Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits) which is solely responsible for host cell attachment, endosomal entry and membrane fusion1–7. GP is thus a primary target for the development of antiviral drugs. Here we report the first unliganded structure of EBOV GP, and complexes with an anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered8–10. Unexpectedly both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the 3-fold axis. Protein-drug interactions, with both GP1 and GP2, are predominately hydrophobic. Residues lining the binding site are highly conserved amongst filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in protein melting temperature upon toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. The results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, therefore preventing fusion between the viral and endosome membranes. Thus these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.

SummaryHantaviruses, a geographically diverse group of zoonotic pathogens, initiate cell infection through the concerted action of Gn and Gc viral surface glycoproteins. Here, we describe the high-resolution crystal structure of the antigenic ectodomain of Gn from Puumala hantavirus (PUUV), a causative agent of hemorrhagic fever with renal syndrome. Fitting of PUUV Gn into an electron cryomicroscopy reconstruction of intact Gn-Gc spike complexes from the closely related but non-pathogenic Tula hantavirus localized Gn tetramers to the membrane-distal surface of the virion. The accuracy of the fitting was corroborated by epitope mapping and genetic analysis of available PUUV sequences. Interestingly, Gn exhibits greater non-synonymous sequence diversity than the less accessible Gc, supporting a role of the host humoral immune response in exerting selective pressure on the virus surface. The fold of PUUV Gn is likely to be widely conserved across hantaviruses.

HmuUV is a bacterial ATP-binding cassette (ABC) transporter that catalyzes heme uptake into the cytoplasm of the gram-negative pathogen Yersinia pestis. We report the crystal structure of HmuUV at 3.0 Å resolution in a nucleotide-free state, which features a heme translocation pathway in an outward-facing conformation, poised to accept a heme from the cognate periplasmic binding protein HmuT. A new assay allowed us to determine in vitro rates of HmuUV-catalyzed heme transport into proteoliposomes and to establish the role of conserved residues in the translocation pathway of HmuUV and at the interface with HmuT. Differences in architecture relative to the related vitamin B(12) transporter BtuCD suggest an adaptation of HmuUV for its smaller substrate. Our study also suggests that type II ABC importers, which include bacterial iron-siderophore, heme and cobalamin transporters, have a coupling mechanism distinct from that of other ABC transporters.

The discovery of African henipaviruses (HNVs) related to pathogenic Hendra virus (HeV) and Nipah virus (NiV) from Southeast Asia and Australia presents an open-ended health risk. Cell receptor use by emerging African HNVs at the stage of host-cell entry is a key parameter when considering the potential for spillover and infection of human populations. The attachment glycoprotein from a Ghanaian bat isolate (GhV-G) exhibits <30% sequence identity with Asiatic NiV-G/HeV-G. Here, through functional and structural analysis of GhV-G, we show how this African HNV targets the same human cell-surface receptor (ephrinB2) as the Asiatic HNVs. We first characterized this virus−receptor interaction crystallographically. Compared with extant HNV-G-ephrinB2 structures, there was significant structural variation in the six-bladed β-propeller scaffold of the GhV-G receptor-binding domain, but not the Greek key fold of the bound ephrinB2. Analysis revealed a surprisingly conserved mode of ephrinB2 interaction that reflects an ongoing evolutionary constraint among geographically distal and phylogenetically divergent HNVs to maintain the functionality of ephrinB2 recognition during virus-host entry. Interestingly, unlike NiV-G/HeV-G, we could not detect binding of GhV-G to ephrinB3. Comparative structurefunction analysis further revealed several distinguishing features of HNV-G function: a secondary ephrinB2 interaction site that contributes to more efficient ephrinB2-mediated entry in NiV-G relative to GhV-G and cognate residues at the very C terminus of GhV-G (absent in Asiatic HNV-Gs) that are vital for efficient receptorinduced fusion, but not receptor binding per se. These data provide molecular-level details for evaluating the likelihood of African HNVs to spill over into human populations.emerging virus | viral attachment | glycoprotein | henipavirus | structure

