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