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JOURNAL OF VIROLOGY Feb 2007 p 1261 1273 Vol 81 No 3 0022 538X 07 08 00H110010 doi 10 1128 JVI 01510 06 Copyright 2007 American Society for Microbiology All Rights Reserved Mutational Analysis of Aminopeptidase N a Receptor for Several Group 1 Coronaviruses Identifies Key Determinants of Viral Host Range H17188 Sonia M Tusell 1 Stephanie A Schittone 2 and Kathryn V Holmes 1 2 Molecular Biology Program 1 and Microbiology Department 2 University of Colorado at Denver and Health Sciences Center Aurora Colorado 80045 Received 14 July 2006 Accepted 31 October 2006 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 substi tution 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 Coronaviruses are important respiratory and enteric patho gens of humans and many animal species 32 40 58 Coro navirus phylogenetic groups 1 2a 2b and 3 differ in host range and pathogenicity 17 32 40 58 Group 1 contains two hu man coronaviruses HCoV 229E and HCoV NL63 that cause acute respiratory tract infections 4 6 56 60 and several important veterinary viruses in pigs porcine epidemic diar rhea virus and transmissible gastroenteritis coronavirus TGEV cause enteric disease and porcine respiratory coro navirus causes respiratory disease 25 26 feline coronavirus FCoV causes enteric or systemic disease in cats 10 27 42 57 and canine coronavirus CCoV causes enteric disease in dogs 44 Human feline canine and porcine group 1 coronaviruses cause transmissible disease within a single host species How ever experimental inoculation of several other species with these coronaviruses can result in viral replication seroconver sion and in some cases nontransmissible disease 2 3 63 64 For serial transmission to occur in a new host species the spike glycoproteins of group 1 coronaviruses need to adapt to their receptor in the new host species by mutation or recombination with another coronavirus that naturally infects the new host species An important determinant of coronavirus host range is the interaction of the H11011200 kDa viral spike S glycoprotein with a receptor glycoprotein on the surface of susceptible cells 18 30 41 49 Several coronavirus receptors have been identified Mouse hepatitis virus in phylogenetic group 2a uses murine carcinoembryonic cell adhesion molecule 1a as a receptor 14 15 62 Human angiotensin converting enzyme 2 hACE 2 is a receptor for severe acute respiratory syndrome SARS CoV in phylogenetic group 2b and HCoV NL63 in group 1 21 36 52 HCoV 229E TGEV FCoV and CCoV in group 1 use aminopeptidase N APN of their natural host species to enter cells 12 28 54 65 In cell culture human APN hAPN is a receptor for only HCoV 229E and porcine APN pAPN is a receptor for only TGEV 12 65 However feline APN fAPN is a receptor for not only FCoV but also HCoV 229E TGEV and CCoV 54 The purpose of this study was to identify key regions and residues in fAPN that determine the host range of these group 1 coronaviruses APN CD13 is a 150 to 160 kDa type II transmembrane glycoprotein expressed as a homodimer on the apical mem branes of epithelial cells in the respiratory and enteric tracts endothelial cells and kidney cells at synaptic junctions and on cells of the immune system monocytes dendritic cells and granulocytes 34 APN is a zinc dependent protease that cleaves N terminal amino acids from biologically active pep tides 34 The APN proteins from human mouse rat rabbit Corresponding author Mailing address University of Colorado Health Sciences Center Microbiology Department Mail Stop 8333 12800 E 19th Avenue P O Box 6511 Aurora CO 80045 Phone 303 724 4231 Fax 303 724 4226 E mail kathryn holmes uchsc edu H17188 Published ahead of print on 8 November 2006 1261 on April 13 2015 by UCSF Library domain II is the trans membrane domain and domain III amino acids aa 40 to 70 of hAPN is the stalk region In hAPN domain IV includes aa 70 to 252 Domains V and VI aa 253 to 580 of hAPN contain the active site of the enzyme and a conserved zinc binding motif HELAH Domain VII aa 581 to 967 at the C terminus of hAPN is predicted to be mainly H9251 helical 51 Until the crystal structures for APN and group 1 coronavirus S glycoproteins are determined the identification of receptor determinants on APN that affect coronavirus host range de pends on the use of mutant and chimeric APN proteins Sev eral regions of APN that are important for entry of group 1 coronaviruses were previously identified using chimeras be tween APN proteins of different species 13 28 Human pig APN chimeras showed that aa 717 to 