Crystallographic data and refinement statistics.

<p>Crystallographic data and refinement statistics.</p

Electron density maps for key regions of the C-terminal extension.

<p>(A) Omit map electron density for C-terminal residues 611–615, which insert into the NA domain active site. (B) Omit map electron density for residues surrounding the interchain disulfide bond at C596. Residues associated with different chains of the observed NA domain dimers are indicated with an A: or B: prefix. (C) Omit map electron density C-terminal extension residues 583–588, which engage a second sialic acid binding site. The omit maps was calculated using the SFCHECK <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002855#ppat.1002855-Vaguine1" target="_blank">[46]</a> program and contoured at 1.57 sigma.</p

The inhibitory C-terminal extension forms a well-defined structure in crystals of the Ulster NA domain.

<p>(A) Crystal structure of the Ulster HN NA domain dimer, shows a dimer-of-dimers tetramer. The C-terminal extension is highlighted in dark blue and red in each pair of dimers. (B) The Ulster HN dimer is shown, with the C-terminal extension in dark blue. The C-terminal extension begins at the base of the β-propeller domain, extends along the outside of the dimer interface, and then rises above the active site before inserting the C-terminus into the receptor-binding site.</p

The C-terminal extension inhibits the NA domain active site.

<p>(A) Top view of a single HN NA domain subunit, showing superpositions with the NDV Kansas low-pH and DANA-bound crystal structures. The additional residues of the C-terminal extension are highlighted in blue and overlap the ligand bound in the active site. The D198 loop is indicated, which changes conformation in the Ulster HN structure, contributing to blocking the active site. (B,C) Detailed comparison of contacts made by the Ulster HN C-terminus and DANA sialic acid within the active site. Dotted lines represent hydrogen bonds and polar contacts identified by Pymol.</p

C596 in the HN C-terminal extension mediates NA domain dimerization.

<p>(A) HN Ulster WT NA domain and HN C596S NA domain were expressed and purified as described in Methods and analyzed by SDS-PAGE under reducing and non-reducing conditions. The gel was stained with Coomassie Brilliant Blue. (B) Sucrose density gradient sedimentation and SDS-PAGE (non-reducing conditions) of Ulster HN NA domain and HN C596S NA domain proteins. (C) EM of WT Ulster HN NA domain, indicates the protein consists primarily of dimers, and the HN C596S NA domain, consists mostly as monomers.</p

Recombinant NDV Ulster F and HN are expressed as F₀ and HN₀ precursors.

<p>(A) Sequence alignment of C-termini of NDV HN strains, La Sota (La), Ulster and Australia-Victoria (AV). Core NA domain residues are colored red, partially conserved C-terminal residues are colored blue. The cysteine at residue 596 is indicated by an asterisk. (B) NDV Ulster F and HN expressed from cDNAs in HeLa cells were metabolically labeled, immunoprecipitated and polypeptides analyzed by SDS-PAGE. Addition of trypsin to the transfected cells led to processing of the precursor proteins (F₀ and HN₀) as shown by the cleavage of F₀ to F₁ and F₂ and the faster mobility of HN as compared to HN₀. (C) Hemadsorption assay on HeLa cells expressing Ulster HN. Cells were overlaid with chicken red blood cells and subsequently washed with PBS⁺ and incubated at. 4°C. pCAGGS is vector control. Ulster HN expressed in HeLa cells binds sialic acid on chicken red blood cells very poorly. Treatment of HN₀ with trypsin causes a large increase in red blood cell binding.</p

Hemadsorption of WT and C596S HN proteins.

<p>Hemadsorption was assayed by measuring the hemoglobin absorbance at 540 nM.</p><p>The values shown are the average of three experiments performed in duplicate.</p

Neuraminidase activity of the Ulster WT and C596S proteins.

<p>The neuraminidase activity of the Ulster WT and C596S proteins was assayed by measuring the fluorescence, at an emission wavelength of 440 nm, produced upon cleavage of the fluorogenic substrate 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid. The fluorescence shown was produced by equivalent amounts of protein.</p

A Chimeric Pneumovirus Fusion Protein Carrying Neutralizing Epitopes of Both MPV and RSV

<div><p>Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are paramyxoviruses that are responsible for substantial human health burden, particularly in children and the elderly. The fusion (F) glycoproteins are major targets of the neutralizing antibody response and studies have mapped dominant antigenic sites in F. Here we grafted a major neutralizing site of RSV F, recognized by the prophylactic monoclonal antibody palivizumab, onto HMPV F, generating a chimeric protein displaying epitopes of both viruses. We demonstrate that the resulting chimeric protein (RPM-1) is recognized by both anti-RSV and anti-HMPV F neutralizing antibodies indicating that it can be used to map the epitope specificity of antibodies raised against both viruses. Mice immunized with the RPM-1 chimeric antigen generate robust neutralizing antibody responses to MPV but weak or no cross-reactive recognition of RSV F, suggesting that grafting of the single palivizumab epitope stimulates a comparatively limited antibody response. The RPM-1 protein provides a new tool for characterizing the immune responses resulting from RSV and HMPV infections and provides insights into the requirements for developing a chimeric subunit vaccine that could induce robust and balanced immunity to both virus infections.</p></div

Heat treatment of wild-type and mutant RSV F does not affect binding of palivizumab and DS-7 neutralizing antibodies.

<p>(a, b) Binding of the anti-HMPV F DS-7 antibody to wild-type (a) and RPM-1 (b) proteins after heating to 50°C for 30 minutes. Both proteins bound with similar affinity to the antibody as the unheated F (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155917#pone.0155917.g003" target="_blank">Fig 3</a>), demonstrating that the DS-7 epitope is not affected by conversion to the post-fusion conformation. (c, d) Binding of palivizumab to heat-treated wild-type (c) and RPM-1 (d) proteins. The wild-type HMPV F protein and RPM-1 mutant showed similar binding interactions with palivizumab as with the unheated F samples, indicating that concversion of pre- to post-fusion conformation had little effect on palivizumab antibody binding.</p