Quantitative measurement of paramyxovirus fusion: differences in requirements of glycoproteins between simian virus 5 and human parainfluenza virus 3 or Newcastle disease virus.
To compare the requirements for paramyxovirus-mediated cell fusion, the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of simian virus 5 (SV5), human parainfluenza virus 3 (HPIV-3), and Newcastle disease virus (NDV) were expressed individually or coexpressed in either homologous or heterologous combinations in CV-1 or HeLa-T4 cells, using the vaccinia virus-T7 polymerase transient expression system. The contribution of individual glycoproteins in virus-induced membrane fusion was examined by using a quantitative assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecyl rhodamine (R18) and a quantitative assay for content mixing based on the cytoplasmic activation of a reporter gene, beta-galactosidase. In these assays, expression of the individual F glycoproteins did not induce significant levels of cell fusion and no cell fusion was observed in experiments when cells individually expressing homologous F or HN proteins were mixed. However, coexpression of homologous F and HN glycoproteins resulted in extensive cell fusion. The kinetics of fusion were found to be very similar for all three paramyxoviruses studied. With NDV and HPIV-3, no cell fusion was detected when F proteins were coexpressed with heterologous HN proteins or influenza virus hemagglutinin (HA). In contrast, SV5 F protein exhibited a considerable degree of fusion activity when coexpressed with either NDV or HPIV-3 HN or with influenza virus HA, although the kinetics of fusion were two- to threefold higher when the homologous SV5 F and HN proteins were coexpressed. Thus, these data indicate that among the paramyxoviruses tested, SV5 has different requirements for cell fusion.
D000818:Animals; D000911:Antibodies, Monoclonal; D002459:Cell Fusion; D002460:Cell Line; D004912:Erythrocytes / Q000502:physiology; D015707:HN Protein / Q000096:biosynthesis / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008561:Membrane Fusion; D009522:Newcastle disease virus / Q000502:physiology*; D010224:Parainfluenza Virus 3, Human / Q000502:physiology*; D018108:Respirovirus / Q000502:physiology*; D013045:Species Specificity; D014162:Transfection; D014616:Vaccinia virus; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000378:metabolism*; D001616:beta-Galactosidase / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D015707:HN Protein; D014759:Viral Envelope Proteins; D001616:beta-Galactosidase