Capturing a Flavivirus Pre-Fusion Intermediate

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

Structure of the St. Louis encephalitis virus postfusion envelope trimer

St. Louis encephalitis virus (SLEV) is a mosquito-borne flavivirus responsible for several human encephalitis outbreaks over the last 80 years. Mature flavivirus virions are coated with dimeric envelope (E) proteins that mediate attachment and fusion with host cells. E is a class II fusion protein, the hallmark of which is a distinct dimer-to-trimer rearrangement that occurs upon endosomal acidification and insertion of hydrophobic fusion peptides into the endosomal membrane. Herein, we report the crystal structure of SLEV E in the posfusion trimer conformation. The structure revealed specific features that differentiate SLEV E from trimers of related flavi- and alphaviruses. SLEV E fusion loops have distinct intermediate spacing such that they are positioned further apart than previously observed in flaviviruses but closer together than Semliki Forest virus, an alphavirus. Domains II and III (DII and DIII) of SLEV E also adopt different angles relative to DI, which suggests that the DI-DII joint may accommodate spheroidal motions. However, trimer interfaces are well conserved among flaviviruses, so it is likely the differences observed represent structural features specific to SLEV function. Analysis of surface potentials revealed a basic platform underneath flavivirus fusion loops that may interact with the anionic lipid head groups found in membranes. Taken together, these results highlight variations in E structure and assembly that may direct virus-specific interactions with host determinants to influence pathogenesis

Development of a highly protective combination monoclonal antibody therapy against Chikungunya virus

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar−/−) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans

EMDataBank.org: unified data resource for CryoEM

Cryo-electron microscopy reconstruction methods are uniquely able to reveal structures of many important macromolecules and macromolecular complexes. EMDataBank.org, a joint effort of the Protein Data Bank in Europe (PDBe), the Research Collaboratory for Structural Bioinformatics (RCSB) and the National Center for Macromolecular Imaging (NCMI), is a global ‘one-stop shop’ resource for deposition and retrieval of cryoEM maps, models and associated metadata. The resource unifies public access to the two major archives containing EM-based structural data: EM Data Bank (EMDB) and Protein Data Bank (PDB), and facilitates use of EM structural data of macromolecules and macromolecular complexes by the wider scientific community

Semi-Automated Image Analysis for the Assessment of Megafaunal Densities at the Arctic Deep-Sea Observatory HAUSGARTEN

Megafauna play an important role in benthic ecosystem function and are sensitive indicators of environmental change. Non-invasive monitoring of benthic communities can be accomplished by seafloor imaging. However, manual quantification of megafauna in images is labor-intensive and therefore, this organism size class is often neglected in ecosystem studies. Automated image analysis has been proposed as a possible approach to such analysis, but the heterogeneity of megafaunal communities poses a non-trivial challenge for such automated techniques. Here, the potential of a generalized object detection architecture, referred to as iSIS (intelligent Screening of underwater Image Sequences), for the quantification of a heterogenous group of megafauna taxa is investigated. The iSIS system is tuned for a particular image sequence (i.e. a transect) using a small subset of the images, in which megafauna taxa positions were previously marked by an expert. To investigate the potential of iSIS and compare its results with those obtained from human experts, a group of eight different taxa from one camera transect of seafloor images taken at the Arctic deep-sea observatory HAUSGARTEN is used. The results show that inter- and intra-observer agreements of human experts exhibit considerable variation between the species, with a similar degree of variation apparent in the automatically derived results obtained by iSIS. Whilst some taxa (e. g. Bathycrinus stalks, Kolga hyalina, small white sea anemone) were well detected by iSIS (i. e. overall Sensitivity: 87%, overall Positive Predictive Value: 67%), some taxa such as the small sea cucumber Elpidia heckeri remain challenging, for both human observers and iSIS

Semi-Automated Image Analysis for the Assessment of Megafaunal Densities at the Arctic Deep-Sea Observatory HAUSGARTEN

Megafauna play an important role in benthic ecosystem function and are sensitive indicators of environmental change. Non-invasive monitoring of benthic communities can be accomplished by seafloor imaging. However, manual quantification of megafauna in images is labor-intensive and therefore, this organism size class is often neglected in ecosystem studies. Automated image analysis has been proposed as a possible approach to such analysis, but the heterogeneity of megafaunal communities poses a non-trivial challenge for such automated techniques. Here, the potential of a generalized object detection architecture, referred to as iSIS (intelligent Screening of underwater Image Sequences), for the quantification of a heterogenous group of megafauna taxa is investigated. The iSIS system is tuned for a particular image sequence (i.e. a transect) using a small subset of the images, in which megafauna taxa positions were previously marked by an expert. To investigate the potential of iSIS and compare its results with those obtained from human experts, a group of eight different taxa from one camera transect of seafloor images taken at the Arctic deep-sea observatory HAUSGARTEN is used. The results show that inter- and intra-observer agreements of human experts exhibit considerable variation between the species, with a similar degree of variation apparent in the automatically derived results obtained by iSIS. Whilst some taxa (e. g. Bathycrinus stalks, Kolga hyalina, small white sea anemone) were well detected by iSIS (i. e. overall Sensitivity: 87%, overall Positive Predictive Value: 67%), some taxa such as the small sea cucumber Elpidia heckeri remain challenging, for both human observers and iSIS

In Vitro and In Vivo Studies Identify Important Features of Dengue Virus pr-E Protein Interactions

Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV) pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors

A Dynamic Landscape for Antibody Binding Modulates Antibody-Mediated Neutralization of West Nile Virus

Neutralizing antibodies are a significant component of the host's protective response against flavivirus infection. Neutralization of flaviviruses occurs when individual virions are engaged by antibodies with a stoichiometry that exceeds a required threshold. From this “multiple-hit” perspective, the neutralizing activity of antibodies is governed by the affinity with which it binds its epitope and the number of times this determinant is displayed on the surface of the virion. In this study, we investigated time-dependent changes in the fate of West Nile virus (WNV) decorated with antibody in solution. Experiments with the well-characterized neutralizing monoclonal antibody (MAb) E16 revealed a significant increase in neutralization activity over time that could not be explained by the kinetics of antibody binding, virion aggregation, or the action of complement. Additional kinetic experiments using the fusion-loop specific MAb E53, which has limited neutralizing activity because it recognizes a relatively inaccessible epitope on mature virions, identified a role of virus “breathing” in regulating neutralization activity. Remarkably, MAb E53 neutralized mature WNV in a time- and temperature-dependent manner. This phenomenon was confirmed in studies with a large panel of MAbs specific for epitopes in each domain of the WNV envelope protein, with sera from recipients of a live attenuated WNV vaccine, and in experiments with dengue virus. Given enough time, significant inhibition of infection was observed even for antibodies with very limited, or no neutralizing activity in standard neutralization assays. Together, our data suggests that the structural dynamics of flaviviruses impacts antibody-mediated neutralization via exposure of otherwise inaccessible epitopes, allowing for antibodies to dock on the virion with a stoichiometry sufficient for neutralization