813 in domain VII of pAPN are essential for TGEV receptor activity 11 while aa 288 to 295 in domain V of hAPN are necessary for HCoV 229E receptor activity 29 The introduction into hAPN of a sequon encoding a potential N glycosylation sequon at aa 291 as found in pAPN abrogates HCoV 229E receptor activity 59 Regions in fAPN that are important for HCoV 229E TGEV and FCoV receptor activity were identified using pig feline and human feline APN chimeras Residues 670 to 840 in domain VII of fAPN are required for TGEV and FCoV re ceptor activity and aa 135 to 297 in domain V of fAPN are required for HCoV 229E receptor activity 19 29 In addi tion aa 643 to 841 in domain VII of canine APN in an hAPN backbone can mediate entry of CCoV TGEV and FCoV 5 In this study chimeras between fAPN and mouse APN mAPN which lacks coronavirus receptor activity were used to identify three small discontinuous regions in fAPN between aa 288 to 290 in domain V and aa 732 to 746 called R1 and aa 764 to 788 called R2 in domain VII that were critical determinants of coronavirus entry and host range Using mu tant APN proteins we identified single residues in fAPN and hAPN that are critical determinants of group 1 coronavirus host range and infection in vitro These results provide a model for the evolution and emergence of coronaviruses as they adapt to recognize species specific differences in the APN pro teins of different host species MATERIALS AND METHODS Cell lines and viruses Felis catus whole fetus Fcwf cells provided by Niels Pedersen University of California at Davis Davis CA a hamster kidney BHK fibroblast cell line a swine testicle cell line provided by David Brian University of Tennessee Knoxville TN canine tumor cell line A 72 provided by Leonard Binn Walter Reed Army Institute for Research Silver Spring MD and human lung fibroblast cell line MRC5 ATCC CCL 171 Manassas VA were grown as previously described 54 59 FCoV genotype II strain 79 1146 CCoV genotype II strain 1 71 provided by Leonard Binn Walter Reed Army Institute for Research TGEV clone E provided by David Brian University of Tennessee and HCoV 229E ATCC VR 740 were propagated and titers of infectious viruses were determined by plaque assay as previously described 54 59 Chimeric and mutant APN plasmids All mouse feline APN m fAPN chi meric and mutant APN cDNA sequences were constructed by standard fusion PCR techniques 24 or by site directed mutagenesis as described previously 59 using Pfu Turbo polymerase Stratagene La Jolla CA All cDNAs encoding chimeric m fAPN mutant and wild type fAPN accession number NM 001009252 54 and mAPN accession number NM 008486 provided by Linda Shapiro University of Connecticut Health Center Farmington CT were cloned into the pcDNA3 1D V5 His TOPO mammalian expression vector Invitrogen Carlsbad CA in frame with the C terminal V5 and six His tags according to the manufacturer s instructions The expression plasmid containing hAPN cDNA 59 was used to introduce mutations into the hAPN DNA sequence All DNA constructs were sequenced by the University of Colorado Cancer Center DNA Sequencing and Analysis Core Facility Transient transfections BHK cells were transfected with plasmids containing cDNA encoding fAPN mAPN hAPN and mutant or chimeric APN proteins using Lipofectamine 2000 Invitrogen according to the manufacturer s instruc tions Twenty four hours after transfection cells were seeded on glass coverslips and 48 h after transfection they were used for virus inoculation or for detection of APN protein expression by immunofluorescence or flow cytometry as de scribed below Generation of cells stably expressing fAPN A plasmid construct containing the fAPN cDNA in the pCiNeo mammalian expression vector Promega Corp Madison WI fAPN pCiNeo was generated by subcloning the fAPN cDNA fragment from the pCR3 expression plasmid Invitrogen described by Tresnan et al 54 into the pCiNeo expression vector using EcoRI and NotI BHK cells were transfected with the fAPN pCiNeo construct using Lipofectamine 2000 Invitrogen according to the manufacturer s instructions and 48 h after trans fection cells were placed under G418 selection GIBCO BRL Grand Island NY Two weeks later cells expressing fAPN were selected by fluorescence activated cell sorting with a mouse anti fAPN RG4 monoclonal antibody MAb from a hybridoma cell line 23 kindly provided by Tsutomu Hohdatsu Kitasato University Japan Cell sorting was done at the University of Colorado Cancer Center Flow Cytometry Core Facility Virus inoculation BHK cells transfected with cDNAs encoding wild type