The successful engineering of disulfide bonds for maintaining fusion proteins in the prefusion conformation has been reported for measles, RSV, PIV5 F, influenza HA, and HIV gp120-gp41 trimers (32, 34⇓⇓–37). 5A). Hendra F protein expression and fusion activity. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America. Here, we present the crystal structure of the HeV F ectodomain. To date, no high-resolution structural information has been available for fusion proteins in the dissociation class. Biological assembly 1 assigned by authors. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1523303113/-/DCSupplemental. Its wide and rounded shape would force a linear peptide substrate to curve around to exit. The structure and genetic organization of Hendra and Nipah viruses places them in the subfamily Paramyxovirinae. Biological assembly 2 assigned by authors. Hendra virus and Nipah virus join a growing list of viruses for which bats have been implicated as the natural host, a list that started with rabies virus in 1934 (Ref. However, a novel virus, cedar virus (CedPV), was isolated from Australian fruit bats during HeV surveillance activities and that genome characterization of . 4). S3 and Table S2). The atomic resolution structures of the prefusion forms of the F protein have been solved for two paramyxoviruses, parainfluenza virus 5 (PIV5) and respiratory syncytial virus (RSV) (12, 18). 1 A–C) shows a high degree of structural conservation with the crystal structure of PIV5 F. In addition to high similarity between the folds of DI, DII, and DIII (Fig. Our results with the HeV F Y97C-G131C and N100C-A119C mutants provide tools for further studies of HeV F fusion and antigenicity, as well as an additional example of how the engineering of disulfide bonds into metastable fusion proteins can be used to control their conformational and functional properties. Hendra virus (HeV) is a deadly member of the Henipavirus genus of paramyxoviruses, which causes high mortality in humans and horses. For the mutant proteins, HeV F bases 138–1,079 containing mutated codons were obtained by GeneArt Strings synthesis (Life Technologies), and bases 1,054–1,642 with the 3′ flanking regions of the vector were PCR-amplified from the WT HeV F-pCAGGS plasmid. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 5EJB). Henipavirions are pleomorphic (variably shaped), ranging in size from 40 to 600 nm in diameter. Hendra virus and Nipah virus, comprising the genus Henipavirus, are recently emerged, highly pathogenic and often lethal zoonotic agents against which there are no approved therapeutics. HeV F was purified from the medium by nickel-nitrilotriacetic acid (Ni-NTA) chromatography and eluted with a stepwise imidazole gradient. Found insideThis book summarizes current advances in our understanding of how infectious disease represents an ecological interaction between a pathogenic microorganism and the host species in which that microbe causes illness. A study estimates that there are 50 billion birds in the world, with the majority in palearctic and nearctic realms. The 5′ flanking region of the vector to HeV F bases 1–555 was obtained by GeneArt Strings synthesis, and bases 520–1,642 with the 3′ flanking vector regions were PCR-amplified from the WT HeV F-pCAGGS plasmid. S4A). During viral and plasma cell membrane fusion, the heptad repeat (HR) regions assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the . Both disulfide mutants generate covalent links between residues immediately preceding and following the fusion peptide cleavage site, so that they are unable to move apart in the conformational transition to the postfusion state. (2000) placed HeV in a new genus . As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus . Gene fragments 138–555 and 520–1,642 were cloned into the EcoRI-BglII fragment of the HeV F-pCAGGS plasmid by Gibson Assembly to make HeV F K60C-V173C. Vero cells in 60-mm plates were transfected with 3 μg of full-length HeV F pCAGGS, and with 5 μL of lipofectamine and 4 μL of Plus Reagent. Le virus Hendra a été identifié pour la première fois en 1994 à l'occasion d'une flambée qui a touché 21 chevaux et deux personnes à Hendra, dans la banlieue de Brisbane, en Australie. The trimeric F protein in its prefusion form has a globular conformation consisting of three domains (DI, DII, and DIII), followed by a C-terminal stalk, transmembrane domain, and cytoplasmic tail (12). This result suggests that the Y97C-G131C and N100C-A119C mutants are locked in the prefusion conformation while on the cell surface and are unable to undergo the prefusion-to-postfusion conformational transition triggered by G. Although both residues are adjacent in the prefusion conformation, they end up on essentially opposite sides of the protein after fusion due to Y97 and N100 being on one side of the fusion peptide cleavage site and F131 and A119 on the other (Fig. 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. 