrane bound attachment (G) and fusion (F) glycoproteins, that are essential for receptor binding and fusion of viral and cellular membranes. The eukaryotic unicellular parasite Leishmania tarentolae has recently been established as a powerful tool to express recombinant proteins with mammalian-like glycosylation patterns, but only few viral proteins have been expressed in this system so far. Here, we describe the purification of a truncated, Strep-tag labelled and soluble version of the HeV attachment protein (sHeV G) expressed in stably transfected L. tarentolae cells. After Strep-tag purification the identity of sHeV G was confirmed by immunoblotting and mass spectrometry. The functional binding of sHeV G to the HeV cell entry receptor ephrin-B2 was confirmed in several binding assays. Generated polyclonal rabbit antiserum against sHeV G reacted with both HeV and Nipah virus (NiV) G proteins in immunofluorescence assay and efficiently neutralised NiV infection, thus further supporting the preserved antigenicity of the purified protein.
Assuntos
Vírus Hendra/química , Leishmania/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Animais , Austrália , Efrina-B2/metabolismo , Vírus Hendra/genética , Vírus Hendra/imunologia , Vírus Hendra/fisiologia , Cavalos , Humanos , Leishmania/metabolismo , Oligopeptídeos/metabolismo , Engenharia de Proteínas , Coelhos , Receptores Virais/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/isolamento & purificação , Ligação Viral , Internalização do VírusRESUMO
Hendra virus (HeV) is one of the two prototypical members of the Henipavirus genus of paramyxoviruses, which are designated biosafety level 4 (BSL-4) organisms due to the high mortality rate of Nipah virus (NiV) and HeV in humans. Paramyxovirus cell entry is mediated by the fusion protein, F, in response to binding of a host receptor by the attachment protein. During posttranslational processing, the fusion peptide of F is released and, upon receptor-induced triggering, inserts into the host cell membrane. As F undergoes a dramatic refolding from its prefusion to postfusion conformation, the fusion peptide brings the host and viral membranes together, allowing entry of the viral RNA. Here, we present the crystal structure of the prefusion form of the HeV F ectodomain. The structure shows very high similarity to the structure of prefusion parainfluenza virus 5 (PIV5) F, with the main structural differences in the membrane distal apical loops and the fusion peptide cleavage loop. Functional assays of mutants show that the apical loop can tolerate perturbation in length and surface residues without loss of function, except for residues involved in the stability and conservation of the F protein fold. Structure-based disulfide mutants were designed to anchor the fusion peptide to conformationally invariant residues of the F head. Two mutants were identified that inhibit F-mediated fusion by stabilizing F in its prefusion conformation.
Assuntos
Vírus Hendra/química , Proteínas Virais de Fusão/química , Sequência de Aminoácidos , Cristalografia por Raios X , Dissulfetos/química , Células HEK293 , Humanos , Dados de Sequência Molecular , Conformação Proteica , Estabilidade ProteicaRESUMO
Hendra virus (family Paramyxoviridae) is a negative sense single-stranded RNA virus (NSRV) which has been found to cause disease in humans, horses, and experimentally in other animals, e.g. pigs and cats. Pteropid bats commonly known as flying foxes have been identified as the natural host reservoir. The Hendra virus nucleocapsid protein (HeV N) represents the most abundant viral protein produced by the host cell, and is highly immunogenic with naturally infected humans and horses producing specific antibodies towards this protein. The purpose of this study was to express and purify soluble, functionally active recombinant HeV N, suitable for use as an immunodiagnostic reagent to detect antibodies against HeV. We expressed both full-length HeV N, (HeV NFL), and a C-terminal truncated form, (HeV NCORE), using a bacterial heterologous expression system. Both HeV N constructs were engineered with an N-terminal Hisx6 tag, and purified using a combination of immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Purified recombinant HeV N proteins self-assembled into soluble higher order oligomers as determined by SEC and negative-stain transmission electron microscopy. Both HeV N proteins were highly immuno-reactive with sera from animals and humans infected with either HeV or the closely related Nipah virus (NiV), but displayed no immuno-reactivity towards sera from animals infected with a non-pathogenic paramyxovirus (CedPV), or animals receiving Equivac® (HeV G glycoprotein subunit vaccine), using a Luminex-based multiplexed microsphere assay.
