Cellular Membrane-Binding Ability of the C-Terminal Cytoplasmic Domain of Human Immunodeficiency Virus Type 1 Envelope Transmembrane Protein gp41

Abstract

The amphipathic ␣-helices located in the cytoplasmic tail of the envelope (Env) transmembrane glycoprotein gp41 of human immunodeficiency virus type 1 have been implicated in membrane association and cytopathicity. Deletion of the last 12 amino acids in the C terminus of this domain severely impairs infectivity. However, the nature of the involvement of the cytoplasmic tail in Env-membrane interactions in cells and the molecular basis for the defect in infectivity of this mutant virus are still poorly understood. In this study we examined the interaction of the cytoplasmic tail with membranes in living mammalian cells by expressing a recombinant cytoplasmic tail fragment and an Escherichia coli ␤-galactosidase/cytoplasmic tail fusion protein, both of them lacking gp120, the gp41 ectodomain, and the transmembrane region. We found through cell fractionation, in vivo membrane flotation, and confocal immunofluorescence studies that the cytoplasmic tail contained determinants to be routed to a perinuclear membrane region in cells. Further mapping showed that each of the three lentivirus lytic peptide (LLP-1, LLP-2, and LLP-3) sequences conferred this cellular membrane-targeting ability. 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 vitro transcription and translation, and posttranslational membrane binding analyses indicated that the ␤-galactosidase/LLP fusion proteins were inserted 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)-associated ␤-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 apposition of the C-terminal cytoplasmic tail plays a crucial role in virus-host interaction.The cytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) envelope (Env) transmembrane (TM) glycoprotein gp41 has multiple functions in the virus life cycle. Mutations, deletions, and truncations in this region may affect virus replication, infectivity, cytopathicity, Env incorporation into virions, cell type-dependent Env stability, and interaction with the viral matrix (MA) protein. A deletion of 144 amino acids, which comprise most of the cytoplasmic tail, from the C terminus of gp41 does not affect virus infectivity, Env assembly into virions, and cytopathogenicity in MT-4 cells (66). The differential virus infectivity of this mutant in permissive cells (MT-4 and M8166) and nonpermissive cells (most T-cell lines and primary cells) can be attributed to the differential requirement of the cytoplasmic tail to incorporate gp120 into virions in different cell types (1, 46). We previously reported that an Env mutant of HIV-1 lacking the whole cytoplasmic tail and the last two amino acids in the TM region can tra...

Photoinduced Reactivity of the HIV-1 Envelope Glycoprotein with a Membrane-Embedded Probe Reveals Insertion of Portions of the HIV-1 Gp41 Cytoplasmic Tail into the Viral Membrane

“…Inhibition of the protease activity encoded in the virus prevents the cleavage of p55 and has been shown to regulate fusion (22,23). Studies with peptides derived from two α helical "lentivirus lytic peptide" domains (LLP-1 and LLP-2) that are highly conserved in HIV-1 have suggested that these domains strongly interact with the cytoplasmic leaflet of plasma membrane (47)(48)(49)(50). The INA-labeling data presented in this study are consistent with this model (Figures 1-3).…”

Effect of Extension of the Cytoplasmic Domain of Human Immunodeficiency Type 1 Virus Transmembrane Protein gp41 on Virus Replication

“…The three highly conserved amphipathic ␣-helical segments, designated lentiviral lytic peptide 1 (LLP)-1, LLP-2, and LLP-3, located in the C terminus of the cytoplasmic tail are thought to be associated with the inner surfaces of viral and cellular membranes (33,45). We previously showed that the multimerization potential and membrane binding ability of the cytoplasmic tail may play a crucial role in virus replication (3,5,7,28) and that the N-terminal segment of LLP-1 contains a structural determinant critical for modulating Env stability (27).…”

Surface Exposure of the HIV-1 Env Cytoplasmic Tail LLP2 Domain during the Membrane Fusion Process

“…The HIV-1 gp41 CT consists of the "Kennedy" polypeptide sequence (aa 731-752) and the lentivirus lytic peptide (LLP1-2, aa 773-862), which contains three highly conserved ␣-helix domains as follows: LLP1 (aa 833-862), LLP2 (aa 773-793), and LLP3 (aa785-807) (11). The HIV-1 gp41 CT has many functions, including interaction with the plasma membrane, decrease of bilayer stability, alteration of membrane ionic permeability, and mediation of cell killing (10,(12)(13)(14)(15)(16). Recent studies have shown that the gp41 CTs of HIV and simian immunodeficiency virus correlate with their infectivity and Env-mediated cell-cell fusion (10,15).…”