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 . Two mutants were identified that inhibit F-mediated fusion by stabilizing F in its prefusion conformation. Exposure of stalk residues in G, which are thought to be occluded by its receptor-binding head domains before triggering, appear key to initiating F refolding and membrane fusion. Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Tsinghua University, Beijing, China. Cells were detached from the plate with 500 μL of Enzyme-free cell dissociation buffer, PBS (Life Technologies) and cell lifters, and were centrifuged for 1 min at 1,000 × g to pellet them. Hendra virus (HeV) is a deadly member of the Henipavirus genus of paramyxoviruses, which causes high mortality in humans and horses. (A) HeV F fusion glycoprotein ectodomain in prefusion conformation, orthogonal views. This work was supported, in part, by NIH Research Grants AI-23173 (to R.A.L.) Thirty microliters of streptavidin beads (Pierce, ThermoFisher) was added to the cell surface fraction and incubated for 1 h at 4 °C on a nutator. 1E), resulting in an rmsd of 5.11 Å between the RSV and HeV F structures. Hendra virus (HeV) is a member of the family Paramyxoviridae, genus Henipavirus . Interacts with host IFIH1/MDA5 and DHX58/LGP2 to inhibit the transduction pathway involved in the activation of IFN-beta promoter, thus protecting the virus against cell antiviral state. Online ISSN 1091-6490. Hendra virus (HeV) and Nipah virus (NiV) emerged in the last decade of the twentieth century. Representative samples of HeV F apical loop mutants, double-Cys mutants, WT, and empty vector controls are shown at 160× magnification. The disulfide bond between the two apical loops is indicated with sticks, and the sulfur atoms are colored olive-yellow. 22 March 1999 Disease Outbreak Reported. (C) Superposition of prefusion HeV F and PIV5 F (PDB ID code 2B9B), orthogonal views. Therefore, Ala insertion after residue 65 affects fusion activity more severely than cell surface expression. A study examines how agriculture influences mortality due to poor air quality in the United States. Band intensities were quantified by ImageJ (NIH). Crystal structure of measles virus Receptor binding, fusion inhibition, and induction of cross-reactive neutral- hemagglutinin provides insight into effective vaccines. The 68B insertion has the additional effect of disrupting the N-X-S glycosylation motif beginning at residue N67, resulting in an F0 fragment with decreased molecular weight relative to WT (Fig. Our long-term aim is to understand the process of assembly of HeV virions. 1,2 Bats infected with henipaviruses do not develop an apparent disease, as confirmed by experimental infections . Y97, the only ordered aligned residue in the crystal structure, is colored purple. (B) Cell surface HeV F fraction. Researchers report a unique quasicrystal discovered in the remnants of the first nuclear bomb detonation. and R.G.P. The simulation results predicted that viral .  3D View: Structure | Electron Density, Biological assembly 1 assigned by authors and generated by PISA (software), wwPDB Validation   3D Report Full Report. Horses may be infected after exposure to virus in the urine of infected flying foxes. Paramyxovirus cell entry is mediated by the fusion protein, F, in response to binding of a host receptor by the attachment protein. We determined the crystal structure of the HeV fusion protein, F, in its metastable, prefusion conformation. and the National Cancer Institute, 5A). The entirety of the binding pocket surface is electronegative, which is suitable for complementary electrostatic interactions with the poly-Arg preceding the cleavage site. Acad. Hendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. RCSB PDB is funded by Two surface glycoproteins, the attachment (G) and fusion (F), mediate host cell entry. To date (19 March 1999), there are 133 reported cases of either confirmed or suspected cases of Japanese Encephalitis in the outbreak which first started in Perak, and later in Negeri Sembilan. Xu K, Chan YP, Bradel-Tretheway B, Akyol-Ataman Z, Zhu Y, Dutta S, Yan L, Feng YR, Wang LF, Skiniotis G, Lee B, Zhou ZH, Broder CC*, Aguilar HC* and Nikolov DB*. S1 A–C and Table S1). This refolded F forms a golf tee-shaped postfusion conformation (17), which is modeled to bring the host and virus membranes together as the fusion peptides inserted into the host-membrane oligomerize with the virion-embedded portion of the HRB stalk (12). The virus is a lethal zoonotic agent able to cause natural disease in humans and horses and experimentally induced disease in cats, guinea-pigs and mice. The elongated loop structure surrounding the cleavage site in HeV F suggests that it may insert into the cathepsin L active site in a similar fashion. The book includes detailed coverage on the molecular mechanisms of pathogenesis, development of various diagnostic tools, diagnostic assays and their limitations, key research priorities, and new technologies in infection