chimeric or mutant APN proteins were inoculated 48 h after transfection with HCoV 229E FCoV TGEV or CCoV diluted in Dulbecco s modified Eagle s minimal essential medium GIBCO BRL supplemented with heat inactivated 10 fetal bovine serum HyClone Logan UT and 2 antibiotic antimycotic GIBCO BLR at a multiplicity of infection MOI of 0 3 to 0 8 After1hof adsorption the inocula were removed and replaced with fresh medium Inocu lated cells were fixed with methanol acetic acid 3 1 for detection of viral antigens as described below Immunofluorescence assay to detect expression of APN proteins or viral antigens To detect APN expression transfected cells on coverslips were fixed with 1 6 paraformaldehyde Ted Pella Inc Redding CA and an immuno fluorescence assay IFA was performed with purified fluorescein conjugated rat anti mAPN R3 242 MAb BD Biosciences Pharmingen San Jose CA purified RG4 MAb or anti hAPN DW1 MAb Incubation with DW1 or RG4 MAbs was followed by incubation with fluorescein conjugated goat anti mouse immuno globulin G IgG Jackson Immunoresearch West Grove PA FCoV TGEV and CCoV antigens were detected with a polyclonal fluorescein conjugated fe line anti FCoV serum FITC FIP VMRD Inc Pullman WA that cross reacts with TGEV and CCoV HCoV 229E antigens were detected with a polyclonal goat anti HCoV 229E serum 59 Immunolabeled cells were analyzed using a Zeiss Axioplan 2 microscope Carl Zeiss Inc New York NY Flow cytometry Surface expression of APN protein was detected with anti fAPN RG4 MAb anti mAPN R3 242 MAb or anti hAPN DW1 MAb Cells were washed and incubated with phycoerythrin conjugated goat anti mouse IgG Jackson Immunoresearch or fluorescein conjugated goat anti rat IgG Jackson Immunoresearch and fixed with 1 6 paraformaldehyde Cells were analyzed at the University of Colorado Cancer Center Flow Cytometry Core Facility Blockade of coronavirus infection with anti fAPN RG4 MAb BHK cells stably expressing fAPN were preincubated with 4 8 H9262g of total protein of purified RG4 MAb or a control mouse MAb control MAb against an irrelevant antigen in medium for 45 min at 4 C and then inoculated with FCoV TGEV CCoV or HCoV 229E at an MOI of 0 1 After virus adsorption for 1 h cells were washed and fresh medium was added containing 16 ng H9262l of RG4 or control MAb Cells were fixed with methanol acetic acid 3 1 16 to 18 h after inoculation and viral antigens were detected by IFA In two experiments BHK cells transiently trans fected with plasmids encoding fAPN or m fAPN chimeric proteins were prein cubated with 14 4 H9262gor4 8H9262g of total protein of RG4 MAb or control MAb and then inoculated with FCoV or HCoV 229E at an MOI of 0 6 or 0 3 Viral antigens were detected as described above Cells expressing viral antigens were counted in five fields at a magnification of H1100340 for all APN constructs except for m fAPN containing aa 582 to 967 of fAPN in the mAPN backbone m fAPN 582 967 for which positive cells were counted in five fields at a magnification of H1100310 For each wild type or chimeric APN protein the number of cells positive for viral antigens in samples treated with the control MAb was set as 100 Samples 1262 TUSELL ET AL J VIROL on April 13 2015 by UCSF Library also data not shown suggesting that mAPN contains all the residues sufficient for HCoV 229E receptor activity but that an N linked glycan at N288 in mAPN may block HCoV 229E entry Two small regions of fAPN called R1 and R2 are required for FCoV and CCoV receptor activity but only R1 of fAPN is required for TGEV receptor activity To further characterize the region in fAPN between aa 704 to 831 that was required for group 1 animal coronavirus receptor activity Fig 2 we aligned the amino acid sequences of APN proteins from dif ferent species and identified residues in this region that were unique to fAPN and might therefore contribute to its receptor activity for the animal coronaviruses Fig 3 By comparing the amino acid sequences of APN proteins that have coronavirus receptor activity feline canine porcine and human APN proteins with sequences of APN proteins that have no known coronavirus receptor activity mouse rat rabbit and bovine APN proteins we identified two areas of high amino acid sequence variation aa 732 to 746 and aa 764 to 788 which we called R1 and R2 respectively Fig 3 Secondary structure predictions indicated that R1 is a loop or unstructured region containing several surface exposed residues N732 K739 N740 T742 and D743 The R2 region is predicted to be mainly H9251 helical with several surface exposed residues Fig 3 We