Human Coronavirus 229E: Receptor Binding Domain and Neutralization by Soluble Receptor at 37°C

Abstract

Truncated human coronavirus HCoV-229E spike glycoproteins containing amino acids 407 to 547 bound to purified, soluble virus receptor, human aminopeptidase N (hAPN). Soluble hAPN neutralized the infectivity of HCoV-229E virions at 37°C, but not 4°C. Binding of hAPN may therefore trigger conformational changes in the viral spike protein at 37°C that facilitate virus entry.Human coronaviruses HCoV-229E in serogroup I and HCoV-OC43 in serogroup II are, after rhinoviruses, the second most important cause of the common cold (7, 13). The first step in HCoV-229E infection is binding of the trimeric 200-kDa viral spike glycoprotein (S) to human aminopeptidase N (hAPN, CD13) on human cells (26). The three-dimensional structures of the spike glycoproteins of the plus-strand RNAcontaining coronaviruses are not yet known. However, the structure of S is predicted to be generally similar to that of type 1 viral fusion proteins of large negative-strand RNA viruses (12,22,25). The approximately 547-amino-acid (aa)-long Nterminal S1 domain of HCoV-229E spike protein has receptorbinding activity (2), and the membrane-anchored S2 domain contains several heptad repeats (3). APNs of the normal host species are also receptors for porcine, feline, and canine coronaviruses in group I (5, 6, 15, 23). The metalloprotease APN is a 150-kDa, class II glycoprotein that is expressed as a dimer on apical membranes of polarized epithelial cells, at synaptic junctions, and on antigen-presenting cells (11,17,19,21).Soluble receptors for many viruses have been used to identify critical receptor-binding sites on the viral attachment proteins, explore the specificity of virus-receptor interactions, and characterize conformational changes in the viral attachment protein that are induced by receptor binding (10,14,18,20,28). Temperature-dependent triggering of such conformational changes in a virus attachment protein by receptors on host cell membranes leads to penetration of the viral genome into the cytoplasm of the host cell. Binding of receptor to the N-terminal domain of a type 1 viral fusion protein at 37°C can trigger a conformational change in the membrane-anchored domain, leading to fusion of the viral envelope with host cell membranes (9,10,14,27). This paper reports the expression in insect cells and characterization of a soluble recombinant hAPN protein (shAPN) and interactions of shAPN with HCoV-229E virions, the S1 domain of the viral spike glycoprotein, and truncated S1 proteins. Characterization of shAPN.A soluble form of hAPN lacking the stalk, transmembrane, and intracellular domains (anchor/ stalk-minus APN), was previously expressed in mammalian cells and shown to be enzymatically active (24). For these studies, we expressed in insect cells the 95-to 98-kDa soluble, anchor/stalk-minus protein (shAPN). The open reading frame included the BiP cleavable signal sequence (MKLCILLAVVA FVGLSGL), a linker (RS), and aa 66 to 967 of hAPN with a C-terminal V5 epitope and His tag. We purified the protein on Ni-nitrilotriacetic acid (NTA) resin...

A New Application of Sequence Fourier Analysis to Specific Ligand-Protein Interactions

“…A similar result was obtained in the VP3/CD155 relationship, but not in VP2 or VP4 (data not shown). Unexpectedly, such a similar result between one of the receptors (i.e., CD155, CD81 or CD120b) and the spike glycoprotein of coronavirus 229E (1158aa) [or SARS (1239aa)] was obtained, while no relation of the latter one with CD13 (967aa) could be successfully elucidated 15) (data not shown).…”

A 193-Amino Acid Fragment of the SARS Coronavirus S Protein Efficiently Binds Angiotensin-converting Enzyme 2

“…Nonetheless, S1 and S2 domains of these latter S proteins can be identified through their homology with the S1 and S2 subunits of cleaved coronavirus S proteins. The S1 domain of all characterized coronaviruses, including that of SARS-CoV, mediates an initial high affinity interaction with a cellular receptor (11)(12)(13).…”

“…The first 330 amino acids of the 769-residue S1 subunit of the MHV S protein is sufficient to bind carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), the cellular receptor for MHV (13)(14)(15). A very different region of the S1 domain of HCoV-229E, between residues 407 and 547, is sufficient to associate with the cellular receptor for this coronavirus, aminopeptidase N (APN, CD13) (11,12,16). Here we show that a 193-amino acid fragment of the SARS-CoV S protein, residues 318 -510, binds the SARSCoV receptor ACE2 and blocks S protein-mediated infection more efficiently than does the full-length S1 domain.…”

Induction of IL-8 Release in Lung Cells via Activator Protein-1 by Recombinant Baculovirus Displaying Severe Acute Respiratory Syndrome-Coronavirus Spike Proteins: Identification of Two Functional Regions

“…The amino acid sequence homology between SARSCoV S protein and other CoV is low (20 -27% identity), except for some conserved regions in the S2 domain (3). The S1 domain of all characterized CoV, including SARS-CoV, mediates the initial high affinity interaction with the cellular receptor (7)(8)(9).…”