Mutational analysis of conserved amino acids in the receptor-binding domain of human parainfluenza virus type 3 hemagglutinin-neuraminidase protein

Objective To determine the functions of conserved amino acids in the receptor-binding domain of human parainfluenza virus type 3(hPIV3) hemagglutinin-neuraminidase(HN) protein. Methods Using a PCR-based site-directed mutagenesis method and the method of homology recombination occurred in vivo to change seven conservative amino acids into alanine respectively, we named them as R192A, D216A, E409A, R424A, R502A, Y530A and E549A. Wild type (wt) and all mutant HN proteins were expressed on the cell surface of BHK-21 cells. Protein folding and processing, cell surface expression, cell fusion efficiency, receptor binding activity and neuraminidase activity were determined. Results WT and each mutant HN protein were folded and processed efficiently and expressed on the cell surface of BHK-21 cells. There was no statistic difference of cell surface expression between WT and each mutant HN protein. Cell fusion efficiency of each mutant protein decreased to some extent, especially R502A to 14.2%. The binding guinea pig erythrocytes activity of each mutant protein was corresponding to its binding human erythrocytes activity. There was different neuraminidase activity among each mutant HN protein. R502A decreased most to 18.6%, but the neuraminidase activity of E549A was similar to that of WT hPIV3 HN (94.9%). Conclusion Conserved amino acids in the receptor-binding domain of hPIV3 HN protein play an important role in cell fusion. R502 is a key amino acid. Key words: Human parainfluenza virus type 3; HN protein; Cell fusion; Hemadsorption; Neuraminidase