Use of site-specific mutagenesis and monoclonal antibodies to map regions of CD46 that interact with measles virus H protein

Researchers at our laboratory have been dissecting the binding domains of t he receptor for the Edmonston laboratory strain of measles virus (CD46) thr ough site-specific mutagenesis. We initially substituted most of the hydrop hilic amino acids in the two external short consensus regions (SCRI and SCR II) of CD46 with the amino acid alanine [Hsu et al (1997) J. Virol. 71:6144 -6154] and found that the glutamic-arginine residues at positions 58 and 59 were particularly sensitive to change. Here we consider the roles of hydro phobic amino acids in the binding between measles virus H protein and CD46. Hydrophobic amino acids in the SCRI and SCRII domains of CD46 were systema tically replaced with serine. The effects of these changes were monitored t hrough the interaction of Sf9 insect cells expressing the H protein and mou se OST-7 cells synthesizing the mutant CD46 molecules. Binding was quantifi ed through a colorimetric assay for beta-galactosidase that was also produc ed by the insect cells. Our results indicate that E45, Y54, 58E/R59, Y68, F 69, Y101, I102, R103, D104, and Y117 seem to be critical residues for the b inding of CD46 to measles virus H protein. The hydrophilic amino acid R59 i n SCR1 and hydrophobic residues Y101, I102, and Y117 in SCR2 seem to be esp ecially important for interaction between H protein and CD46. In addition, we mapped the antigenic epitopes of five monoclonal antibodies that are kno wn to inhibit the binding between H protein and CD46. Three of these antibo dies recognized regions in SCRI, and two reacted with amino acids in SCR2. For the most part, the determinants recognized by the monoclonal antibody c orresponded to the amino acids that were most sensitive to change in the bi nding process. The SCR1 and SCR2 domains of CD46 were modeled from an analo gous region in another complement regulatory protein, factor H, whose three -dimensional structure has been previously reported. Amino acids implicated in binding seem to lie on one planar face of the SCR1 and SCR2 domains. Th ese studies serve as a prelude to understanding the structural interactions that occur between CD46 and the measles virus H protein. (C) 1999 Academic Press.