Assuntos
Vírus Hendra/química , Vírus Hendra/imunologia , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Vírus Hendra/genética , Vírus Hendra/ultraestrutura , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/virologia , Cavalos , Humanos , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/ultraestrutura , Plasmídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/ultraestrutura , SuínosRESUMO
The Hendra virus is a member of the Henipavirus genus within the Paramyxoviridae family. The nucleoprotein, which consists of a structured core and of a C-terminal intrinsically disordered domain (N(TAIL)), encapsidates the viral genome within a helical nucleocapsid. N(TAIL) partly protrudes from the surface of the nucleocapsid being thus capable of interacting with the C-terminal X domain (XD) of the viral phosphoprotein. Interaction with XD implies a molecular recognition element (MoRE) that is located within N(TAIL) residues 470-490, and that undergoes α-helical folding. The MoRE has been proposed to be embedded in the hydrophobic groove delimited by helices α2 and α3 of XD, although experimental data could not discriminate between a parallel and an antiparallel orientation of the MoRE. Previous studies also showed that if the binding interface is enriched in hydrophobic residues, charged residues located close to the interface might play a role in complex formation. Here, we targeted for site directed mutagenesis two acidic and two basic residues within XD and N(TAIL). ITC studies showed that electrostatics plays a crucial role in complex formation and pointed a parallel orientation of the MoRE as more likely. Further support for a parallel orientation was afforded by SAXS studies that made use of two chimeric constructs in which XD and the MoRE were covalently linked to each other. Altogether, these studies unveiled the multiparametric nature of the interactions established within this complex and contribute to shed light onto the molecular features of protein interfaces involving intrinsically disordered regions.
Assuntos
Vírus Hendra/química , Modelos Moleculares , Nucleoproteínas/química , Proteínas Virais/química , Vírus Hendra/genética , Interações Hidrofóbicas e Hidrofílicas , Mutagênese Sítio-Dirigida , Proteínas do Nucleocapsídeo , Nucleoproteínas/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Virais/genéticaRESUMO
Nipah and Hendra viruses are recently emerged paramyxoviruses belonging to the Henipavirus genus. The Henipavirus phosphoprotein (P) consists of a large intrinsically disordered domain and a C-terminal domain (PCT) containing alternating disordered and ordered regions. Among these latter is the P multimerization domain (PMD). Using biochemical, analytical ultracentrifugation and small-angle X-ray scattering (SAXS) studies, we show that Hendra virus (HeV) PMD forms an elongated coiled-coil homotrimer in solution, in agreement with our previous findings on Nipah virus (NiV) PMD. However, the orientation of the N-terminal region differs from that observed in solution for NiV PMD, consistent with the ability of this region to adopt different conformations. SAXS studies provided evidence for a trimeric organization also in the case of PCT, thus extending and strengthening our findings on PMD. The present results are discussed in light of conflicting reports in the literature pointing to a tetrameric organization of paramyxoviral P proteins.
Assuntos
Vírus Hendra/química , Vírus Hendra/fisiologia , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , UltracentrifugaçãoRESUMO
Hendra virus (HeV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The HeV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid. Recruitment of the viral polymerase onto the nucleocapsid template relies on the interaction between the C-terminal domain, N(TAIL), of N and the C-terminal X domain, XD, of the polymerase co-factor phosphoprotein (P). Here, we provide an atomic resolution description of the intrinsically disordered N(TAIL) domain in its isolated state and in intact nucleocapsids using nuclear magnetic resonance (NMR) spectroscopy. Using electron microscopy, we show that HeV nucleocapsids form herringbone-like structures typical of paramyxoviruses. We also report the crystal structure of XD of P that consists of a three-helix bundle. We study the interaction between N(TAIL) and XD using NMR titration experiments and provide a detailed mapping of the reciprocal binding sites. We show that the interaction is accompanied by α-helical folding of the molecular recognition element of N(TAIL) upon binding to a hydrophobic patch on the surface of XD. Finally, using solution NMR, we investigate the interaction between intact nucleocapsids and XD. Our results indicate that monomeric XD binds to N(TAIL) without triggering an additional unwinding of the nucleocapsid template. The present results provide a structural description at the atomic level of the protein-protein interactions required for transcription and replication of HeV, and the first direct observation of the interaction between the X domain of P and intact nucleocapsids in Paramyxoviridae.
