Virus particles formed in infected cells transmit the viral genome and the infection from cell to cell. Viruses must therefore enter into host cells to initiate the replicative infective cycle. In their extracellular transit stage, via distinct proteins or motifs, virus particles attach to specific cell surface molecules (receptors) to penetrate into cells. Receptor-binding virus proteins are targets for immune responses that would prevent virus entry and infection. The study of virus-receptor interactions and virus neutralisation is important for the development of therapies preventing virus infections.

The coronaviruses are a large family of enveloped viruses mostly responsible of enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10-20% of all common colds [1]. Coronavirus infections cause important economic losses, and high mortality rates were witnessed in recent outbreaks. Coronavirus particles display large surface projections (17–20 nm in length) comprised of homotrimers of the spike glycoproteins (S), which generate a characteristic crown-like appearance (Figure 15). These spike glycoproteins mediate particle attachment to cell surface receptors and entry into host cells. In addition, they contain antigenic sites, recognised by antibodies neutralising coronaviruses. The cell surface aminopeptidase N (APN), a target for cancer therapy, is a major cell entry receptor for coronaviruses. A subset of coronaviruses, the alphacoronavirus, attach to the APN to penetrate into host cells. A fragment of the S protein, the receptor binding domain (RBD), bears the APN recognition motifs. These motifs overlap with antigenic sites recognised by neutralising antibodies that prevent coronaviruses binding to APN. We employed X-ray crystallography with the aim to identify how alphacoronaviruses attach to the APN and how neutralising antibodies prevent coronaviruses binding to the receptor and infection. We determined the crystal structures of the RBD of porcine coronavirus in complex with either its APN receptor or with a neutralising antibody (nAb) (Figure 15). Diffraction data extending to about 3Å resolution for structure determination was collected at beamline ID29.

Fig. 15: Structural view of coronavirus (CoV) binding to its host cell aminopeptidase N (APN) receptor and its inhibition by neutralising antibodies. Ribbon representations of the crystal structures of a porcine coronavirus spike (CoV S) receptor-binding domain (RBD), shown in blue, in complex with the cell surface dimeric pig APN ectodomain, or with a CoV neutralising antibody (nAb, green). APN domains are orange (DI), yellow (DII), red (DIII) and green (DIV). The N-terminus of the APN connects to the membrane. Two coronavirus residues (Tyr and Trp) exposed to the APN and nAb contact tip of the RBD are shown as red spheres. The coronavirus RBD was fitted into the cryo-EM (EMD-1423) structure of a trimeric CoV S fragment [2], shown as a grey surface. An EM-micrograph of CoV particles with their characteristic projections formed by homotrimers of the S glycoprotein that binds to the APN for cell entry is also shown.

The APN is a type II membrane protein and thus, the N-terminus of its ectodomain is located near the cell membrane (Figure 15). The APN ectodomain structure revealed this to be composed of four domains (DI to DIV). APN forms dimers on the cell surface and in the crystals. The dimerisation surface comprises about 1000 Å2 of DIV (green in Figure 15). In the virus-receptor complex, each RBD molecule (blue in Figure 15) contacts the membrane distal region of a single APN molecule. The protruding tip of the RBD, which bears two exposed aromatic residues (Tyr and Trp) in loops (red in Figure 15), docks into recessed APN surfaces. The Tyr residue fits between an a helix and a glycan, whereas the Trp sits in a cavity formed by DII and DIV of APN (not shown). The receptor binding tip of the RBD is also targeted by the crystallised nAb (Figure 15), which mostly binds to the Tyr residue. Other coronavirus neutralising antibodies recognise also the APN-binding tip of the RBD, showing that this region is a major antigenic site in the envelope S. These results indicate that antibodies neutralise coronavirus infections by blocking virus biding to its APN receptor. Efficient coronavirus vaccines must elicit immune responses focused toward the identified receptor binding motifs in the envelope S protein. These structural results provide a compelling view of coronavirus cell entry and immune neutralisation, and may aid the design of antivirals or coronavirus vaccines. APN is also a target for cancer therapy and its structure could facilitate the development of anti-cancer drugs.

 

Principal publication and authors

J. Reguera (a,b), C. Santiago (a), G. Mudgal (a), D. Ordoño (a), L. Enjuanes (a) and J.M. Casasnovas (a), PLoS Pathogens 8, e1002859 (2012).

(a) Centro Nacional de Biotecnología, CNB-CSIC, Madrid (Spain)

(b) Present address: European Molecular Biology Laboratory, Grenoble Outstation (France)

 

References

[1] P.S. Masters, Adv. Virus Res. 66, 193-292 (2006).

[2] D.R. Beniac, A. Andonov, E. Grudeski and T.F. Booth, Nat. Struct. Mol. Biol. 13, 751-752 (2006).