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Abstract
BackgroundThe gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP) that is only active in the cytoplasm. The tag protein (HaloTag) and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system.ResultsIn the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells) supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells), the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability.ConclusionsIt is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.
Highlights
The gp41 subunit of the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD)
green fluorescent protein (GFP) is located in the cytoplasm it folds into an active form, whereas when it is translocated into the periplasm it is non-functional [31]
These data suggested that there was no topological shift of GFP reporter in these regions; the Kennedy sequence and lentiviral lytic peptide (LLP) regions are not exposed to the periplasmic region
Summary
The gp subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). The transmembrane protein gp mediates the membrane fusion between the host and viral membranes. It is composed of an ectodomain (extracellular domain), a cytoplasmic domain, and a. The transmembrane domain of gp was first deduced from the hydropathy plot of Env as a hydrophobic domain [10] This transmembrane domain, referred to as the membrane-spanning domain (MSD), is composed of 23 highly conserved amino acid residues corresponding to amino acid residues 684 to 706 in the HXB2 strain (Figure 1A, B). The transmembrane portion of the single MSD model is expected to cross the membrane twice and one of LLPs, LLP2, is a putative third MSD (Figure 1C)
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