Assuntos
Vírus Hendra/química , Proteínas do Nucleocapsídeo/química , Fosfoproteínas/química , Cristalografia por Raios X , Vírus Hendra/genética , Vírus Hendra/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Vírus Hendra/química , Proteínas Virais de Fusão/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Reações Cruzadas , Cristalografia por Raios X , Vírus Hendra/genética , Vírus Hendra/imunologia , Infecções por Henipavirus/genética , Infecções por Henipavirus/imunologia , Humanos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologiaRESUMO
Hendra virus is a highly pathogenic paramyxovirus classified as a biosafety level four agent. The fusion (F) protein of Hendra virus is critical for promoting viral entry and cell-to-cell fusion. To be fusogenically active, Hendra virus F must undergo endocytic recycling and cleavage by the endosomal/lysosomal protease cathepsin L, but the route of Hendra virus F following internalization and the recycling signals involved are poorly understood. We examined the intracellular distribution of Hendra virus F following endocytosis and showed that it is primarily present in Rab5- and Rab4-positive endosomal compartments, suggesting that cathepsin L cleavage occurs in early endosomes. Hendra virus F transmembrane domain (TMD) residues S490 and Y498 were found to be important for correct Hendra virus F recycling, with the hydroxyl group of S490 and the aromatic ring of Y498 important for this process. In addition, changes in association of isolated Hendra virus F TMDs correlated with alterations to Hendra virus F recycling, suggesting that appropriate TMD interactions play an important role in endocytic trafficking.
Assuntos
Endocitose , Vírus Hendra/metabolismo , Infecções por Henipavirus/fisiopatologia , Infecções por Henipavirus/virologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Endossomos/metabolismo , Vírus Hendra/química , Vírus Hendra/genética , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Alinhamento de Sequência , Proteínas Virais de Fusão/genéticaRESUMO
While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that ß-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion.
Assuntos
Vírus Hendra/metabolismo , Infecções por Henipavirus/virologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Vírus Hendra/química , Vírus Hendra/genética , Humanos , Dados de Sequência Molecular , Dobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteínas Virais de Fusão/genéticaRESUMO
Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed beta-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to alpha-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60 degrees ) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses.
Assuntos
Vírus Hendra/fisiologia , Infecções por Henipavirus/virologia , Proteínas do Envelope Viral/química , Montagem de Vírus , Linhagem Celular , Dimerização , Vírus Hendra/química , Vírus Hendra/genética , Humanos , Conformação Molecular , Estrutura Secundária de Proteína , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Hendra virus (HeV) is an emerging paramyxovirus capable of infecting and causing disease in a variety of mammalian species, including humans. The virus infects its host cells through the coordinated functions of its fusion (F) and attachment (G) glycoproteins, the latter of which is responsible for binding the virus receptors ephrinB2 and ephrinB3. In order to identify the receptor binding site, a panel of G glycoprotein constructs containing mutations was generated using an alanine-scanning mutagenesis strategy. Based on a predicted G structure, charged amino acids residing in regions that could be homologous to those in the measles virus H attachment glycoprotein known to be involved in its protein receptor interaction were targeted. Using a coprecipitation-based assay, seven single-amino-acid substitutions in HeV G were identified as having significantly impaired binding to both the ephrinB2 and ephrinB3 viral receptors: D257A, D260A, G439A, K443A, G449A, K465A, and D468A. The impairment of receptor interaction conferred a concomitant diminution in their abilities to promote membrane fusion when coexpressed with F. The G glycoprotein mutants were also recognized by three or more conformation-dependent monoclonal antibodies of a panel of five, were expressed on the cell surface, and retained their abilities to bind and coprecipitate F. Interestingly, some of these mutant G glycoproteins coprecipitated with F more efficiently than wild-type G. Taken together, these data provide strong biochemical and functional evidence that some of these residues could be part of a conformation-dependent, discontinuous, and overlapping ephrinB2 and -B3 binding domain within the HeV G glycoprotein.
