Role of the cytoplasmic domain of the Newcastle disease virus fusion protein in association with lipid rafts.

To explore the association of the Newcastle disease virus (NDV) fusion (F) protein with cholesterol-rich membrane domains, its localization in detergent-resistant membranes (DRMs) in transfected cells was characterized. After solubilization of cells expressing the F protein with 1% Triton X-100 at 4 degrees C, ca. 40% of total, cell-associated F protein fractionated with classical DRMs with densities of 1.07 to l.14 as defined by flotation into sucrose density gradients. Association of the F protein with this cell fraction was unaffected by the cleavage of F(0) to F(1) and F(2) or by coexpression of the NDV attachment protein, the hemagglutinin-neuraminidase protein (HN). Furthermore, elimination by mutation, of potential palmitate addition sites in and near the F-protein transmembrane domain had no effect on F-protein association with DRMs. Rather, specific deletions of the cytoplasmic domain of the F protein eliminated association with classical DRMs. Comparisons of deletions that affected fusion activity of the protein and deletions that affected DRM association suggested that there is no direct link between the cell-cell fusion activity of the F protein and DRM association. Furthermore, depletion of cholesterol from cells expressing F and HN protein, while eliminating DRM association, had no effect on the ability of these cells to fuse with avian red blood cells. These results suggest that specific localization of the F protein in cholesterol-rich membrane domains is not required for cell-to-cell fusion. Paramyxovirus F-protein cytoplasmic domains have been implicated in virus assembly. The results presented here raise the possibility that the cytoplasmic domain is important in virus assembly at least in part because it directs the protein to cholesterol-rich membrane domains.