IDENTIFICATION OF A CONTIGUOUS 6-RESIDUE DETERMINANT IN THE MHV RECEPTOR THAT CONTROLS THE LEVEL OF VIRION BINDING TO CELLS

Murine carcinoembryonic antigens serve as receptors for the binding an d entry of the enveloped coronavirus mouse hepatitis virus (MHV) into cells. Numerous receptor isoforms are now known, and each has extensiv e differences in its amino terminal immunoglobulin-like domain (NTD) t o which MHV binds via its protruding spike proteins. Some of these rec eptor alterations may affect the ability to bind viral spikes. To iden tify individual residues controlling virus binding differences, we hav e used plasmid and vaccinia virus vectors to express two forms of MHV receptor differing only in their NTD. The two receptors, designated bi liary glycoproteins (Bgp) 1(a) and 1(NTD)(b), varied by 29 residues in the 107 amino acid NTD, When expressed from cDNAs in receptor-negativ e HeLa cells, these two Bgp molecules were displayed on cell surfaces to equivalent levels, as both were equally modified by a membrane-impe rmeant biotinylation reagent. Infectious center assays revealed that t he 1(a) isoform was 10 to 100 times more effective than 1(NTD)(b) in i ts ability to confer sensitivity to MHV (strain A59) infection. Bgp1(a ) was also more effective than Bgp1(NTD)(b) in comparative virus adsor ption assays, binding 6 times more MHV (strain A59) and 2.5 times more MHV (strain JHMX). Bgp1(a) was similarly more effective in promoting the capacity of viral spikes to mediate intercellular membrane fusion as judged by quantitation of syncytia following cocultivation of spike and receptor-bearing cells. To identify residues influencing these di fferences, we inserted varying numbers of 1(b) residues into the Bgp1( a) background via restriction fragment exchange and site-directed muta genesis. Analysis of the resulting chimeric receptors showed that resi dues 38 to 43 of the NTD were key determinants of the binding and fusi on differences between the two receptors. These residues map to an exp osed loop (C-C' loop) in a structural model of the closely related hum an carcinoembryonic antigen. (C) 1997 Academic Press.