Project:  Lipid-raft association of the HIV glycoprotein gp41.
                    A Fluorescence Lifetime Imaging Microscopy study of protein co-clustering in plasma-membrane microdomains.  

Roland Schwarzer 

The HIV envelope protein complex mediates host cell infection by binding cellular receptors and later on triggering membrane fusion. Hitherto, little is known about the role of those proteins during assembly and budding of the newly synthesized virus particles. It has been reported that membranes of HIV-infected cells as well as virions are enriched in cholesterol, hence it was suggested that HIV-1 may take advantage of cholesterol and sphingolipids during budding. Both findings point to an important function of lipid microdomains, the so called rafts, in the late virus lifecycle

Figure 1: Schematic presentation of suggested plasma membrane compartmentation (from website of the National Institute of General Medical Sciences.)	Figure 2: Fluorescence Lifetime (FLIM) image of Chinese Hamster Ovary cells transfected with fluorescent HIV gp41 and marker proteins to detect protein clustering and raft localization via FRET.

This work focuses on the transmembrane part of the viral spike protein complex formed by the envelope proteins, the glycoprotein gp41. We apply Fluorescence Lifetime Imaging Microscopy (FLIM) to detect Förster Resonance Energy Transfer (FRET) between a raft marker, a GPI-anchored cyan fluorescent protein (CFP), and gp41 fusion proteins labeled with yellow fluorescent proteins (YFP) to elucidate raft clustering. Since energy transfer is highly dependent on the distance between the participating molecules, efficient FRET can be considered as a strong indication for close proximity of raft marker and fusion proteins and, therefore for colocalization in lipid microdomains. By combining obtained FRET data with acceptor fluorescence intensity analysis this method provides reliable clustering information independent of expression level and fluorophore concentration. Several gp41 chimera were produced to address the role of different protein domains for raft association but also intracellular distribution and trafficking. The impact of truncations of the cytoplasmic tail as well as mutations of, the cholesterol recognition amino acid consensus (CRAC) domain, intrinsic trafficking signals and a palmitoylation site were studied in this context.

Results:

Wildtype and several truncated gp41 constructs show FRET upon co-expression with the raft marker indicating co-clustering in plasma membrane microdomains. The influence of protein palmitoylation and the CRAC domain on this raft-affinity will be further investigated.

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