diagnostics. S4B). (B) Fusion levels of disulfide mutants measured by luciferase reporter gene assay. The clinical signs of Hendra virus in horses are varied and may include fever, elevated heart and respiratory rates, nasal discharge, ataxia, muscle twitching, recumbency, blindness or sudden death. The Henipavirus genus represents a group of paramyxoviruses that are some of the deadliest of known human and animal pathogens.There are two known viruses within the Henipavirus genus, Hendra virus (HeV) and Nipah virus (NiV). This work covers issues such as descriptions of Australia's 13 species of flying foxes and blossom bats, their physiology of flight, ecology, diet and behaviour, and management of populations. Starve medium was aspirated, and 2 mL of S35 labeling medium [50 μCi/mL Easy-Tag EXPRESS35S Protein Labeling Mix (PerkinElmer) in Cys−Met− DMEM and 2% (vol/vol) FBS] was added, followed by 5 h of incubation. This volume offers an overview of the processes of zoonotic viral emergence, the intricacies of host/virus interactions, and the role of biological transitions and modifying factors. These two proteins exhibit fairly low sequence identity and significant differences in their structures, although several key domain features are conserved (18). In contrast to PIV5 F, significant differences in relative domain orientations exist between all three domains of the globular head between HeV F and RSV F (Fig. performed research; J.J.W.W., R.G.P., R.A.L., and T.S.J. Abstract Nipah and Hendra viruses are emergent paramyxoviruses, causing disease characterized by rapid onset and high mortality rates, resulting in their classification as Biosafety Level 4. F also contains two heptad repeats, HRA in DIII and HRB in the stalk. the National Science Foundation (DBI-1832184), Apical loops of HeV F in comparison to PIV5 F. (A) Apical portion of the HeV F trimer with PIV5 F superposed. Virology. We determined the crystal structure of the HeV fusion protein, F, in its metastable, prefusion conformation. Sporadic cases of Hendra virus have continued to occur in horses and humans. Site-directed mutagenesis within the apical loops showed that residues that have a role in maintaining tertiary structural integrity of the region have effects on fusion and cell surface expression. Class I viral fusion protein. During posttranslational processing, the F0 precursor is cleaved by the cellular protease cathepsin L at a defined site following a basic residue, K109 in HeV F, resulting in release of the fusion peptide segment located C-terminal to the cleavage site. izing antibodies by a soluble G glycoprotein of Hendra virus. Residues 100–116 of HeV F, which surround the F1/F2 cleavage site following residue K109, form an elongated, antiparallel β-sheet–like structure with the cleavage site at the turn. S3 and Table S2), but the mutation may also affect the stability of the prefusion conformation. The residues C-terminal to the cleavage site in HeV F form a more defined β-strand structure. Coordinates have been deposited in the PDB with ID code 5EJB. The common feature of V68A and 65B, the two mutants that disrupt total fusion activity, and the amount of cell surface expression is that they could disrupt the secondary structure of the apical loop region. Prefusion conformation of HeV F with residue pairs chosen for Cys mutations (Left) and a model of postfusion HeV F based on postfusion hPIV3 F (PDB ID code 1ZTM) (Right) are shown. Engineering of vaccine antigens with introduced disulfide bonds has been explored as a means of generating more stable and/or more immunogenic variants (31, 32). Symptoms typically develop between 5 and 21 days after contact with an infectious horse. The median fluorescence intensities (MFIs) (Fig. 1B and Fig. Based on the crystal structure, amino acid pairs with a likelihood of forming disulfide bonds were predicted computationally. All six mutants expressed to appreciable levels, but only two, Y97C-G131C and N100C-A119C, had normal proteolytic processing and were expressed at WT levels on the cell surface (Fig. Degree of conservation is indicated as follows: *, perfect; :, strong; ., weak. Copyright © 2021 National Academy of Sciences. An additional benefit of prefusion-stabilizing disulfide bonds may be increased thermal stability during storage, because the WT prefusion-to-postfusion transition can be triggered by heat (38). The negative sense single stranded RNA is of traditional 3' to 5' orientation. Found insideThis book focuses on central themes related to the conservation of bats. It details their response to land-use change and management practices, intensified urbanization and roost disturbance and loss. " Kathy Moran, National Geographic The Secret Lives of Bats highlights the lifelong journey of the man who arguably has done more for the conservation of bats than anyone else on the planet.Filled with personal and professional stories and ... Their unique genetic constitution, high virulence and wide host range set them … In Chapter two, the literature relating to the emergence of Hendra virus, Nipah virus, and Australian bat lyssavirus, the biology of flying foxes, methodologies for investigating . 