Analysis Of The Interaction Of Measles Virus Hemagglutinin Protein With Its Receptors And Their Roles In MV Infection

Measles virus Hemagglutinin protein (H) is the key molecule mediating MV infection in host cells. CD46 is the first identified MV receptor which specifically interact with H protein. Our data showed that two important amino acid residues in MV H protein govern the binding to CD46 receptor and hemadsorption. The expression of CD46 was not downregulated by the infection of MV strain S-191 both on mRNA level and cellular surface protein level. SLAM (signaling lymphocytic activation molecule) is a newly identified MV receptor. Based on our previous results, we study on the interaction of H and SLAM and further analysis the functional domain on both molecules. A mechanism of MV infection is also suggested.The site-directed mutagenesis of the H genes were performed to confirm that the substitution of Asn -Tyr at position 481 and Ser -Gly at position 546 in the H protein were responsible for hemadsorption alteration. Anti-CD46 monoclonal antibody (M75 and M160) study identified that these two substitutions also governed the MV H protein's interaction with CD46 receptor. Our data showed that two important amino acid residues in MV H protein govern the binding to CD46 receptor and hemadsorption. The novel amino acid residue at position546 in MV H protein, which was critical for hemadsorption and CD46 binding, was firstly reported.Previous research showed that the expression of measles virus receptor CD46 wasdownregulated after expression of measles virus haemagglutinin protein on the surface of the virus infected cell or triggered by infected cell-to-cell contact. We reported here that the mRNA level of CD46 in MV infected cells was not changed which tested by real-time quantitative PCR. To further analysis the surface expression alteration of CD46 after MV infection, flow cytometric analysis and indirect immunofluorescence were used to detect the protein level of CD46. Altogether, our results provided a demonstration that the expression of CD46 was not downregulated by the infection of MV strain S-191 both on mRNA level and cellular surface protein level. Previous results reported the "downregulation" of CD46 expression on cell surface may because H protein masks the antibody recognition site on CD46 which resulting in "downregulation" of the expression of CD46.Several truncated SLAM mutants were constructed and subcloned into yeast two-hybrid vector pGAD424. These truncated mutants were designed to delete transmembrane and intracellular domain, signal peptide domain, retain N-terminal domain, and retain C-terminal domain. After co-transforming each mutant with pGBT-tH, the SD/-His/-Leu/-Trp selective medium growth test and B-galactosidase filter assay showed that all mutants without mutant 1 can interact with H protein in HF7c. These results demonstrated that the transmembrane and intracellular domain of SLAM (amino acids 237-335) and the signal peptide (amino acids 1-26) had no contribution to interact with H since amino acids 27-236 (SLAM mutant 3) showed full activity. The N-terminal part (amino acids 27-135) of SLAM mutant 3, that is SLAM mutant 2, could exert the activity alone while the C-terminal part (amino acids 136-236) of SLAM mutant 3, or SLAM mutant 1, couldn't if without the N-terminal domain. It indicated that the N-terminal domain (amino acids 27-135, or SLAM mutant 2) was essential and enough to interact with H protein.Amino acids 27-135 of SLAM was subcloned into prokaryotic expressing vector pRsetB to expression and purify and used to screen a 10-mer phage display peptide library in this study. After 4 rounds of screening and sequence analysis, the deduced amino acid sequence of screened peptides SGFDPLITHA and SDWDPLFTHK showed highly homologous with amino acid 429-438 of MV H (SGFGPLITHG). Peptides SGFDPLITHA and SDWDPLFTHK specifically inhibited binding of H to SLAM and further inhibit MV infection suggests that these peptides can be developed to MV blocking reagents and amino acid 429-438 in H protein is functionally involved in receptor binding and may constitute part of the receptor-bindin...