Cellular membrane-binding ability of the C-terminal cytoplasmic domain of human immunodeficiency virus type 1 envelope transmembrane protein gp41

The amphipathic a-helices located in the cytoplasmic tail of the envelope ( Env) transmembrane glycoprotein gp41 of human immunodeficiency virus type I have been implicated in membrane association and cytopathicity. Deletion o f the last 12 amino acids in the C terminus of this domain severely impairs infectivity. However, the nature of the involvement of the cytoplasmic tai l in Env-membrane interactions in cells and the molecular basis for the def ect in infectivity of this mutant virus are still poorly understood. In thi s study we examined the interaction of the cytoplasmic tail with membranes in living mammalian cells by expressing a recombinant cytoplasmic tail frag ment and an Escherichia coli beta -galactosidase/cytoplasmic tail fusion pr otein, both of them lacking gp120, the gp41 ectodomain, and the transmembra ne region. We found through cell fractionation, in vivo membrane flotation, and confocal immunofluorescence studies that the cytoplasmic tail containe d determinants to be routed to a perinuclear membrane region in cells. Furt her mapping showed that each of the three lentivirus lytic peptide (LLP-1, LLP-2, and LLP-3) sequences conferred this cellular membrane-targeting abil ity. Deletion of the last 12 amino acids from the C terminus abolished the ability of the LLP-1 motif to bind to membranes. High salt extraction, in v itro transcription and translation, and posttranslational membrane binding analyses indicated that the beta -galactosidase/LLP fusion proteins were in serted into membranes via the LLP sequences. Subcellular fractionation and confocal microscopy studies revealed that each of the LLP motifs, acting in a position-independent manner, targeted non-endoplasmic reticulum (ER)-ass ociated beta -galactosidase and enhanced green fluorescence protein to the ER. Our study provides a basis for the involvement of the gp41 cytoplasmic tail during Env maturation and also supports the notion that the membrane a pposition of the C-terminal cytoplasmic tail plays a crucial role in virus- host interaction.