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

Ancient DNA Resolves Identity and Phylogeny of New Zealand's Extinct and Living Quail (Coturnix sp.)

BACKGROUND: The New Zealand quail, Coturnix novaezealandiae, was widespread throughout New Zealand until its rapid extinction in the 1870's. To date, confusion continues to exist concerning the identity of C. novaezealandiae and its phylogenetic relationship to Coturnix species in neighbouring Australia, two of which, C. ypsilophora and C. pectoralis, were introduced into New Zealand as game birds. The Australian brown quail, C. ypsilophora, was the only species thought to establish with current populations distributed mainly in the northern part of the North Island of New Zealand. Owing to the similarities between C. ypsilophora, C. pectoralis, and C. novaezealandiae, uncertainty has arisen over whether the New Zealand quail is indeed extinct, with suggestions that remnant populations of C. novaezealandiae may have survived on offshore islands. METHODOLOGY/PRINCIPAL FINDINGS: Using fresh and historical samples of Coturnix sp. from New Zealand and Australia, DNA analysis of selected mitochondrial regions was carried out to determine phylogenetic relationships and species status. Results show that Coturnix sp. specimens from the New Zealand mainland and offshore island Tiritiri Matangi are not the New Zealand quail but are genetically identical to C. ypsilophora from Australia and can be classified as the same species. Furthermore, cytochrome b and COI barcoding analysis of the New Zealand quail and Australia's C. pectoralis, often confused in museum collections, show that they are indeed separate species that diverged approximately 5 million years ago (mya). Gross morphological analysis of these birds suggests a parallel loss of sustained flight with very little change in other phenotypic characters such as plumage or skeletal structure. CONCLUSION/SIGNIFICANCE: Ancient DNA has proved invaluable for the detailed analysis and identification of extinct and morphologically cryptic taxa such as that of quail and can provide insights into the timing of evolutionary changes that influence morphology

Introduced Mammalian Predators Induce Behavioural Changes in Parental Care in an Endemic New Zealand Bird

The introduction of predatory mammals to oceanic islands has led to the extinction of many endemic birds. Although introduced predators should favour changes that reduce predation risk in surviving bird species, the ability of island birds to respond to such novel changes remains unstudied. We tested whether novel predation risk imposed by introduced mammalian predators has altered the parental behaviour of the endemic New Zealand bellbird (Anthornis melanura). We examined parental behaviour of bellbirds at three woodland sites in New Zealand that differed in predation risk: 1) a mainland site with exotic predators present (high predation risk), 2) a mainland site with exotic predators experimentally removed (low risk recently) and, 3) an off-shore island where exotic predators were never introduced (low risk always). We also compared parental behaviour of bellbirds with two closely related Tasmanian honeyeaters (Phylidonyris spp.) that evolved with native nest predators (high risk always). Increased nest predation risk has been postulated to favour reduced parental activity, and we tested whether island bellbirds responded to variation in predation risk. We found that females spent more time on the nest per incubating bout with increased risk of predation, a strategy that minimised activity at the nest during incubation. Parental activity during the nestling period, measured as number of feeding visits/hr, also decreased with increasing nest predation risk across sites, and was lowest among the honeyeaters in Tasmania that evolved with native predators. These results demonstrate that some island birds are able to respond to increased risk of predation by novel predators in ways that appear adaptive. We suggest that conservation efforts may be more effective if they take advantage of the ability of island birds to respond to novel predators, especially when the elimination of exotic predators is not possible

In Situ Characterization of Binary Mixed Polymer Brush-Grafted Silica Nanoparticles in Aqueous and Organic Solvents by Cryo-Electron Tomography

