Neutral operator with variable parameter and third-order neutral differential equation

Additional file 1. The TRS sequences of structural genes in the HP-PRRSV/SD16 genome (GenBank: JX087437)

Structural Bases of Coronavirus Attachment to Host Aminopeptidase N and Its Inhibition by Neutralizing Antibodies

<div><p>The coronaviruses (CoVs) are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10–20% of all common colds. A subset of CoVs uses the cell surface aminopeptidase N (APN), a membrane-bound metalloprotease, as a cell entry receptor. In these viruses, the envelope spike glycoprotein (S) mediates the attachment of the virus particles to APN and subsequent cell entry, which can be blocked by neutralizing antibodies. Here we describe the crystal structures of the receptor-binding domains (RBDs) of two closely related CoV strains, transmissible gastroenteritis virus (TGEV) and porcine respiratory CoV (PRCV), in complex with their receptor, porcine APN (pAPN), or with a neutralizing antibody. The data provide detailed information on the architecture of the dimeric pAPN ectodomain and its interaction with the CoV S. We show that a protruding receptor-binding edge in the S determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the pAPN ectodomain. Comparison of the RBDs of TGEV and PRCV to those of other related CoVs, suggests that the conformation of the S receptor-binding region determines cell entry receptor specificity. Moreover, the receptor-binding edge is a major antigenic determinant in the TGEV envelope S that is targeted by neutralizing antibodies. Our results provide a compelling view on CoV cell entry and immune neutralization, and may aid the design of antivirals or CoV vaccines. APN is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs.</p> </div

Sequence alignment of homologous CoV RBD.

<p>Alignment was carried out with the T-Coffee program (<a href="http://www.ebi.ac.uk/" target="_blank">http://www.ebi.ac.uk/</a>). RBD sequences of TGEV, canine and feline CoVs (<i>Alphacoronavirus</i> 1), human CoVs (<i>Alphacoronavirus</i>), Bulbul-CoV (putative <i>Deltacoronavirus</i>) and IBV (<i>Gammacoronavirus</i>). Sequence of the TGEV RBD is numbered and its β-strands are shown with arrows. Residues in the two turns at the tip of the TGEV RBD β-barrel structure are indicated with a double T (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#ppat-1002859-g005" target="_blank">Figure 5</a>).</p

Crystal structure of the TGEV RBD in complex with the TGEV-neutralizing mAb 1AF10.

<p><b>A.</b> Ribbon diagram of the Fab fragment of the 1AF10 mAb bound to the TGEV RBD. Blue surface and ribbon representation is shown for the RBD domain, with β-strands and the terminal ends (n and c) where the remaining CoV S locates labeled. The antibody-binding regions at the tip are colored in red (β1–β2 region) and orange (β3–β4 and β5–β6). The 1AF10-Fab fragment is shown with heavy chain in grey and light chain in green. Variable (V_H and V_L) and constant (C_H and C_L) Ig domains of the Fab fragment are also indicated. <b>B.</b> Detailed view of the interaction, showing the RBD tip and the RBD-binding regions of the Fab variable domains. Buried regions are colored as in A for the RBD, and in dark green for the mAb. <b>C.</b> The RBD epitope for 1AF10. Surface and ribbon representation of the 1AF10 variable domains and stick drawing of buried residues at the interface, shown with carbons in orange or magenta for the RBD, and in green for the 1AF10 mAb. In this and following figures, nitrogens are in blue and oxygens in red, intermolecular hydrogen bonds are shown as dashed red lines, whereas intramolecular hydrogen bonds are black. <b>D.</b> Ligplot representation of 1AF10 mAb interaction with the RBD β1–β2 turn. RBD residues in the turn (magenta) and interacting mAb residues (green).</p

Structure of the RBD of TGEV and conformation of the receptor-binding edge in <i>Alphacoronavirus</i>.

<p><b>A</b>. Ribbon diagram of RBD protein structure with β-strands in light or dark blue, coils in orange, and helix in red. A β-bulge at β-strand 5 is shown in magenta. N- and C-terminal ends on the terminal side of the structure are indicated in lowercase letters. The Asn residues at glycosylation sites and the attached glycans defined in the structure are shown as a ball-and-stick model, with carbons in yellow. Cysteine residues and disulfide bonds are shown as green cylinders. Side chains of the pAPN-binding Tyr and Trp residues in the loops at the β-barrel domain tip are shown in red in panels A to C. <b>B</b>. Stereo view of superimposed <i>Alphacoronavirus</i> RBD structures. The pAPN-binding RBD of TGEV is in blue and the ACE2-binding RBD of HCoV-NL63 (PDB ID 3KBH) is in orange. <b>C</b>. Surface representation of the TGEV and HCoV-NL63 RBD structures in B, with receptor-binding residues in pink or red. <b>D</b>. Structure-based sequence alignment of <i>Alphacoronavirus</i> RBD structures shown in C. β-strands are marked (bars) above or beneath their sequences. TGEV sequence is numbered. ACE2 receptor-binding residues reported for HCoV-NL63 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#ppat.1002859-Wu1" target="_blank">[11]</a>, as well as pAPN receptor-binding residues for TGEV (Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#ppat.1002859.s006" target="_blank">Table S2</a>) are colored as in C. Residues absent in the RBD structures are in grey, and the thrombin recognition sequence at the end of the TGEV RBD is in lowercase letters.</p

Determinants of TGEV S antigenic site A.

