Mutational analysis of aminopeptidase N, a receptor for several group 1 coronaviruses, identifies key determinants of viral host range

Abstract

Feline coronavirus (FCoV), porcine transmissible gastroenteritis coronavirus (TGEV), canine coronavirus (CCoV), and human coronavirus HCoV-229E, which belong to the group 1 coronavirus, use aminopeptidase N (APN) of their natural host and feline APN (fAPN) as receptors. Using mouse-feline APN chimeras, we identified three small, discontinuous regions, amino acids (aa) 288 to 290, aa 732 to 746 (called R1), and aa 764 to 788 (called R2) in fAPN that determined the host ranges of these coronaviruses. Blockade of infection with anti-fAPN monoclonal antibody RG4 suggested that these three regions lie close together on the fAPN surface. Different residues in fAPN were required for infection with each coronavirus. HCoV-229E infection was blocked by an N-glycosylation sequon present between aa 288 to 290 in murine APN. TGEV required R1 of fAPN, while FCoV and CCoV required both R1 and R2 for entry. N740 and T742 in fAPN and the homologous R741 in human APN (hAPN) were key determinants of host range for FCoV, TGEV, and CCoV. Residue N740 in fAPN was essential only for CCoV receptor activity. A conservative T742V substitution or a T742R substitution in fAPN destroyed receptor activity for the pig, dog, and cat coronaviruses, while a T742S substitution retained these receptor activities. Thus, the hydroxyl on T742 is required for the coronavirus receptor activity of fAPN. In hAPN an R741T substitution caused a gain of receptor activity for TGEV but not for FCoV or CCoV. Therefore, entry and host range of these group 1 coronaviruses depend on the ability of the viral spike glycoproteins to recognize small, species-specific amino acid differences in the APN proteins of different species.

FIG. 1.

BHK cells transfected with fAPN but not mAPN are susceptible to infection with FCoV, TGEV, CCoV, and HCoV-229E. Surface expression of fAPN or mAPN proteins on paraformaldehyde-fixed BHK cells transiently transfected with plasmids encoding fAPN or mAPN was detected by immunolabeling with anti-fAPN RG4 or anti-mAPN R3-242 MAb, respectively. Viral antigens in cells inoculated with FCoV, TGEV, CCoV, or HCoV-229E were detected after 24 h by immunolabeling with antiviral antibodies.

FIG. 2.

HCoV-229E and the animal coronaviruses FCoV and TGEV require different regions of fAPN for receptor activity. BHK cells were transiently transfected with plasmids encoding m/fAPN chimeric proteins. The numbers in parentheses indicate fAPN residues present in the m/fAPN protein. At 48 h posttransfection, the surface expression of the chimeric m/fAPN_1-582 and m/fAPN_251-582 proteins was detected with anti-fAPN RG4 MAb, and the surface expression of all other m/fAPN chimeric proteins was detected with anti-mAPN R3-242 MAb. At 24 h after inoculation, viral antigens were detected with antiviral antibodies.

FIG. 3.

Alignment of aa 701 to 821 of APN proteins of different species. Align Plus 5 in the Clone Manager 7 software suite (Scientific and Educational Software) was used to align amino acid sequences of APN proteins from different species using a BLOSUM 62 scoring matrix. Accession numbers for feline, canine, porcine, human, bovine, mouse, rat, and rabbit APN are P79171, XP536190, P15145, P15144, P79098, P97449, P15684, and P15541, respectively. APN proteins that have coronavirus receptor activity are indicated in black, and APN proteins with no known coronavirus receptor activity are in gray. Two regions of high variability between APN sequences of different species, R1 and R2, are underlined. The R3 region corresponds to an N-glycosylation sequon that is absent in fAPN but present in APN sequences of several other species. The PROFsec and PROFacc software programs (47) were used to predict the secondary structure and solvent accessibility, respectively, of aa 701 to 821 of fAPN. The predicted secondary structure of this region of fAPN is shown above the sequence alignment. Predicted helical regions are shown as thick lines, and predicted loops or unstructured regions are thin lines. Gray dots indicate residues in R1, R2, and R3 that are predicted to be surface exposed. Arrowheads indicate residues N740 and T742 of fAPN that are required for FCoV, TGEV, and/or CCoV receptor activity.

FIG. 4.

Two small discontinuous regions, R1 and R2, of fAPN are required for FCoV and CCoV receptor activity, but only R1 of fAPN is required for TGEV receptor activity. The mutant m/fAPN_R1(T742V),R2 protein is an m/fAPN_R1,R2 protein with a T742V substitution. Surface expression of the m/fAPN proteins 48 h after transfection was detected with anti-mAPN R3-242 MAb. Viral antigens were detected with antiviral antibody 10 to 12 h after inoculation.

FIG. 5.

Amino acid residues T742 and N740 in fAPN are key determinants of host range for FCoV, TGEV, and CCoV but not for HCoV-229E. The surface expression of mutant fAPN proteins with the single amino acid substitution N740D, N740Q, T742V, T742S, or T742R at 48 h after transfection was analyzed by flow cytometry with anti-fAPN RG4 MAb. In the flow cytometry panels, the dotted line indicates mock-transfected cells, the gray line indicates cells expressing the mutant fAPN protein, and the black line shows cells expressing the wild-type fAPN protein. Ninety-two percent of transfected cells expressed wild-type fAPN. The percentage of cells expressing the mutant fAPN protein is shown for each overlay. Viral antigens were detected with antiviral antibody 10 to 12 h after inoculation.

FIG. 6.

The single amino acid substitution R741T in hAPN mediates receptor activity for TGEV but not for FCoV, TGEV, or CCoV. The surface expression of wild-type hAPN and the hAPN_R741T proteins was detected with anti-hAPN DW1 MAb, and the expression of wild-type mAPN and mAPN_V740T proteins was detected with anti-mAPN R3-242 MAb. Viral antigens were detected with antiviral antibody 10 to 12 h after inoculation.

FIG. 7.

Summary of the receptor activities of wild-type, chimeric, and mutant APN proteins. The receptor activities for the APN proteins are summarized for each group 1 coronavirus. For m/fAPN chimeras, amino acids in parenthesis are the fAPN amino acids present in the chimeric protein (see text for and R1, R2, and R3). This figure includes some data presented by Tusell and Holmes (55). Triangles indicate single amino acid substitutions in the APN with amino acids present in fAPN (black), mAPN (white), or hAPN (gray); white-outlined triangles represent a single amino acid substitution that is not present in fAPN, mAPN, or hAPN. NA, not applicable; ND, not done.