We present an in situ cryo-electron microscopy (cryoEM) study of mixed poly­(acrylic acid) (PAA)/polystyrene (PS) brush-grafted 67 nm silica nanoparticles in organic and aqueous solvents. These organic–inorganic nanoparticles are predicted to be environmentally responsive and adopt distinct brush layer morphologies in different solvent environments. Although the self-assembled morphology of mixed PAA/PS brush-grafted particles has been studied previously in a dried state, no direct visualization of microphase separation was achieved in the solvent environment. CryoEM allows the sample to be imaged in situ, that is, in a frozen solvated state, at the resolution of a transmission electron microscope. Cryo-electron tomograms (cryoET) were generated for mixed PAA/PS brush-grafted nanoparticles in both <i>N,N</i>-dimethylformamide (DMF, a nonselective good solvent) and water (a selective solvent for PAA). Different nanostructures for the mixed brushes were observed in these two solvents. Overall, the brush layer is more compact in water, with a thickness of 18 nm, as compared with an extended layer of 27 nm in DMF. In DMF, mixed PAA/PS brushes are observed to form laterally separated microdomains with a ripple wavelength of 13.8 nm. Because of its lower grafting density than that of PAA, PS domains form more or less cylindrical or truncated cone-shaped domains in the PAA matrix. In water, PAA chains are found to form a more complete shell around the nanoparticle to maximize their interaction with water, whereas PS chains collapse into the core of surface-tethered micelles near the silica core. The cryoET results presented here confirm the predicted environmentally responsive nature of PAA/PS mixed brush-grafted nanoparticles. This experimental approach may be useful for the design of future mixed brush-grafted nanoparticles for nano- and biotechnology applications

Class averages of radial averaged particle images of the WNV/E16 Fab complex at pH 6.0.

<p>The classification suggests the presence of at least four different radial expansion intermediates that represent advancing stages of the E protein layer expansion (IM-1 through 4). The number of images grouped in each class is indicated in percent below the respective class average. Most of the particles clustered in expansion stage IM-4, in which the outer protein layer, presumably composed of E and Fab molecules, reaches its maximum radius separated by an about 60 Å-wide gap from the outer lipid leaflet. A representative class average of the complex at pH 8 is shown on the left. The red lines indicate the radial limits of the nucleocapsid core (NC), the lipid bilayer (LB), the E protein layer (E) and the Fab molecules (Fab) in the pH 8 structure. Areas of high density are depicted with black pixels, low density areas are shown in white.</p

CryoEM image reconstruction of WNV in complex with E16 antibody fragments at pH 8.

<p>(A) Stereoscopic view of a surface rendering of WNV (green) complexed with E16 Fab (blue) at 23 Å resolution <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000672#ppat.1000672-Kaufmann1" target="_blank">[14]</a>, viewed down an icosahedral twofold axis. The black triangular outline identifies an icosahedral asymmetric unit. (B) Same as A, but depicting the complex of WNV and E16 scFv. E16 scFv binds WNV in a similar fashion as E16 Fab. Only two of three epitopes in the asymmetric unit are utilized. The fivefold proximal epitope is occluded because of steric hindrance.</p

Equatorial slices of cryoEM image reconstructions of WNV in complex with E16 antibody fragments in different pH environments.

<p>(A) WNV/Fab complex (top half) and WNV/scFv complex (bottom half) at pH 8, rendered at 23 Å resolution. Red arcs (1 through 5) specify the outer radii of the nucleocapsid core (154 Å), lipid bilayer (205 Å), E glycoprotein shell (247.5 Å), scFv molecules (278 Å), and Fab fragments (318.5 Å) from the viral center. The positions of the icosahedral two-, three- and fivefold axes are indicated with black arrows and numbers. (B) WNV/Fab complex (top half) and WNV/scFv complex (bottom half) at pH 6, rendered to 25 Å resolution. The red arcs (6 and 7) specify the outer radius of the expanded E/scFv (317.5 Å) and E/Fab (347 Å) protein layer, respectively. The low pH triggered radial expansion of the E/scFv or E/Fab protein shell resulted in a ∼60 Å wide shell of low density between the lipid bilayer and the expanded outer protein layer, as indicated by the green arrow. The scale bars represent 100 Å.</p