Assuntos
Aminoácidos/metabolismo , Vírus Hendra/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo , Substituição de Aminoácidos/genética , Aminoácidos/genética , Sítios de Ligação/genética , Linhagem Celular , Efrina-B2/metabolismo , Efrina-B3/metabolismo , Células HeLa , Vírus Hendra/química , Vírus Hendra/genética , Humanos , Valor Preditivo dos Testes , Ligação Proteica , Conformação Proteica , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
The Nipah and Hendra viruses are highly pathogenic paramyxoviruses that recently emerged from flying foxes to cause serious disease outbreaks in humans and livestock in Australia, Malaysia, Singapore and Bangladesh. Their unique genetic constitution, high virulence and wide host range set them apart from other paramyxoviruses. These characteristics have led to their classification into the new genus Henpavirus within the family Paramyxoviridae and to their designation as Biosafety Level 4 pathogens. The fusion protein, an enveloped glycoprotein essential for viral entry, belongs to the family of class I fusion proteins and is characterized by the presence of two heptad repeat (HR) regions, HR1 and HR2. These two regions associate to form a fusion-active hairpin conformation that juxtaposes the viral and cellular membranes to facilitate membrane fusion and enable subsequent viral entry. The Hendra and Nipah virus fusion core proteins were crystallized and their structures determined to 2.2 A resolution. The Nipah and Hendra fusion core structures are six-helix bundles with three HR2 helices packed against the hydrophobic grooves on the surface of a central coiled coil formed by three parallel HR1 helices in an oblique antiparallel manner. Because of the high level of conservation in core regions, it is proposed that the Nipah and Hendra virus fusion cores can provide a model for membrane fusion in all paramyxoviruses. The relatively deep grooves on the surface of the central coiled coil represent a good target site for drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation.
Assuntos
Vírus Hendra/química , Proteínas do Envelope Viral/química , Proteínas Virais de Fusão/química , Sequência de Aminoácidos , Cristalização , Fusão de Membrana , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae. Each has a broad species tropism and can cause disease with high mortality in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) envelope glycoproteins (Envs). Seven Fabs, m101 to -7, were selected for their significant binding to a soluble form of Hendra G (sG) which was used as the antigen for panning of a large naïve human antibody library. The selected Fabs inhibited, to various degrees, cell fusion mediated by the HeV or NiV Envs and virus infection. The conversion of the most potent neutralizer of infectious HeV, Fab m101, to immunoglobulin G1 (IgG1) significantly increased its cell fusion inhibitory activity: the 50% inhibitory concentration was decreased more than 10-fold to approximately 1 microg/ml. The IgG1 m101 was also exceptionally potent in neutralizing infectious HeV; complete (100%) neutralization was achieved with 12.5 microg/ml, and 98% neutralization required only 1.6 microg/ml. The inhibition of fusion and infection correlated with binding of the Fabs to full-length G as measured by immunoprecipitation and less with binding to sG as measured by enzyme-linked immunosorbent assay and Biacore. m101 and m102 competed with the ephrin-B2, which we recently identified as a functional receptor for both HeV and NiV, indicating a possible mechanism of neutralization by these antibodies. The m101, m102, and m103 antibodies competed with each other, suggesting that they bind to overlapping epitopes which are distinct from the epitopes of m106 and m107. In an initial attempt to localize the epitopes of m101 and m102, we measured their binding to a panel of 11 G alanine-scanning mutants and identified two mutants, P185A and Q191 K192A, which significantly decreased binding to m101 and one, G183, which decreased binding of m102 to G. These results suggest that m101 to -7 are specific for HeV or NiV or both and exhibit various neutralizing activities; they are the first human monoclonal antibodies identified against these viruses and could be used for treatment, prophylaxis, and diagnosis and as research reagents and could aid in the development of vaccines.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus Hendra/imunologia , Vírus Nipah/imunologia , Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Especificidade de Anticorpos , Reações Cruzadas , Relação Dose-Resposta Imunológica , Epitopos/imunologia , Glicoproteínas/imunologia , Vírus Hendra/química , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Testes de Neutralização , Biblioteca de Peptídeos , Solubilidade , Proteínas do Envelope Viral/imunologiaRESUMO
Interferon (IFN) can activate Signal Transducer and Activator of Transcription (STAT) proteins to establish a cellular antiviral response and inhibit virus replication. Many viruses have evolved strategies to inhibit this antiviral mechanism, but paramyxoviruses are unique in their abilities to directly target the IFN-responsive STAT proteins. Hendra virus and Nipah virus (Henipaviruses) are recently emerged paramyxoviruses that are the causative agents of fatal disease outbreaks in Australia and peninsular Malaysia. Similar to other paramyxoviruses, Henipaviruses inhibit IFN signal transduction through a virus-encoded protein called V. Recent studies have shown that Henipavirus V proteins target STAT proteins by inducing the formation of cytoplasmically localized high molecular weight STAT-containing complexes. This sequestration of STAT1 and STAT2 prevents STAT activation and blocks antiviral IFN signaling. As the V proteins are important factors for host evasion, they represent logical targets for therapeutics directed against Henipavirus epidemics.