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.
Highlights
Of enveloped viruses requires the selective recruitment of viral components at distinct sites of the host cell membranes from which viruses bud
Our results on transmembrane domain (TMD)-HA clustering in living cells are compatible with recent reports on the localization of HA on the cell surface using immunoelectron microscopy and fluorescence photoactivation localization microscopy (FPALM) [9, 10, 37]
We have no quantitative data, our results show that localization of TMD and CT sequence of HA (TMD-HA) dimers into raft domains reflects the behavior of fulllength HA, at least on the qualitative level
Summary
Of enveloped viruses requires the selective recruitment of viral components at distinct sites of the host cell membranes from which viruses bud. 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. 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]. 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
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