<p><b>A.</b> Binding of TGEV-neutralizing, site A-specific mAbs to RBD mutants. Relative binding (%) of mutants to wild type SA protein is shown for TGEV S-specific mAbs (top; described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#ppat-1002859-g001" target="_blank">Figure 1C</a>) and a control anti-HA antibody (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#s4" target="_blank">Materials and Methods</a>). RBD regions in which mutations locate are shown (bottom; see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#ppat-1002859-g005" target="_blank">Figure 5D</a>). Mean and standard deviation of data from at least three experiments. <b>B.</b> Antigenic site A in the TGEV RBD and epitopes for antibodies. Surface and ribbon representation of the RBD with the 1AF10 contact regions colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#ppat-1002859-g002" target="_blank">Figure 2B</a>. Three antibody-binding residues (Tyr528, Trp571 and Asn632) in the loops at the RBD tip, as well as TGEV Lys524, Arg577 and Gly529 residues associated with Aa, Ab and Ac subsites <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#ppat.1002859-Gebauer1" target="_blank">[24]</a>, respectively. Lines indicate epitopes for mAbs specific for each of the three antigenic subsites: Aa in yellow, Ab in green and Ac in red.</p

Data collection and refinement statistics.

<p>The crystal structures contain two independent molecules in the asymmetric unit. Ligands included in the structures are two acetate molecules in the TGEV RBD-1AF10 structure and zinc ions in the pAPN structure, one in each molecule of the asymmetric unit. Statistics for the highest-resolution shell are in parentheses.</p

APN-binding domain and epitopes for neutralizing mAbs overlap in TGEV and PRCV S proteins.

<p><b>A</b>. Scheme of TGEV and PRCV S proteins showing the S1, S2, transmembrane (T) and cytoplasmic (Cy) regions. Location of the C, B, D and A antigenic sites <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#ppat.1002859-Sanchez1" target="_blank">[21]</a>, and the pAPN RBD (bar with N and C-terminal residues) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#ppat.1002859-Godet1" target="_blank">[22]</a> are shown. Length is indicated for mature S1 regions. <b>B.</b> A short, soluble S protein variant containing the TGEV RBD region binds to cell surface pAPN. Binding of a bivalent SA-Fc fusion protein (SA) to BHK-pAPN (open histograms), alone (left) or in the presence of the site A-specific 6AC3 mAb (right), as analyzed in FACS. Filled histograms correspond to an unrelated Fc fusion protein. <b>C.</b> Binding of site A-specific TGEV-neutralizing mAb to the SA protein. mAb binding to plastic-bound SA protein, monitored by optical density (OD). Site A mAbs are specific for the Aa (1BB1), Ab (1DE7) and Ac (6AC3, 1AF10) subsites <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#ppat.1002859-Gebauer1" target="_blank">[24]</a>. An anti-HA mAb that binds to the HA tag in the SA protein was used as control. <b>D.</b> Site A-specific mAbs prevent SA protein binding to pAPN. Binding of the SA protein to BHK-pAPN cells in panel B was monitored alone or in the presence of site A (shown in C) or site D-specific (1DG3) mAb, and the binding ratio determined (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#s4" target="_blank">Materials and Methods</a>). <b>C, D</b>. Mean and standard deviation for three experiments.</p

Crystal structure of the PRCV RBD bound to the pAPN ectodomain.

<p><b>A.</b> The dimeric PRCV RBD-pAPN complex in the crystals. Ribbon drawings of the pAPN molecules are shown with domains (D) in orange (N-terminal DI), yellow (DII), red (DIII) and green (C-terminal DIV), and the N-terminal end (n) near the cell membrane. The RBD is shown as ribbon and surface drawings in blue and cyan, with the pAPN-binding Tyr and Trp residues at the RBD tip in red. Location of the remaining CoV S is indicated at the RBD terminal end. Glycans are shown as sticks with carbons in yellow and the zinc ion at the pAPN catalytic site as a cyan sphere. <b>B.</b> Detail of the RBD β1–β2 region with the exposed Tyr residue interacting with the pAPN. Side chains of RBD and pAPN residues engaged in the interaction are shown as sticks with carbons in magenta or green, respectively. NAG7361 glycan N-linked to pAPN Asn736 is shown with carbons in yellow and the electron density map, determined without the glycan, shown as a blue mesh contoured at 3 sigma. <b>C.</b> Detail of the RBD β3–β4 region with the Trp residue interacting with the pAPN. In <b>B</b> and <b>C</b>, RBD residues are numbered following the TGEV sequence shown in <b>D</b>, and intermolecular hydrogen bonds are shown as dashed red lines. <b>D</b>. Structure-based sequence alignment of the TGEV and PRCV RBDs. β-strands are marked with bars. TGEV sequence is numbered. In red, 1AF10 mAb- (for TGEV) and pAPN receptor-binding residues (for PRCV) identified by the structures. Residues absent in the RBD structures are in grey, and the thrombin recognition sequences at the end of recombinant porcine CoV RBDs are in lowercase letters.</p

Important virus-receptor binding motifs.

<p><b>A.</b> Binding of TGEV RBD mutants to cell surface pAPN. Relative binding (%) was determined for mutant to wild type SA proteins (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#s4" target="_blank">Materials and Methods</a>). Substituted residues at the RBD tip that contact pAPN in the PRCV RBD-pAPN structure are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#ppat-1002859-g003" target="_blank">Figure 3</a>, except for the V617Ngly mutant, with a glycan at RBD position 617 in the β5-β5b loop, outside the RBD tip (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002859#ppat-1002859-g003" target="_blank">Figure 3D</a>). <b>B.</b> TGEV RBD binding to cell surface pAPN glycosylation mutants. Relative binding of the SA protein and the anti-HA mAb to HA-tagged pAPN proteins with (pAPN) or without the glycan linked to Asn736 (N736A and T738V). Mean and standard deviation for three experiments.</p