Project:  Lateral Sorting of Influenza Virus Hemagglutinin in Membranes  

Silvia Scolari 

Hemagglutinin (HA) is a homotrimeric glycoprotein embedded in the envelope of Influenza virus. It mediates binding of the virus to the host cell as well as fusion between the viral envelope and the endosomal membrane. HA is supposed to entrap in Rafts reflecting liquid-ordered lipid domains enriched of sphingomyelin and cholesterol. In order to elucidate the role of the HA transmembrane domain in lipid raft localization we expressed constructs harboring the transmembrane domain and the cytoplasmic tail but lacking the N-terminal ectodomain of HA in the plasma membrane of mammalian cells (CHO-K1). The N-terminus of the transmembrane domain was tagged with YFP (HA-YFP). We studied Foerster’s energy transfer (FRET) between the artificial HA-YFP and a GPI anchored CFP as a raft marker by fluorescence lifetime imaging microscopy (FLIM) (Fig.1).

First results suggest that HA constructs are indeed sorted and enriched into cholesterol dependent lipid domains indicated by enhanced FRET efficiency. This is supported by the observation that cholesterol depletion of the plasma membrane caused a significant decrease of FRET. Likewise, deletion of the three highly conserved palmitoylation sites of HA is also accompanied by a reduction of FRET efficiency. Taken together, the results are in agreement with sorting of HA constructs into cholesterol-enriched lipid domains.

Fig.1: Principle of FRET measurements between yfp-tagged Influenza HA transmembrane domain and a cfp-bound raft marker.

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