The NDV F protein expressed in the absence of other viral proteins localizes to a DRM fraction of cells, a cell fraction thought to represent lipid raft association in cells. That F protein is associated with DRMs was shown by the flotation of F protein into a sucrose density gradient after cell disruption with Triton X-100 at 4°C into fractions that also contain the ganglioside GM1, a component of lipid rafts. Furthermore, the flotation of F protein into these gradients was inhibited by conditions known to disrupt lipid raft domains and DRMs: cholesterol depletion, solublization of cells with octylglucoside, and solubilization of cells with Triton X-100 at room temperature (36
). These results confirm the association of F protein with DRMs with densities of l.07 to 1.14.
Because NDV F protein expressed in the absence of other viral proteins is localized in DRMs, some intrinsic property of the F protein itself is likely to be responsible for this localization. With the goal of determining the functional significance of raft association, we sought to determine what property of the F protein was responsible for DRM localization.
Paramyxovirus F proteins, synthesized as a precursor F0
, must be proteolytically cleaved for fusion activity, and this cleavage is reported to result in significant conformational changes in the F protein (12
). However, cleavage had no role in DRM association. A point mutation in the F-protein cleavage site that eliminated cleavage of the molecule had little effect on DRM localization of the protein. Furthermore, coexpression of F protein with HN protein, which is also reported to result in conformational differences in the F protein (14
), had no effect on F-protein localization in DRMs. Similarly, coexpression of the measles virus attachment protein with measles virus F protein had no effect on the F-protein association with DRMs (18
Many proteins associated with lipid rafts are modified by covalent addition of the fatty acid palmitate, and the DRM association of many of these proteins is attributed to this modification (23
). This saturated fatty acid likely has a preference for the liquid-ordered lipids characteristically found in lipid rafts. Indeed, the DRM association of several viral glycoproteins can be prevented by eliminating this modification. Mutations of palmitate addition sites in the HIV Env protein (29
), the murine leukemia virus Env protein (15
), influenza virus HA (23
), and the Ebola virus glycoprotein(3
) all eliminated their DRM associations. The F proteins of many paramyxoviruses, including the F protein of NDV (8
), are modified by palmitate. However, mutation of the candidate palmitoylation sites in the F protein had no effect on localization of the fully glycosylated protein in DRMs.
Mutations in the CT domain of the NDV F protein did, however, significantly affect localization with DRMs with densities of l.07 to 1.14 g/ml. Deletion of the entire domain (31 amino acids) (d523-553) or deletion of the most carboxyl-terminal 14 amino acids (d540-553) virtually eliminated classical DRM association, whereas deletion of the last seven amino acids (d547-553) had little effect on DRM association. This result might indicate that amino acids 540 to 547 are crucial. However, deletion of only seven amino acids (d540-546) was not sufficient to disrupt association with classical DRMs. Furthermore, the deletion mutant d525-531, which contains the seven amino acids from 540 to 547, was defective in association with these DRMs. These results argue that neither a specific linear sequence nor a specific length is critical for DRM association. Rather, a conformational determinant of the CT domain may be important for this property of the F protein, a determinant disrupted by the loss of the last 13 amino acids or by the loss of 6 amino acids near the TM domain. Alternatively, mutations in the CT domain of the F protein may have an indirect role in classical DRM localization due to effects on the conformation of other regions in the protein. However, with the exception of d523-553, which is poorly cleaved, no structural defects have been detected in any of the CT mutant proteins. Indeed, all are normally cleaved, and all are precipitated with a conformation-specific monoclonal antibody (35
). Thus, these results are most consistent with the notion that the conformation of the CT domain itself is important for classical DRM localization and that mutations that affect this conformation inhibit DRM association. For instance, this domain may interact with host proteins or lipids specific to the intracellular side of lipid rafts.
The percentage of total cell-associated NDV F protein in the DRM fraction of cells prepared as we have described above was quite similar to that reported for the measles virus F protein (18
). It is interesting that only a fraction of total measles virus or NDV F proteins are found in DRMs. It is possible that paramyxovirus F proteins are not tightly associated with this cell fraction. Indeed, increased amounts of Sendai F protein were found in classical DRMs after solubilization in lower concentrations of Triton X-100 (1
). Alternatively, F protein may be in two different populations or two different domains on cell surfaces. Indeed, cell surface F protein participates in two different pathways, virion assembly and cell-cell fusion (14
). We have previously reported that mutations in the CT domain of the NDV F protein can affect cell-cell fusion as measured by syncytium formation (35
). To determine the role of lipid raft association in cell-cell fusion, we compared the fusion activities of two of these mutants, d540-546 and d525-531. Fusion activities of the mutant proteins were measured by syncytium formation as well as content mixing. Our results, presented here as well as previously (35
), showed no obvious correlation with classical DRM association and fusion activity. F-d525-531 mutant protein is not associated with these DRMs but has approximately half the syncytium-forming activity and nearly 60% the content mixing activity of wild-type protein. In contrast, mutant F-d540-546 is associated with DRMs at nearly wild-type levels but has lower fusion activities than F-d525-531. Thus, the lipid raft association of the F protein does not appear to be directly correlated with cell-cell fusion or syncytium formation. Our finding that cholesterol depletion of cells expressing HN and F proteins fused normally to avian RBC supports this conclusion.
The lipid raft association directed by the F-protein CT domain may, however, be important for virus assembly, as has been suggested in numerous virus systems (reviewed in reference 4
). In several systems, it has been reported that mutations of F-protein CT domains affected virus assembly (7
). In other studies, F proteins are reported to associate with lipid raft domains (1
) as we report here. Our results raise the possibility that the paramyxovirus F-protein CT domain may be involved in virus assembly at least in part because it directs the protein to lipid raft domains, a possibility currently under investigation.
In summary, we have found that the NDV F protein is localized in classical DRMs, i.e., DRMs with a density of 1.07 to 1.14. Furthermore, the CT domain of the F protein is important for association with these DRMs. The association of F protein with heavy-density DRMs, as previously defined in neutrophils (25
), is currently under investigation.