Mutational analysis of virus specific amino acids in the fusion active domain of paramyxovirus fusion protein / 中华微生物学和免疫学杂志

ABSTRACT

Objective To identify the effects of virus specific amino acids in the fusion active domains of paramyxovirus fusion proteins on the specific membrane fusion. Methods Site-directed mutagenesis was used to obtain mutants in the identified fusion active domains of Newcastle disease virus (NDV) fusion protein (F) and human parainfluenza virus (hPIV) fusion protein (F). All the mutant F genes were co-expressed with their homol-ogous or heterogenous hemagglutinin-neuraminidase (HN) genes in eukaryocytes. The fusion functions of mutants were assayed by Giemsa staining and reporter gene method. The expression efficiencies of mutants were assayed by fluorescence-activated cell sorter (FACS). Results In the NDV F mutants, N150D-L152D had 46.31% fusion activity of wide type. The fusion activities of N257D-N258D-Q259E, G271D-N272D and Q279E-Q281E almost disappeared, and they had only 1.25%, 3.14% and 2.23% of fusion activities, respectively, compared with wide type. N296D-N297D had 97.68% fusion activity of wide type. In the hPIV F mutants, D143A-E145A had 32.63% fusion activity of wide type. The fusion activity of E223Q-K224A almost disappeared, and it had only 1.91% fusion activity of wide type. K263A-R265A, D268A-D270A and R475A-R476A had 14.63%, 19.52% and 28.95% of fusion activities respectively compared with wild type. The analysis of FACS indicated that proteins of NDV F N257D-N258D-Q259E, G271D-N272D, Q279E-Q281E and hPIV F E223Q-K224A were not expressed on the cell surface, while proteins of the rest mutants were expressed nearly as the same as the wide types. Con-clusion As to NDV F, the amino acids of N257, N258, Q259, G271, N272, Q279 and Q281 were significant to the specific membrane fusion, and N150 and L152 were also important, but N296 and N297 were not. For hPIV F, the amino acids of E223 and K224 were significant to the specific membrane fusion, and D143, E145, K263, 11265, D268, D270, R475 and R476 were also important.