Oligomeric structure of gp41, the transmembrane protein of human immunodeficiency virus type 1 (original) (raw)

Analysis of a highly immunodominant epitope in the human immunodeficiency virus type 1 transmembrane glycoprotein, gp41, defined by a human monoclonal antibody

Journal of Virology, 1990

A human monoclonal antibody, 41-7 [immunoglobulin G1(kappa)], directed against the transmembrane glycoprotein gp41 of the human immunodeficiency virus type 1 (HIV-1) has been produced by direct fusion of lymph node cells from an HIV-1-infected individual with a human B-lymphoblastoid cell line. The minimal essential epitope for 41-7 was mapped to a conserved seven-amino acid sequence, N-CSGKLIC-C, located within the N-terminal part of gp41. Antibodies blocking the binding of 41-7 could be detected in the serum of all HIV-1-infected individuals tested, irrespective of the stage of the infection. The epitope is located externally to the plasma membrane, and it is accessible to antibody in the native conformation of the glycoprotein. Despite this, no neutralizing activity of 41-7 could be demonstrated in vitro. These data indicate, directly and indirectly, that this immunodominant epitope on gp41, although exposed on the viral surface, elicits antibodies lacking antiviral activity and,...

A region of the C-terminal tail of the gp41 envelope glycoprotein of human immunodeficiency virus type 1 contains a neutralizing epitope: evidence for its exposure on the surface of the virion

Journal of General Virology, 2003

The ∼150 amino acid C-terminal tail of the gp41 transmembrane glycoprotein of human immunodeficiency virus type 1 (HIV-1) is generally thought to be located inside the virion. However, we show here that both monoclonal IgG and polyclonal epitope-purified IgG specific for the746ERDRD750epitope that lies within the C-terminal tail neutralized infectious virus. IgG was mapped to the C-terminal tail by its failure to neutralize tail-deleted virus, and by sequencing of antibody-escape mutants. The fact that antibody does not cross lipid membranes, and infectious virus is by definition intact, suggested that ERDRD was exposed on the surface of the virion. This was confirmed by reacting virus and IgG, separating virus and unbound IgG by centrifugation, and showing that virus was neutralized to essentially the same extent as virus that had been in constant contact with antibody. Epitope exposure on virions was independent of temperature and therefore constitutive. Monoclonal antibodies spec...

Mutational Analysis of Conserved Domains within the Cytoplasmic Tail of gp41 from Human Immunodeficiency Virus Type 1: Effects on Glycoprotein Incorporation and Infectivity

Journal of Virology, 2000

The transmembrane (TM) glycoprotein gp41 of human immunodeficiency virus type 1 possesses an unusually long (∼150 amino acids) and highly conserved cytoplasmic region. Previous studies in which this cytoplasmic tail had been deleted partially or entirely have suggested that it is important for virus infectivity and incorporation of the gp120-gp41 glycoprotein complex into virions. To determine which regions of the conserved C-terminal domains are important for glycoprotein incorporation and infectivity, several small deletions and amino acid substitutions which modify highly conserved motifs were constructed in the infectious proviral background of NL4.3. The effects of these mutations on infectivity and glycoprotein incorporation into virions produced from transfected 293-T cells and infected H9 and CEM×174 cells were determined. With the exception of a mutation deleting amino acids QGL, all of the constructs resulted in decreased infectivity of the progeny virus both in a single-r...

The extracellular domain of immunodeficiency virus gp41 protein: Expression in Escherichia coli , purification, and crystallization

Protein Science, 1997

The env gene of SIV and HIV-I encodes a single glycoprotein gp160, which is processed to give a noncovalent complex of the soluble glycoprotein gp120 and the transmembrane glycoprotein gp41. The extracellular region (ectodomain), minus the N-terminal fusion peptide, of gp41 from HIV-1 (residues 27-154) and SIV (residues 27-149) have been expressed in Escherichia coli. These insoluble proteins were solubilized and subjected to a simple purification and folding scheme, which results in high yields of soluble protein. Purified proteins have a trimeric subunit composition and high a-helical content, consistent with the predicted coil-coil structure. SIV gp41 containing a double cysteine mutation was crystallized. The crystals are suitable for X-ray structure determination and, preliminary analysis, together with additional biochemical evidence, indicates that the gp41 trimer is arranged as a parallel bundle with threefold symmetry.

Effects of amino acid changes in the extracellular domain of the human immunodeficiency virus type 1 gp41 envelope glycoprotein

Journal of Virology, 1993

Changes were introduced into conserved amino acids within the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein. The effect of these changes on the structure and function of the HIV-1 envelope glycoproteins was examined. The gp41 glycoprotein contains an amino-terminal fusion peptide (residues 512 to 527) and a disulfide loop near the middle of the extracellular domain (residues 598 to 604). Mutations affecting the hydrophobic sequences between these two regions resulted in two phenotypes. Some changes in amino acids 528 to 562 resulted in a loss of the noncovalent association between gp41 and the gp120 exterior glycoprotein. Amino acid changes in other parts of the gp41 glycoprotein (residues 608 and 628) also resulted in subunit dissociation. Some changes affecting amino acids 568 to 596 resulted in envelope glycoproteins partially or completely defective in mediating membrane fusion. Syncytium formation was more sensitive than ...

Evidence against Extracellular Exposure of a Highly Immunogenic Region in the C-Terminal Domain of the Simian Immunodeficiency Virus gp41 Transmembrane Protein

Journal of Virology, 2012

The generally accepted model for human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein topology includes a single membrane-spanning domain. An alternate model has been proposed which features multiple membrane-spanning domains. Consistent with the alternate model, a high percentage of HIV-1-infected individuals produce unusually robust antibody responses to a region of envelope, the so-called "Kennedy epitope," that in the conventional model should be in the cytoplasm. Here we show analogous, robust antibody responses in simian immunodeficiency virus SIVmac239-infected rhesus macaques to a region of SIVmac239 envelope located in the C-terminal domain, which in the conventional model should be inside the cell. Sera from SIV-infected rhesus macaques consistently reacted with overlapping oligopeptides corresponding to a region located within the cytoplasmic domain of gp41 by the generally accepted model, at intensities comparable to those observed for immunodominant areas of the surface component gp120. Rabbit serum raised against this highly immunogenic region (HIR) reacted with SIV envelope in cell surface-staining experiments, as did monoclonal anti-HIR antibodies isolated from an SIVmac239infected rhesus macaque. However, control experiments demonstrated that this surface staining could be explained in whole or in part by the release of envelope protein from expressing cells into the supernatant and the subsequent attachment to the surfaces of cells in the culture. Serum and monoclonal antibodies directed against the HIR failed to neutralize even the highly neutralization-sensitive strain SIVmac316. Furthermore, a potential N-linked glycosylation site located close to the HIR and postulated to be outside the cell in the alternate model was not glycosylated. An artificially introduced glycosylation site within the HIR was also not utilized for glycosylation. Together, these data support the conventional model of SIV envelope as a type Ia transmembrane protein with a single membrane-spanning domain and without any extracellular loops.