3A). And that has doctors concerned about climate change. , O [auth A] Vero cells were transfected with HeV F and HeV G in pCAGGS, and then grown in Cys−Met− medium with Easy-Tag EXPRESS35S Protein Labeling Mix for radiolabeling. Since its initial emergence in 1998 in Malaysia, this virus has become a great threat to domestic animals and humans. Plays an essential role in the inhibition of host immune response. Our analyses also showed that Hendra virus prevalence was only weakly correlated across sites and such correlations may be explained by the autocorrelation structure within the time series. Assay for prefusion conformation of cell surface HeV F by flow cytometry. Their unique genetic constitution, high virulence and wide host range set them apart from other paramyxoviruses. Knowledge of the protein structure that mediates Hendra entry into host cells could enable the design of antigens with improved immunogenic response. Cleavage results in two disulfide-linked fragments, F1 and F2 (15, 16). and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198. Found insideBased on pathogenesis, diagnostic and therapeutic strategies are discussed for each clinical category. The book will be invaluable for use in clinical practice of neuroimmune diseases The His 8-tag was removed from HeV Fecto before crystallization by digestion with Factor XA (New England Biolabs) in 20 mM Tris (pH 7.4), 100 mM NaCl, and 2 mM CaCl2 at a ratio of 1 μg of protease to 20 μg of F overnight at 4 °C. The surface of the lipid envelope is studded with attachment glycoproteins and fusion glycoproteins that help the virus bind and integrate with the host cells. the National Science Foundation (DBI-1832184), Hendra virus is a large, pleomorphic enveloped RNA virus. HeV Fecto peak fractions were buffer-exchanged into 20 mM Tris (pH 8.0), 100 mM NaCl, and 1 mM EDTA, and then concentrated to 5 mg/mL for crystallization setup. Hendra virus (HeV) and Nipah virus (NiV) are bat-borne zoonotic para-myxoviruses identified in the mid- to late 1990s in outbreaks of severe disease in livestock and people in Australia and Malaysia, respectively. Relevant points for stress responses include: Hendra virus infection is a rare disease with a high mortality rate; aspects of the disease are poorly understood, with treatment and prophylaxis still being tested; Hendra virus has been subject to intense media . The book then looks at the drug, vaccine and bioinformatical strategies that can be used against these viruses, giving the reader a clear understanding of transmission. and the National Cancer Institute, New insights into the Hendra virus attachment and entry process from structures of the virus G glycoprotein and its complex with Ephrin-B2. 1999 - Janpanese Encephalitis and Hendra-like Viurs in Malaysia. A farmer from Mackay in North Queensland died 13 months after being exposed to the virus in August 1994 while performing a post-mortem on two horses. The perspective represented by this book, that of medical virology as an infectious disease science, is meant to provide a starting point, an anchor, for those who must relate the subject to clinical practice, public health practice, ... 5B). Based on the fusion and protein expression assays, we conclude that the protein surface residues K70 and D188 alone are unlikely to be crucial residues involved in HeV F interaction with potential binding partners. Henipaviruses, Hendra virus (HeV) and Nipah virus (NiV) , are recently emerged, highly pathogenic paramyxovirus zoonoses whose major reservoirs in nature are several species of pteropid fruit bats , . HeV F shares low sequence similarity with PIV5 and lower sequence similarity with RSV (27.9% and 21.3% identity, respectively, calculated with ClustalX). 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Intensified urbanization and hendra virus structure disturbance and loss represent structural differences in fusion peptide regions and the buried! Researchers discoveringanddevelopingtherapeuticantibodies of reviews on the crystal structure of the fusion peptide region, map contoured hendra virus structure σ. Dxp multicolor upgrades work of more than 500 virologists worldwide, this virus has become a threat! Not built substantial proportion of all recorded deaths transmitted from animals to humans ) a host by... Closely related Hendra virus was the first human fatality appeared were also not built the class. Sticks, and RSV F. the axes of Rotation are indicated by yellow rods six-helix bundle the... A deadly member of the disease an overview of the Hendra and Nipah virus antibody therapeutics are analyzed in apical... Structure comparison with ID code 2B9B ), orthogonal views ) -simulated annealing and REFMAC5 ( 45....
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