key: cord-1013064-5s405fdn
authors: Yeager, Curtis L.; Ashmun, Richard A.; Williams, Richard K.; Cardellichio, Christine B.; Shapiro, Linda H.; Look, A. Thomas; Holmes, Kathryn V.
title: Human aminopeptidase N is a receptor for human coronavirus 229E
date: 1992
journal: Nature
DOI: 10.1038/357420a0
sha: 6fcf7a9b95a53e1ab89b02f3bf6336457c18dca7
doc_id: 1013064
cord_uid: 5s405fdn

HUMAN coronaviruses (HCV) in two serogroups represented by HCV-229E and HCV-OC43 are an important cause of upper respiratory tract infections(1). Here we report that human aminopeptidase N, a cell-surface metalloprotease on intestinal, lung and kidney epithelial cells(2–5), is a receptor for human coronavirus strain HCV-229E, but not for HCV-OC43. A monoclonal antibody, RBS, blocked HCV-229E virus infection of human lung fibroblasts, immunoprecipitated aminopeptidase N and inhibited its enzymatic activity. HCV-229E-resistant murine fibroblasts became susceptible after transfection with complementary DNA encoding human aminopeptidase N. By contrast, infection of human cells with HCV-OC43 was not inhibited by antibody RBS and expression of aminopeptidase N did not enhance HCV-OC43 replication in mouse cells. A mutant aminopeptidase lacking the catalytic site of the enzyme did not bind HCV-229E or RBS and did not render murine cells susceptible to HCV-229E infection, suggesting that the virus-binding site may lie at or near the active site of the human aminopeptidase molecule.

HUMAN coronaviruses (HCV) in two serogroups represented by HCV-229E and HCV-OC43 are an important cause of upper respiratory tract infections i • Here we report that human aminopeptidase N, a cell-surface metalloprotease on intestinal, lung and kidney epithelial cells~5, is a receptor for human coronavirus strain HCV-229E, but not for HCV-OC43. A monoclonal antibody, RBS, blocked HCV-229E virus infection of human lung fibroblasts, immunoprecipitated aminopeptidase N and inhibited its enzymatic activity. HCV-229E-resistant murine fibroblasts became susceptible after transfection with complementary DNA encoding human aminopeptidase N. By contrast, infection of human cells with HCV-OC43 was not inhibited by antibody RBS and expression of aminopeptidase N did not enhance HCV-OC43 replication in mouse cells. A mutant aminopeptidase lacking the catalytic site of the enzyme did not bind HCV-229E or RBS and did not render murine cells susceptible to HCV-229E infection, suggesting that the virus-binding site may lie at or near the active site of the human aminopeptidase molecule.

To develop a monoclonal antibody against the HCV-229E receptor, we produced hybridomas against deoxycholatesolubilized membrane proteins of two HCV-229E-susceptible human cell lines (WI38 lung fibroblasts and HL60 myeloid leukaemia cells). A monoclonal antibody designated RBS protected WI38 and RD human cell lines from HCV-229E-induced cytopathic effects and protected WI38 cells from virus infection ( Fig. la-c) . RBS pretreatment reduced the number of HCV-229E-infected WI-38 cells at 10 h post-infection by 96%, compared with cells pretreated with control mouse ascites. By contrast, RBS did not inhibit replication of HCV-OC43 in WI38 or RD cells, indicating that the receptor specificities of HCV-OC43 and HCV-229E are different.

Susceptibility to HCV-229E infection in mouse-human somatic cell hybrids depends on a gene located on human chromosome 15 (ref. 6) . A promising candidate for the HCV-II To whom correspondence should be addressed. t Binding of antibodies to hAPN mut -3T3 cells was measured by flow cytometry, as outlined in the legend to Fig. 3 . The mutant lacks peptidase activity; thus, assays for chemical inhibition were not applicable (NA).

