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 virusreceptor 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.Human coronaviruses (HCoV) in two serogroups represented by HCoV-229E (serogroup 1) and HCoV-OC43 (serogroup 2) are an important cause of upper respiratory tract infection. Serological studies suggest that they cause from 15 to 30% of human upper respiratory infections or colds (25). Although there is no animal model for the pathogenesis of HCoV respiratory infections, HCoV-229E and HCoV-OC43 have been administered intranasally to human volunteers, and this has shown that people can be repeatedly infected by the same strain (5). There is also in vitro and in vivo evidence for infection of cells of the central nervous system (CNS) by HCoV (39,51,68,69). Coronaviruses have a lipid membrane that has large peplomers that protrude from the virion and give it the appearance of a corona. These peplomers or spike glycoproteins are the viral attachment proteins. The 27-kb genomic RNA of HCoV-229E is positive sense, has a 5Ј cap, is polyadenylated at the 3Ј terminus, and is infectious when transfected into cells of a wide variety of species (25). In contrast, infection by HCoV-229E virions is limited to cells of human or feline origin in vitro, demonstrating that the receptor for HCoV-229E is a major determinant in species specificity (25).The cellular receptor for HCoV-229E is human aminopeptidase N (hAPN). HCoV-229E infection ...

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