| Abstract: | Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy to study the interaction of the cytoplasmic and transmembrane domain (TMD) of HA with agly co sylphos pha tidyl ino si tol (GPI)-anchored peptide, an established marker for rafts in the exoplasmic leaflet of living mammalian plasma membranes. Cyan fluorescent protein (CFP) was fused to GPI, whereas the HA sequence was tagged with yellow fluorescent protein (YFP) on its exoplasmic site (TMD-HA-YFP), avoiding any interference of fluorescent proteins with the proposed role of the cytoplasmic domain in lateral organization of HA. Constructs were expressed in Chinese hamster ovary cells (CHO-K1) for which cholesterol-sensitive lipid nanodomains and their dimension in the plasma membrane have been described (Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and Mayor, S. (2004) Cell 116, 577–589). Upon transfection in CHO-K1 cells, TMD-HA-YFP is partially expressed as a dimer. Only dimers are targeted to the plasma membrane. Clustering of TMD-HA-YFP with GPI-CFP was observed and shown to be reduced upon cholesterol depletion, a treatment known to disrupt rafts. No indication for association of TMD-HA-YFP with GPI-CFP was found when palmitoylation, an important determinant of raft targeting, was suppressed. Clustering of TMD-HA-YFP and GPI-CFP was also observed in purified plasma membrane suspensions by homoFRET. We concluded that the pal mit oy lated TMD-HA alone is sufficient to recruit HA to cholesterol-sensitive nanodomains. The corresponding construct of the spike protein E2 of Semliki Forest virus did not partition preferentially in such domains.Assembly of enveloped viruses requires the selective recruitment of viral components at distinct sites of the host cell membranes from which viruses bud. One of the most intensely studied enveloped viruses with respect to assembly is the influenza virus, in which budding takes place at the plasma membrane of epithelial cells. Three membrane proteins are embedded in the influenza virus envelope: hemagglutinin (HA),3 which mediates binding of the virus to the host cell and fusion with cell target membrane (1); neuraminidase; and the proton channel M2. The inner viral membrane leaflet is covered by the matrix protein M1, which is supposed to mediate binding of the eight viral RNA-nucleoprotein complexes harboring the genetic information of the virus. Several studies support a role of lipid domains as a platform for enrichment of viral components. HA, the most abundant envelope protein of the influenza virus, has been found to be enriched in detergent-resistant membrane (DRM) fractions (2–4). Typical lipid components of those fractions are saturated phospholipids, glycosphingolipids, and cholesterol, which are known to form liquid-ordered domains (5). This has led to the idea that so-called lipid rafts, which resemble liquid-ordered domains, could function as assembly sites. Support for this hypothesis was given by the observation that the lipid composition of the influenza virus envelope is more similar to that of a raft than to the overall plasma membrane (2, 6).As it has been shown that DRM fractions may not represent the native state of lipid domains, in particular of rafts (7, 8), subsequent efforts have focused on other techniques to assess the lateral organization of HA. Electron microscopy studies using immunogold labeling (4, 9), Förster resonance energy transfer (FRET) measurements between fluorescent HA antibodies in fixed cells (9), and investigations on photoactivatable HA in living cells (10) have revealed cholesterol-sensitive clustering of HA in the plasma membrane of mammalian cells at lengths between 20 and 900 nm.A specific problem encountered in studying the lateral organization of proteins in the plasma membrane is that lipid domains as rafts are typically organized at a submicroscopic level. Indeed, several attempts to image raft domains in biological membranes have suggested that rafts are very small and highly dynamic (11, 12). A guiding study in the characterization of lipid domains in biological membranes has been performed by Mayor and colleagues (13) on the plasma membrane of CHO-K1 cells. Based on homoFRET measurements they have shown that about 20–40% of GFP-tagged glycosylphosphatidylinositol (GPI) (for review see Brown and Rose (14)) and other GPI-anchored proteins are organized with about three to four copies in small cholesterol-sensitive clusters. Mathematical modeling of those experimental data is consistent with a domain diameter of about 5 nm (13).In the present study we investigated the lateral organization of the C terminus of HA, corresponding to the transmembrane domain (TMD), and the cytoplasmic tail (CT) of the protein in the plasma membrane of CHO-K1 cells, taking advantage of the well characterized spatial arrangement of the raft marker GPI in those cells (see above). Lateral organization was studied essentially by fluorescence lifetime imaging microscopy (FLIM)-based FRET between CFP (donor) and YFP (acceptor). For this purpose, we replaced the ectodomain of HA by YFP and studied FRET between this construct and GPI-CFP. As a complementary approach, we performed ensemble measurements in suspensions of plasma membranes purified from cells expressing fluorescent GPI and HA constructs. We measured homoFRET by time-resolved fluorescence anisotropy, providing information on the aggregation/clustering state of the fluorescent constructs, which is important in rationalizing the FLIM-FRET data. |