* Confluent monolayers of WI38 cells in 96-well plates were pretreated with dilutions of antibodies or inhibitors in medium for 1 h, and then challenged with 1 x 10 3 p.f.u. per well of HCV-229E. After 1 h of adsorption, the inoculum was removed, and the cells were incubated with fresh medium containing antibodies or inhibitors for 48 h, at which time the mDnolayers were examined for virus-induced cytopathic effects. Such effects were evident in HCV-229E-infected controls pretreated with normal serum, but not in mock-infected controls. Plus signs, HCV-229E-induced cytopathic effects were inhibited by antibodies up to a dilution of 1: 200. All incubations were at 37°C. § Inhibitors were tested at the following concentrations: bestatin, 1 mg ml-1 ; l,10-phenanthroline, 1.5 mM; 2,2' -dipyridyl, 2.5 mM; actinonin, 2.7 mM. Antibodies were tested at concentrations that saturated available binding sites in flow cytometric assays. • This exopeptidase removes amino-terminal residues to complete the digestion of short peptides in the gut and helps break down neurotransmitter peptides in the brain 2 ,3,5,8. hAPN is identical to CD13, a glycoprotein identified on granulocytes, monocytes and their bone marrow progenitors 9 ,1O. Porcine aminopeptidase N is a receptor for transmissible gastroenteritis virus, a porcine corona virus in the same serogroup as HCV-229E (ref. 11). Because aminopeptidase from humans, pigs and other mammals are structurally similar 9 , 12-14, we investigated whether HCV-229E and RBS would bind specifically to hAPN and whether expression of hAPN by murine cells would make them susceptible to infection with HCV-229E.

Murine NIH3T3 cells transfected with hAPN cDNA in a retroviral vector 9 (hAPN-3T3) and untransfected NIH3T3 cells were chaUenged with HCV-229E and HCV-OC43 to determine their susceptibility to virus infection. Although the control NIH3T3 cells were resistant to HCV-229E infection (Fig. Id) , the hAPN-transfected mouse cells were susceptible to infection with this virus (Fig. Ie) . By contrast, hAPN-3T3 cells were no more susceptible than NIH3T3 cells to infection with HCV-OC43 (data not shown). Thus, expression of hAPN confers HCV-229E susceptibility, but not HCV-OC43 susceptibility, on murine cells.

We analysed binding of RBS to membrane preparations from hAPN-3T3 or parental NIH3T3 fibroblasts. The antibody bound to membranes of hAPN-3T3 but not to those of NIH3T3 cells (Fig. 2a) , indicating that RBS recognized hAPN. Similarly, HCV-229E virions bound more strongly to hAPN-3T3 membranes than to NIH3T3 membranes (Fig. 2b) , and RBS competi-tively inhibited virus binding to hAPN-3T3 membranes (Fig.  2c) , suggesting that RBS and HCV-229E may recognize adjacent or overlapping epitopes of the peptidase. Some, but not all, antibodies that bind to extracellular epitopes of hAPN inhibit enzymatic activiti 5 • We compared the ability of RBS to inhibit the enzymatic activity of hAPN with that of hAPN/CD13- Hybridoma supernatants (1.624) were tested for the ability to inhibit HCV-299E infection of WI38 cells and one. RBS. was positive. The RBS hybridoma was subcloned three times. and ascites fluid produced by these cells in BALB/c mice was used as the source of antibody. Cells grown on coverslips were pretreated for 1 h at 37°C with a 1: 10 dilution of RBS. control ascites or control medium. and then challenged with 1 x 10 4 p.f.u.

HCV-229E. Cells were incubated at 37 ac for 10 h (a-c) or for 18 h (d-f) and fixed in cold acetone. Antisera remained in the plates throughout the experiment. Intracellular HCV-229E antigens were detected with goat anti-HCV-229E and rhodamine-labelled rabbit anti-goat immunoglobulin. specific antibodies MY7 and WMIS. RBS blocked hAPN activity as efficiently as WMIS and better than MY7 (Fig. 2d) .

