Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41.

Abstract

The human immunodeficiency virus transmembrane glycoprotein gp4l has at its amino terminus a strongly hydrophobic stretch of 28 amino acids flanked by a highly conserved series of polar amino acids. To investigate the role in syncytium formation of the hydrophobic amino terminus of gp4l and the polar border of this hydrophobic region, we introduced eight single-amino acid substitutions and one double-amino acid substitution in the amino-terminal 31 amino acids of gp4l. The mutant envelope glycoproteins were expressed from two distinct human immunodeficiency virus type 1 envelope glycoprotein expression vectors; the effects of the mutations on syncytium formation, envelope glycoprotein transport, secretion, and CD4 receptor-binding were analyzed. Results showed that polar substitutions throughout the hydrophobic amino terminus of gp4l greatly reduced or blocked syncytium formation mediated by the human immunodeficiency virus type 1 envelope glycoproteins, as did nonconservative mutations in the polar border of the hydrophobic amino terminus. Mutations at gp4l amino acids 15, 26, and 29 also significantly increased the extent of gpl20 secretion into the extracellular medium. None of the mutations detectably affected envelope glycoprotein processing or envelope glycoprotein binding to CD4.Enveloped viruses, under appropriate conditions, induce cell fusion, or syncytium formation, upon infection of permissive cell types. Early descriptions of such virus-induced cell fusion involved the orthomyxoviruses and paramyxoviruses (1-3), and one of the early retroviral plaque assays depended upon the ability of murine leukemia viruses to induce syncytium formation in the XC cell line (4). Later work indicated that expression of the orthomyxovirus envelope proteins was necessary and sufficient to induce cell fusion (5). More recently, syncytium formation has been described as a characteristic of human immunodeficiency virus (HIV) infection of CD4+ cells, and, as demonstrated for the orthomyxoviruses and paramyxoviruses (3), the envelope glycoproteins have been shown to be responsible for this cell-fusion property (6, 7). The syncytium formation induced by HIV leads to cell death in culture and may contribute to the depletion of CD4+ helper T cells in AIDS patients (6-10). Membrane fusion is also an essential step in the infection process (11).HIV envelope glycoproteins, like those of other retroviruses, are synthesized as a polyprotein precursor that is cleaved to generate the surface and transmembrane (TM) envelope proteins (12,13 (17,18). Because of their role in fusion, the hydrophobic amino-terminal regions of the orthomyxovirus and paramyxovirus TM proteins were termed "fusion peptides." Several studies suggested that the analogous hydrophobic regions at the amino termini of retroviral TM envelope proteins were also involved in the cell-fusion properties of these proteins. Linker insertion mutations introduced after AA 10 of the HIV-1 gp41 glycoprotein blocked cell fusion (19), as did point mutations at positions...

Bimodal down-regulation of CD4 in cells expressing human immunodeficiency virus type 1 Vpu and Env

“…The HIV sequence in pKF1 was derived from the pNL4-3 molecular clone (Adachi et al, 1986). A StuIBamHI fragment from the env region of pKFI was replaced with the corresponding restriction fragment from pHenv41.2; this is derived from the BH10 clone of HIV-1, which has a Val to Glu mutation at the second amino acid of gp41 (Freed et al, 1990). The resulting pKF41.2 retroviral vector was used to transduce HeLa-CD4 cells.…”

Synthesis, Enhanced Fusogenicity, and Solid State NMR Measurements of Cross-Linked HIV-1 Fusion Peptides

“…The free peptide causes fusion of liposomes and erythrocytes, and numerous mutational studies have shown strong correlations between fusion peptide-induced liposome fusion and viral/host cell fusion (5,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide to Lipid Distances Reveal the Intimate Contact of β Strand Peptide with Membranes and the Proximity of the Ala-14−Gly-16 Region with Lipid Headgroups

“…The ~170-residue ectodomain of gp41 lies outside HIV and is subdivided into a more C-terminal "soluble ectodomain" and a ~20-residue N-terminal fusion peptide (HFP) which is apolar and fairly conserved. The HFP is believed to interact with the target cell membrane after gp120 binds to cellular receptors and fusion is greatly disrupted by mutation or deletion of the HFP (10)(11)(12)(13).…”