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Hantaviruses are zoonotic pathogens that cause severe hemorrhagic fever and pulmonary syndrome. The outer membrane of the hantavirus envelope displays a lattice of two glycoproteins, Gn and Gc, which orchestrate host cell recognition and entry. Here, we describe the crystal structure of the Gn glycoprotein ectodomain from the Asiatic Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Structural overlay analysis reveals that the HTNV Gn fold is highly similar to the Gn of Puumala virus (PUUV), a genetically and geographically distinct and less pathogenic hantavirus found predominantly in northeastern Europe, confirming that the hantaviral Gn fold is architecturally conserved across hantavirus clades. Interestingly, HTNV Gn crystallized at acidic pH, in a compact tetrameric configuration distinct from the organization at neutral pH. Analysis of the Gn, both in solution and in the context of the virion, confirms the pH-sensitive oligomeric nature of the glycoprotein, indicating that the hantaviral Gn undergoes structural transitions during host cell entry. These data allow us to present a structural model for how acidification during endocytic uptake of the virus triggers the dissociation of the metastable Gn-Gc lattice to enable insertion of the Gc-resident hydrophobic fusion loops into the host cell membrane. Together, these data reveal the dynamic plasticity of the structurally conserved hantaviral surface.IMPORTANCE Although outbreaks of Korean hemorrhagic fever were first recognized during the Korean War (1950 to 1953), it was not until 1978 that they were found to be caused by Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Here, we describe the crystal structure of HTNV envelope glycoprotein Gn, an integral component of the Gn-Gc glycoprotein spike complex responsible for host cell entry. HTNV Gn is structurally conserved with the Gn of a genetically and geographically distal hantavirus, Puumala virus, indicating that the observed α/β fold is well preserved across the Hantaviridae family. The combination of our crystal structure with solution state analysis of recombinant protein and electron cryo-microscopy of acidified hantavirus allows us to propose a model for endosome-induced reorganization of the hantaviral glycoprotein lattice. This provides a molecular-level rationale for the exposure of the hydrophobic fusion loops on the Gc, a process required for fusion of viral and cellular membranes.

Transmission of hemorrhagic fever New World arenaviruses from their rodent reservoirs to human populations poses substantial public health and economic dangers. These zoonotic events are enabled by the specific interaction between the New World arenaviral attachment glycoprotein, GP1, and cell surface human transferrin receptor (hTfR1). Here, we present the structural basis for how a mouse-derived neutralizing antibody (nAb), OD01, disrupts this interaction by targeting the receptor-binding surface of the GP1 glycoprotein from Junín virus (JUNV), a hemorrhagic fever arenavirus endemic in central Argentina. Comparison of our structure with that of a previously reported nAb complex (JUNV GP1-GD01) reveals largely overlapping epitopes but highly distinct antibody-binding modes. Despite differences in GP1 recognition, we find that both antibodies present a key tyrosine residue, albeit on different chains, that inserts into a central pocket on JUNV GP1 and effectively mimics the contacts made by the host TfR1. These data provide a molecular-level description of how antibodies derived from different germline origins arrive at equivalent immunological solutions to virus neutralization. . These viruses are endemic to rodent populations in rural areas of Argentina, Bolivia, and Venezuela, respectively, and viral spillover into human populations can result in severe hemorrhagic fever (HF) (3). Novel pathogenic NW arenaviruses continue to be identified (4-6), underscoring a wide-scale need for effective vaccines and therapeutics.JUNV, the etiological agent of Argentine hemorrhagic fever (AHF), constitutes one of the most dangerous NW arenaviruses, putting an estimated 5 million people at risk (7,8). JUNV infection typically exhibits a rapid onset of disease (7-14 d) and high mortality rates (15-30%) (7, 9, 10). There are no internationally approved drugs for preventing or treating NW arenavirus HF. However, the successful development of a live, attenuated JUNV vaccine, Candid#1, has proven that AHF can be controlled (11). Similarly, virus-neutralizing immune plasma from convalescent individuals has been successfully used for the treatment of AHF (10, 12).The JUNV envelope surface is decorated by trimeric multifunctional glycoprotein complex (GPC) spikes. Each protomer in the trimer consists of: a myristoylated (13) stable signal peptide, an attachment subunit (GP1), and a transmembrane fusion subunit (GP2) (14-19). Host-cell entry of JUNV and other clade B arenaviruses is initiated by the interaction between the arenaviral GP1 and the host transferrin receptor (TfR1) (20). The GP1 subunit interacts with the apical domain of TfR1, distal from natural transferrin and hereditary hemochromatosis protein recognition sites (21, 22). A primary determinant of zoonotic spread of clade B arenaviruses is the ability of the GP1 to recognize the human TfR1 ortholog (20, 23).The JUNV GPC spike comprises the primary target for neutralizing immune responses (24) and three GPC-specific mousederived nAbs-GD01, OD01, and GB03-have shown prom...

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