Flow cytometric results indicated that both RBS and MY7 bound to hAPN-3T3 cells (Fig. 3a, b) , but not to untransfected NIH3T3 cells. RBS also immunoprecipitated the mature lS0K hAPN glycoprotein and its 130K intracellular precursor 16 For measurements of enzymatic activity on intact cells. 2 x 10 5 cells were washed in PBS. resuspended in isotonic defined medium (50 mM phosphate buffer. pH 7.4) and incubated at 37°C for 1 h in an excess of RBS. WM15. MY7 or UPCIO (control mouse myeloma protein). After incubation. and while still at 37 ac. the APN substrate. alanine-p-nitroaniline. was added to a final concentration of 6 mM. Samples were periodically removed and chilled to 4°C to arrest enzymatic activity. After centrifugation. free p-nitroaniline present in the cell-free supernatants was detected by absorbance measurement at 405 nm. Degradation of substrate in the absence of cells over the same time period was minimal. All measurements were made in triplicate. hAPN-3T3 cells (data not shown). RBS did not bind to NIH3T3 cells transfected with a cDNA encoding a mutant hAPN glycoprotein (hAPN mut -3T3; Fig. 3C ) 17 lacking a 39-amino-acid region (amino acids 360-398) which includes the His-Glu-X-X-His sequence that mediates zinc binding and catalytic activity in a series of related metalloproteases ls . MY7 bound to hAPN mut -3T3 cells (Fig. 3d) , confirming expression of the mutant protein on the cell surface. But hAPN mut -3T3 cells were not susceptible to HCV-229E infection (Fig. If) , indicating that both RBS and HCV-229E recognize epitopes that were eliminated or altered in the mutant polypeptide.

We investigated whether the ability of antibodies to block HCV-229E infectivity correlated with their ability to block enzymatic activity or to bind to the mutant hAPN polypeptide. RBS and WMI5 both blocked enzyme activity and virus infection, but failed to bind to hAPN mut -3T3 cells (Table 1) . By contrast, although MY7 also inhibited HCV-229E infection, it only partially inhibited enzyme activity and recognized the mutant hAPN glycoprotein. Infection of W138 cells by HCV-229E was not prevented by either actinonin or bestatin, small inhibitory molecules that bind competitively to the active site of hAPN I9 -21 . By contrast, HCV-229E infection was inhibited by 1,10phenanthroline and 2,2' -dipyridyl, which block aminopeptidase N activity by cheiating the zinc required by enzymatically active hAPN molecuJes ls . These chelators may alter the conformation We have shown that human coronavirus HCV-229E uses human aminopeptidase N as a receptor and that antibodies directed against the active site of this enzyme prevent virus infection of human cells. A serologically unrelated human respiratory corona virus, HCY-OC43, does not use hAPN as a receptor, and its receptor is unknown. HCV-OC43 is related to murine coronavirus MHV-A59, whose receptor 22 -24 is a member of the carcinoembryonic antigen family of glycoproteins in the immunoglobulin superfamily. Thus, two related coronaviruses, HCY-229E and TGEY, use aminopeptidase N glycoproteins of their normal host species as receptors, whereas two coronaviruses in a different serogroup do not use this aminopeptidase as their receptors. Possibly, coronaviruses related to HCY-229E, such as canine or feline coronaviruses, also use as receptors aminopeptidase or related metalloproteases, such as aminopeptidase A (refs 25, 26) or neutral endopeptidase 26 -3o • Because of their abundance at the apical surfaces of epithelial cells in the alimentary and respiratory tracts, these metalloproteases are promlsmg candidates for receptors of other groups of mammalian viruses or for bacteria or fungi. 0

Molecular and Cellular Basis of Digestion

Leukocyte Typing IV

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Oveksler and D. Wessner for discussions. and J. Gilbert for review