Mutational analysis of the virus and monoclonal antibody binding sites in MHVR, the cellular receptor of the murine coronavirus mouse hepatitis virus strain A59

Abstract

The primary cellular receptor for mouse hepatitis virus (MHV), a murine coronavirus, is MHVR (also referred to as Bgp1a or C-CAM), a transmembrane glycoprotein with four immunoglobulin-like domains in the murine biliary glycoprotein (Bgp) subfamily of the carcinoembryonic antigen (CEA) family. Other murine glycoproteins in the Bgp subfamily, including Bgp1b and Bgp2, also can serve as MHV receptors when transfected into MHV-resistant cells. Previous studies have shown that the 108-amino-acid N-terminal domain of MHVR is essential for virus receptor activity and is the binding site for monoclonal antibody (MAb) CC1, an antireceptor MAb that blocks MHV infection in vivo and in vitro. To further elucidate the regions of MHVR required for virus receptor activity and MAb CC1 binding, we constructed chimeras between MHVR and other members of the CEA family and tested them for MHV strain A59 (MHV-A59) receptor activity and MAb CC1 binding activity. In addition, we used site-directed mutagenesis to introduce selected amino acid changes into the N-terminal domains of MHVR and these chimeras and tested the abilities of these mutant glycoproteins to bind MAb CC1 and to function as MHV receptors. Several recombinant glycoproteins exhibited virus receptor activity but did not bind MAb CC1, indicating that the virus and MAb binding sites on the N-terminal domain of MHVR are not identical. Analysis of the recombinant glycoproteins showed that a short region of MHVR, between amino acids 34 and 52, is critical for MHV-A59 receptor activity. Additional regions of the N-terminal variable domain and the constant domains, however, greatly affected receptor activity. Thus, the molecular context in which the amino acids critical for MHV-A59 receptor activity are found profoundly influences the virus receptor activity of the glycoprotein.

FIG. 1

Comparison of the amino acid sequences of the N-terminal domains of the murine biliary glycoproteins MHVR (also referred to as Bgp1^a or C-CAM), Bgp1^b, and Cea10. For Bgp1^b and Cea10, only amino acids that differ from MHVR are shown.

FIG. 2

Functions associated with the N-terminal domain recombinant chimeric anchored glycoproteins. All chimeras also contained the second, third, fourth, transmembrane, and cytoplasmic domains of MHVR (not shown). Unshaded regions represent MHVR sequences, black regions represent Cea10 sequences, and gray regions represent Bgp1^b sequences. Selected amino acid positions are indicated above. Receptor activity and MAb CC1 receptor blockade activity were determined by immunofluorescence as described in Materials and Methods. MAb CC1 binding was determined by immunoblot assay of cell lysates following infection with vaccinia virus vTF7-3 and transfection of recombinant plasmid. ND, not done.

FIG. 3

Detection of MHV antigens or chimeric glycoproteins in hamster cells transfected with plasmid containing the cDNA of MHVR, Cea10, or representative MHVR/Cea10 chimeras. BHK-21 cells grown on glass coverslips were transfected with recombinant MHVR cDNA in which the N-terminal domain consisted of Cea10 (A and D), MHVR (B and E), or MHVR_1–52/Cea10_53–108 (C and F). Following transfection, cells in panels A, B, C, E, and F were inoculated with MHV-A59, incubated for 16 h, and fixed in cold acetone. Cells in panel E and F were pretreated with MAb CC1. Viral antigens in the cytoplasm were detected with anti-MHV serum and rhodamine-labeled goat anti-mouse IgG. Cells in panel D were transfected, and surface expression of the transfected molecule was confirmed by immunofluorescence with the anti-MHVR polyclonal antibody 655.

FIG. 4

Functions associated with the N-terminal recombinant chimeric anchored glycoproteins with and without ClaI restriction enzyme sites. All chimeras also contained the second, third, fourth, transmembrane, and cytoplasmic domains of MHVR (not shown). Unshaded regions represent MHVR sequences, and black regions represent Cea10 sequences. Mutated amino acids are indicated. Receptor activity and MAb CC1 receptor blockade activity were determined by immunofluorescence as described in Materials and Methods. MAb CC1 binding was determined by immunoblot assay of cell lysates following infection with vaccinia virus vTF7-3 and transfection of recombinant plasmid. ND, not done.

FIG. 5

Functions associated with recombinant chimeric anchored glycoproteins containing various constant domains. All chimeras also contained the transmembrane and cytoplasmic domains of MHVR (not shown). Domains are numbered (1 = N-terminal domain). The arrow indicates approximate location of amino acid 70 in the N-terminal domain. Unshaded regions represent MHVR sequences, and gray regions represent Bgp1^b sequences. Receptor activity and MAb CC1 receptor blockade activity were determined by immunofluorescence as described in Materials and Methods. MAb CC1 binding was determined by immunoblot assay of cell lysates following infection with vaccinia virus vTF7-3 and transfection of recombinant plasmid. ND, not done.

FIG. 6

Recognition of MHVR, Bgp1^b, Cea10, or representative chimeric glycoproteins by anti-MHVR antibodies. Cell lysates from BHK-21 cells infected with vaccinia virus vTF7-3 and transfected with cDNA encoding MHVR, Bgp1^b, Cea10, or recombinant glycoproteins were separated by electrophoresis on sodium dodecyl sulfate–8 or 10% polyacrylamide gels, transferred to nitrocellulose, and incubated with either anti-MHVR MAb CC1 (A) or rabbit polyclonal anti-MHVR antibody 655 (B). Bound antibodies were detected by incubation of immunoblots with ¹²⁵I-staphylococcal protein A. Sizes are indicated in kilodaltons. R, amino acids from MHVR; C, amino acids from Cea10; 1^b, amino acids from Bgp1^b.

FIG. 7

Structural model of the N-terminal domain of MHVR. Structure was determined based on sequence similarities between the N-terminal domains of MHVR and CD4. Beta sheets are indicated by wide ribbons and labeled. Numbers indicate amino acid positions. The amino acid region critical for MHV-A59 receptor activity is in black. A putative salt bridge between residues R₆₄ and D₈₂ is indicated.