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      Capturing a Flavivirus Pre-Fusion Intermediate

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          During cell entry of flaviviruses, low endosomal pH triggers the rearrangement of the viral surface glycoproteins to a fusion-active state that allows the release of the infectious RNA into the cytoplasm. In this work, West Nile virus was complexed with Fab fragments of the neutralizing mAb E16 and was subsequently exposed to low pH, trapping the virions in a pre-fusion intermediate state. The structure of the complex was studied by cryo-electron microscopy and provides the first structural glimpse of a flavivirus fusion intermediate near physiological conditions. A radial expansion of the outer protein layer of the virion was observed compared to the structure at pH 8. The resulting ∼60 Å-wide shell of low density between lipid bilayer and outer protein layer is likely traversed by the stem region of the E glycoprotein. By using antibody fragments, we have captured a structural intermediate of a virus that likely occurs during cell entry. The trapping of structural transition states by antibody fragments will be applicable for other processes in the flavivirus life cycle and delineating other cellular events that involve conformational rearrangements.

          Author Summary

          West Nile Virus (WNV) and other related viruses such as dengue virus enter their host cell by a process that involves fusion between the viral membrane and the membrane of cellular vesicles (endosomes) resulting in the release of the viral genome into the cytoplasm of the cell. This fusion event is initiated by low pH in the endosomes. Little is known regarding structural changes within the viral particle that render the viral surface proteins capable of fusion. In the present study, we used antibody fragments as a tool to trap virions in a pre-fusion intermediate state and examined these particles by cryo-electron microscopy. We showed that low pH triggered a radial displacement of the virion's external protein layer. The surface proteins moved away from the viral membrane, a shift made possible by the outward extension of a small alpha-helical region of the surface protein. The process gives the proteins greater sideways freedom for their reorganization into the fusion-active state. Our results provide a first structural glimpse into the low pH-induced conformational rearrangement of the flavivirus particle that occurs prior to fusion of viral and endosomal membranes. Such dissection of the fusion process highlights targets for the development of antiviral strategies.

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          Most cited references 18

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          EMAN: semiautomated software for high-resolution single-particle reconstructions.

          We present EMAN (Electron Micrograph ANalysis), a software package for performing semiautomated single-particle reconstructions from transmission electron micrographs. The goal of this project is to provide software capable of performing single-particle reconstructions beyond 10 A as such high-resolution data become available. A complete single-particle reconstruction algorithm is implemented. Options are available to generate an initial model for particles with no symmetry, a single axis of rotational symmetry, or icosahedral symmetry. Model refinement is an iterative process, which utilizes classification by model-based projection matching. CTF (contrast transfer function) parameters are determined using a new paradigm in which data from multiple micrographs are fit simultaneously. Amplitude and phase CTF correction is then performed automatically as part of the refinement loop. A graphical user interface is provided, so even those with little image processing experience will be able to begin performing reconstructions. Advanced users can directly use the lower level shell commands and even expand the package utilizing EMAN's extensive image-processing library. The package was written from scratch in C++ and is provided free of charge on our Web site. We present an overview of the package as well as several conformance tests with simulated data. Copyright 1999 Academic Press.
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            Viral membrane fusion

            Infection by viruses having lipid-bilayer envelopes proceeds through fusion of the viral membrane with a membrane of the target cell. Viral ‘fusion proteins’ facilitate this process. They vary greatly in structure, but all seem to have a common mechanism of action, in which a ligand-triggered, large-scale conformational change in the fusion protein is coupled to apposition and merger of the two bilayers. We describe three examples—the influenza virus hemagglutinin, the flavivirus E protein and the vesicular stomatitis virus G protein—in some detail, to illustrate the ways in which different structures have evolved to implement this common mechanism. Fusion inhibitors can be effective antiviral agents.
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              The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution.

              The crystallographically determined structure of a soluble fragment from the major envelope protein of a flavivirus reveals an unusual architecture. The flat, elongated dimer extends in a direction that would be parallel to the viral membrane. Residues that influence binding of monoclonal antibodies lie on the outward-facing surface of the protein. The clustering of mutations that affect virulence in various flaviviruses indicates a possible receptor binding site and, together with other mutational and biochemical data, suggests a picture for the fusion-activating, conformational change triggered by low pH.

                Author and article information

                Role: Editor
                PLoS Pathog
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                November 2009
                November 2009
                26 November 2009
                : 5
                : 11
                [1 ]Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America
                [2 ]MacroGenics Inc., Rockville, Maryland, United States of America
                [3 ]Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America
                [4 ]Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, United States of America
                [5 ]Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, United States of America
                [6 ]Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America
                University of North Carolina, United States of America
                Author notes

                Conceived and designed the experiments: BK MGR. Performed the experiments: BK PRC HAH. Analyzed the data: BK RJK MSD MGR. Contributed reagents/materials/analysis tools: SJ DHF MSD. Wrote the paper: BK RJK MSD MGR.

                Kaufmann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 5
                Research Article
                Biochemistry/Macromolecular Assemblies and Machines
                Biophysics/Macromolecular Assemblies and Machines
                Computational Biology/Macromolecular Structure Analysis
                Infectious Diseases/Viral Infections
                Virology/Host Invasion and Cell Entry
                Virology/Virion Structure, Assembly, and Egress

                Infectious disease & Microbiology


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