identified a region called R3 aa 819 to 821 of fAPN which contained a potential N glycosylation sequon in canine mouse rabbit and human APN proteins but not in feline porcine rat or bovine APN proteins Fig 3 Since the amino acid sequences of the R1 and R2 regions are highly variable among APN proteins of different species and are predicted to contain surface exposed residues we an alyzed the roles of these regions in coronavirus receptor activ ity We also tested whether a potential N linked glycan at amino acid residue N819 in the R3 of mAPN could block entry of FCoV TGEV or CCoV The R1 R2 and R3 regions of mAPN were replaced with the corresponding fAPN regions All m fAPN chimeric proteins were expressed on the cell sur face of transfected cells Fig 4 Cells expressing the chimeric m fAPN proteins carrying the R1 R2 and R3 regions of fAPN m fAPN R1 R2 R3 or m fAPN R1 R2 were susceptible to infec tion with FCoV TGEV and CCoV Fig 4 Thus the R3 region of fAPN was not necessary for infection with these animal coronaviruses The m fAPN R1 chimeric protein had receptor activity for TGEV but not for FCoV or CCoV although this protein had less TGEV receptor activity than m fAPN R1 R2 Fig 4 and data not shown Therefore while R1 is sufficient for TGEV infection interactions with R2 may increase the efficiency of TGEV infection However the R2 region of fAPN alone in an mAPN backbone was not sufficient for entry of these pig cat and dog coronaviruses Fig 4 These experiments identified two small regions of 15 aa R1 and 25 aa R2 of fAPN in an mAPN backbone that were necessary and sufficient for FCoV and CCoV infection whereas only R1 of fAPN was sufficient for TGEV infection Asparagine 740 and threonine 742 in R1 of fAPN are im portant determinants of host range for FCoV TGEV and CCoV but not for HCoV 229E To identify specific residues in APN that determine the host ranges of FCoV TGEV and CCoV we substituted single amino acids in the R1 region of the chimeric m fAPN R1 R2 protein with the corresponding res idues from mAPN The mutant m fAPN R1 R2 proteins with the single amino acid substitution K739N D743N H744R or TABLE 2 Blockade of FCoV and HCoV 229E infection with the anti fAPN RG4 MAb in cells expressing fAPN or chimeric m fAPN proteins a APN protein HCoV 229E infection FCoV infection Control MAb RG4 MAb Control MAb RG4 MAb fAPN H11002H11001H11002H11001 m fAPN 251 967 H11002H11001H11002H11001 m fAPN 251 582 H11002H11001NA NA m fAPN 582 967 NA NA H11002H11002 b m fAPN 1 582 H11002H11001NA NA a Infection with FCoV or HCoV 229E was detected by immunofluorescence with antiviral antibodies 16 to 18 h after inoculation H11001 antibody blockade of virus infection H110215 of cells infected H11002 no antibody blockade of virus infec tion H1135090 of cells infected NA not applicable the m fAPN chimeric protein has no receptor activity for the virus b This antibody does not bind to this m fAPN chimera TABLE 1 Mapping the binding epitopes of anti fAPN RG4 and anti mAPN R3 242 MAbs a APN protein RG4 MAb binding R3 242 MAb binding IFA FC IFA FC Mock transfected H11002H11002H11002H11002 fAPN H11001H11001H11002H11002 mAPN H11002H11002H11001H11001 m fAPN 251 967 H11001H11001H11001H11001 m fAPN 251 582 H11001H11001H11001H11001 m fAPN 582 967 H11002H11002H11001H11001 m fAPN 1 582 H11001H11001H11002H11002 a BHK cells transiently transfected with plasmids encoding fAPN mAPN or chimeric m fAPN proteins were tested for reactivity with anti fAPN RG4 or anti mAPN R3 242 MAbs by IFA and flow cytometry FC VOL 81 2007 CORONAVIRUS HOST RANGE DETERMINANTS ON APN 1265 on April 13 2015 by UCSF Library also data not shown Therefore the three discontinuous regions of fAPN aa 288 to 290 R1 and R2 that are key determinants of the host range of the human feline canine and porcine coronaviruses probably lie close together on the surface of the fAPN protein forming part of a large interface that contains the binding sites for RG4 MAb and for the spike glycoproteins of these four group 1 corona viruses Cocrystals between the receptor binding domains RBDs of the viral attachment proteins for herpes simplex virus human immunodeficiency virus and SARS CoV with their cellular receptors HveA CD4 and ACE2 respectively identified numerous contact residues in the large H110111 500 to 1 700 2 binding interfaces between the viral RBDs and re ceptor proteins 8 9 31 35 The cocrystal of the SARS CoV RBD with its hACE 2 receptor showed that 18 residues in the receptor contact 14 residues in the spike 35 Mutational 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 a