The entry of measles virus (MV) into human cells is mediated by the initial attachment of the viral hemagglutinin (HA) to the complement regulatory protein CD46. Two subdomains, one each within CD46 short consensus repeats (SCRs) 1 and 2, are responsible for this interaction. However, little is known about the regions within MV HA needed for a high-affinity CD46 interaction. To better define the HA-CD46 interaction, we took three approaches: chimeric domain swapping, peptide scanning, and alanine scanning mutagenesis. Chimeras of MV HA and the closely related rinderpest virus (RPV) HA were generated and tested for cell surface expression and the ability to hemadsorb CD46+ red blood cells (RBC). Exchanges with the N terminus of RPV were tolerated as MV HA could be replaced with RPV HA up to amino-acid position 154. However, both larger swaps with RPV and a small RPV HA replacement at the C terminus aborted cell-surface expression. Peptide scanning with 51 overlapping peptides derived from three MV HA regions showed one peptide, corresponding to MV HA amino acids 468-487, blocked hemagglutination of African green monkey (AGM) RBCs and inhibited MV infection of Chinese hamster ovary cells (CHO) expressing human CD46. Alanine scanning mutants mapped sites on the MV HA that were not required for trafficking to the cell surface or function in hemagglutination as well as a novel site required for CD46 interaction, amino acids 473-477., (Copyright 1999 Academic Press.)