Structures of enveloped virions determined by cryogenic electron microscopy and tomography : Advances in Virus Research

Enveloped viruses enclose their genomes inside a lipid bilayer which is decorated by membrane proteins that mediate virus entry. These viruses display a wide range of sizes, morphologies and symmetries. Spherical viruses are often isometric and their envelope proteins follow icosahedral symmetry. Filamentous and pleomorphic viruses lack such global symmetry but their surface proteins may display locally ordered assemblies. Determining the structures of enveloped viruses, including the envelope proteins and their protein-protein interactions on the viral surface, is of paramount importance. These structures can reveal how the virions are assembled and released by budding from the infected host cell, how the progeny virions infect new cells by membrane fusion, and how antibodies bind surface epitopes to block infection. In this chapter, we discuss the uses of cryogenic electron microscopy (cryo-EM) in elucidating structures of enveloped virions. Starting from a detailed outline of data collection and processing strategies, we highlight how cryo-EM has been successfully utilized to provide unique insights into enveloped virus entry, assembly, and neutralization.Peer reviewe

Structural Basis for a Convergent Immune Response against Ebola Virus

Ebola virus disease is a severe health problem in Africa. Vaccines that display the Zaire ebolavirus glycoprotein spike complex are a prime component for the effort to combat it. The V(H)3-15/V(lambda)1-40-based class of antibodies was recently discovered to be a common response in individuals who received the Ebola virus vaccines. These antibodies display attractive properties, and thus likely contribute to the efficacy of the vaccines. Here, we use cryo-EM to elucidate how three V(H)3-15/V(lambda)1-40 antibodies from different individuals target the virus and found a convergent mechanism against a partially conserved site on the spike complex. Our study rationalizes the selection of the V(H)3-15/V(lambda)1-40 germline genes for specifically targeting this site and highlights Ebolavirus species-specific sequence divergences that may restrict breadth of V(H)3-15/V(lambda)1-40-based humoral response. The results from this study could help develop improved immunization schemes and further enable the design of immunogens that would be efficacious against a broader set of Ebolavirus species

Mapping of the Lassa virus LAMP1 binding site reveals unique determinants not shared by other old world arenaviruses

<div><p>Cell entry of many enveloped viruses occurs by engagement with cellular receptors, followed by internalization into endocytic compartments and pH-induced membrane fusion. A previously unnoticed step of receptor switching was found to be critical during cell entry of two devastating human pathogens: Ebola and Lassa viruses. Our recent studies revealed the functional role of receptor switching to LAMP1 for triggering membrane fusion by Lassa virus and showed the involvement of conserved histidines in this switching, suggesting that other viruses from this family may also switch to LAMP1. However, when we investigated viruses that are genetically close to Lassa virus, we discovered that they cannot bind LAMP1. A crystal structure of the receptor-binding module from Morogoro virus revealed structural differences that allowed mapping of the LAMP1 binding site to a unique set of Lassa residues not shared by other viruses in its family, illustrating a key difference in the cell-entry mechanism of Lassa virus that may contribute to its pathogenicity.</p></div

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Mapping of the LAMP1 binding site on GP1_LASV.

<p>(A) Structural comparison of the loop preceding β5. A yellow dashed line marks an apparent hydrogen bond. (B) Representative images of LAMP1 pull-down experiments by the indicated GP1_LASV-Fc mutants, each one selected from three independent repeats. The panel is composed of three intact membranes as designated by the horizontal lines. (C) Superimposition of the β–hairpins of GP1_LASV and GP1_MORV presented as Cα traces and side-chains only. (D) Ribbon representation of the β–hairpins and their close vicinity in GP1_MORV and GP1_LASV. (E) Surface representation of GP1_LASV. Residues that were mutated and critically affected, weakly affected, or had no affect on LAMP1 binding are colored purple, pink, and blue, respectively, using the same color scheme used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006337#ppat.1006337.g002" target="_blank">Fig 2E</a>. The histidine triad is red. (F) The crystal structure of GP1_LASV (white surface) docked into the EM density of the trimeric spike complex of LASV at pH 5.0 (EMDB: 3292), shown as green mesh at 1.5 σ. The histidine triad is red and all residues identified as important for LAMP1 binding are purple. The crevices between the 3 GP1 subunits are marked with black arrows.</p

GP1_LCMV and GP1_MORV do not interact with LAMP, although GP1_MORV adopts the same global fold as GP1_LASV.

<p>(A) Pull-down assay by the indicated GP1-Fc fusion proteins. The presence of LAMP1 is detected by anti-LAMP1 antibody, and Fc levels are shown using anti-Fc for load control. The pull-down assay was independently repeated three times, and a representative image is shown. (B) SPR analysis of the indicated GP1-Fc analytes. Each analyte was injected at 500 nM over immobilized distal domain of LAMP1. (C) A phylogenetic tree based on the sequences of the GPCs from the indicated OW mammarenaviruses. The scale bar represents a substitution rate of 0.4 per site. (D) Graphical representation of the 16 chains, each colored differently, that make the asymmetric unit. The chains are traced as tubes with a radius proportional to B-factor. N-linked glycans are shown as spheres. (E) Cα traces of all 16 chains superimposed. N-linked glycans are shown with lines. The cysteine residues that make the three disulfide bonds (marked with a ‘D’) are shown with spheres and are labeled, and secondary structure elements are numbered. (F) Multiple sequence alignment of GP1s from the indicated OW mammarenaviruses showing the secondary structure elements as observed in the crystal structure of GP1_MORV. The numbering of the amino acids is based on the sequence of GPC_MORV. Fully conserved residues are highlighted with red background, and partially conserved residues are shown in red. Numbers below the sequences mark the locations and connectivity of the disulfide bridges. The symbol ‘Ψ’ marks the locations of N-linked glycans seen in the crystal structure. We used ESPript [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006337#ppat.1006337.ref021" target="_blank">21</a>] (<a href="http://espript.ibcp.fr/" target="_blank">http://espript.ibcp.fr</a>) for generating this graphical representation.</p

qCMA: A Desktop Application for Quantitative Collective Cell Migration Analysis

Collective migration is an important cellular trait, which is intensely studied by both basic and translational researchers. Investigation of the underlying mechanisms necessitates high-throughput assays and computational algorithms capable of generating reproducible quantitative measurements of cell migration. We present a desktop tool that can be used easily by any researcher, to quantify both fluorescent and phase-contrast images produced in the course of commonly used gap closure ("scratch," "wound healing") collective migration assays. The software has a simple graphical interface that allows the user to tune the relevant parameters and process large numbers of images (or movies). The output contains segmented images and the numerical values inferred from them, allowing easy quantitative analysis of the results

Rational design of universal immunotherapy for TfR1-tropic arenaviruses

International audienceCertain arenaviruses that circulate in rodent populations can cause life-threatening hemor-rhagic fevers when they infect humans. Due to their efficient transmission, arenaviruses pose a severe risk for outbreaks and might be exploited as biological weapons. Effective countermeasures against these viruses are highly desired. Ideally, a single remedy would be effective against many or even all the pathogenic viruses in this family. However, despite the fact that all pathogenic arenaviruses from South America utilize transferrin receptor 1 (TfR1) as a cellular receptor, their viral glycoproteins are highly diversified, impeding efforts to isolate cross-neutralizing antibodies. Here we address this problem using a rational design approach to target TfR1-tropic arenaviruses with high potency and breadth. The pan-reactive molecule is highly effective against all arenaviruses that were tested, offering a universal therapeutic approach. Our design scheme avoids the shortcomings of previous immu-noadhesins and can be used to combat other zoonotic pathogens