Molecular determinants of species specificity in the coronavirus receptor aminopeptidase N (CD13): influence of N-linked glycosylation

Abstract

Aminopeptidase N (APN), a 150-kDa metalloprotease also called CD13, serves as a receptor for serologically related coronaviruses of humans (human coronavirus 229E [HCoV-229E]), pigs, and cats. These virus-receptor interactions can be highly species specific; for example, the human coronavirus can use human APN (hAPN) but not porcine APN (pAPN) as its cellular receptor, and porcine coronaviruses can use pAPN but not hAPN. Substitution of pAPN amino acids 283 to 290 into hAPN for the corresponding amino acids 288 to 295 introduced an N-glycosylation sequon at amino acids 291 to 293 that blocked HCoV-229E receptor activity of hAPN. Substitution of two amino acids that inserted an N-glycosylation site at amino acid 291 also resulted in a mutant hAPN that lacked receptor activity because it failed to bind HCoV-229E. Single amino acid revertants that removed this sequon at amino acids 291 to 293 but had one or five pAPN amino acid substitution(s) in this region all regained HCoV-229E binding and receptor activities. To determine if other N-linked glycosylation differences between hAPN, feline APN (fAPN), and pAPN account for receptor specificity of pig and cat coronaviruses, a mutant hAPN protein that, like fAPN and pAPN, lacked a glycosylation sequon at 818 to 820 was studied. This sequon is within the region that determines receptor activity for porcine and feline coronaviruses. Mutant hAPN lacking the sequon at amino acids 818 to 820 maintained HCoV-229E receptor activity but did not gain receptor activity for porcine or feline coronaviruses. Thus, certain differences in glycosylation between coronavirus receptors from different species are critical determinants in the species specificity of infection.

FIG. 1

Amino acid alignment of a small region of hAPN, fAPN, and pAPN. The predicted amino acid sequences of hAPN, fAPN, and pAPN were aligned using Clustal W (version 1.4) and the Blosum similarity matrix. hAPN amino acids 278 to 306, aligned with fAPN amino acids 277 to 305 and pAPN amino acids 273 to 301, are shown. The numbers above the sequence correspond to the hAPN glycoprotein, and the consensus sequence is shown on the bottom line. Identical amino acids are shown on dark gray background, similar amino acids are shown on a light gray background, and dissimilar amino acids are shown on a white background.

FIG. 2

Mutations in amino acids 288 to 295 of hAPN blocked HCoV-229E infection. CMT93 cell lines were generated by transfection with wt-hAPN (A and B), p6-hAPN (C and D), or pCi-neo (E and F), and the hAPN-expressing lines (A to D) were sorted by FACS for similar expression levels of hAPN protein. hAPN protein was shown by indirect IFA with MAb WM47 (A, C, and E) and infection by HCoV-229E was shown by indirect IFA, 22 h postinoculation, with anti-spike glycoprotein MAb 511H.6 (B, D, and F).

FIG. 3

Single amino acid reversions that remove an N²⁹¹ glycosylation signal in p6-hAPN result in HCoV-229E receptor activity. Immunoblots show hAPN expression (A) and HCoV-229E nucleocapsid expression (B) in transiently transfected BHK-21 cells. Cell lysates were isolated from BHK-21 cells transiently transfected with wt-hAPN (lane 1), p6-hAPN (lane 2), p6-hAPN/N291E (lane 3), p6-hAPN/T293Q (lane 4), pCi-neo (lane 5), or untransfected BHK-21 cells (lane 6). The cells were inoculated at an MOI of 1 with HCoV-229E 24 h after transfection, and cell lysates were prepared 20 h postinoculation. The proteins were separated by SDS–10% PAGE and transferred to Immobilon-P. (A) hAPN protein expression (arrow) was identified by reactivity with polyclonal mouse antiserum to hAPN; (B) HCoV-229E nucleocapsid (N) protein expression (arrow), was shown by polyclonal goat antiserum to HCoV-229E.

FIG. 4

Addition of an N-linked glycosylation site at amino acid 291 of hAPN blocked HCoV-229E receptor activity. Cell lysates isolated from stably transfected CMT93 cell lines that were sorted by FACS to express similar levels of hAPN were analyzed by immunoblotting. Cell lysates were isolated from cells expressing wt-hAPN (lane 1), p6-hAPN (lane 2), hAPN/N²⁹¹KT (lane 3), hAPN/E²⁹¹KT (lane 4), hAPN/N²⁹¹KQ (lane 5), or a line created with the empty expression vector pCi-neo (lane 6). The cells were inoculated with HCoV-229E at an MOI of 2, and the cell lysates were prepared 21.5 h postinoculation. The proteins were separated by SDS–10% PAGE and transferred to Immobilon-P, and HCoV-229E nucleocapsid (N) protein expression (arrow) was shown using polyclonal goat antiserum to HCoV-229E.

FIG. 5

Wild-type and mutant hAPN glycoproteins expressed at similar levels on the plasma membrane are differentially infected by HCoV-229E. Cell lines that expressed similar levels of wt-hAPN (A and B), p6-hAPN (C and D), hAPN/N²⁹¹KT (E and F), hAPN/E²⁹¹KT (G and H), hAPN/N²⁹¹KQ (I and J), or a line created by transfection with pCi-neo (K and L) were inoculated with HCoV-229E at an MOI of 1. hAPN expression was identified by indirect IFA with MAb WM47 (A, C, E, G, I, and K), and HCoV-229E spike glycoprotein expression was identified with anti-spike glycoprotein MAb 511H.6 (B, D, F, H, J, and L).

FIG. 6

Binding of HCoV-229E was inhibited by the addition of a glycosylation signal at amino acid 291 of hAPN. CMT93 cell lines that had similar levels of hAPN surface expression were mixed with ³H-labeled virus, the cells were washed and lysed, and ³H was counted as described in Materials and Methods. The average ³H-labeled virus bound by the wt-hAPN was 1.38 × 10⁴ cpm and was defined as 100% binding. The average total counts bound by cells transfected with pCi-neo was defined as 0% binding. The average of the other cell lines was reported as a percentage of wild-type binding activity, and the error bars represent the standard deviation of the three replicates.

FIG. 7

The influence of a glycosylation signal at N⁸¹⁸ of hAPN and p6-hAPN on receptor activity for serogroup 1 coronaviruses of humans, pigs, and cats. Nucleocapsid (N) proteins (arrows) were identified by immunoblotting with goat polyclonal antisera to HCoV-229E (A) or feline polyclonal antisera to FIPV that cross-reacts with TGEV (B and C). FCWF (lane 1) or CMT93 cell lines that express wt-hAPN (lane 2), p6-hAPN (lane 3), hAPN/N818E (lane 4), p6-hAPN/N818E (lane 5), and p6-hAPN/T820E (lane 6) or cell lines created with the empty expression vector pCi-neo (lane 7) were inoculated with HCoV-229E (A), TGEV (B), or FECoV 79-1863 (C).

FIG. 8

Amino acid sequence alignment from amino acids 288 to 295 of the hAPN expression plasmids summarizes the consequence of an N²⁹¹ glycosylation sequon on HCoV-229E receptor activity. The predicted amino acids of hAPN expression plasmids were aligned using Clustal W (version 1.4) and the Blosum similarity matrix. The numbers above the sequences correspond to amino acids of the hAPN glycoprotein. Identical amino acids are shown on a dark gray background.