Peptides corresponding to the heptad repeat motifs in the transmembrane protein (gp41) of human immunodeficiency virus type 1 elicit antibodies to receptor-activated conformations of the envelope glycoprotein.

Two heptad repeat regions in the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane subunit (gp41) self-assemble into a six-helix bundle structure that is critical for virus entry. Immunizations with peptides corresponding to these regions generated antibodies specific to the receptor-activated conformations of gp41.

Structure-function studies of the self-assembly domain of the human immunodeficiency virus type 1 transmembrane protein gp41.

The coiled-coil region of the human immunodeficiency virus type 1 transmembrane protein (gp41) makes up the interior core of the six-helix bundle structure of the gp41 self-assembly domain. We extended our previous study of this domain (Y. Weng and C. D. Weiss, J. Virol. 72:9676-9682, 1998) by analyzing 23 additional mutants at positions that lie at the interface of the interior core and outer helices. We found nine new functional mutants. For most mutants, the activity could be explained by the ability of the modeled mutants to stabilize the six-helix bundle structure. The present study provides insights into the envelope glycoprotein fusion mechanism and information for rational drug and vaccine design.

Mutational analysis of residues in the coiled-coil domain of human immunodeficiency virus type 1 transmembrane protein gp41.

The envelope glycoprotein (Env) of human immunodeficiency virus mediates virus entry into cells by undergoing conformational changes that lead to fusion between viral and cellular membranes. A six-helix bundle in gp41, consisting of an interior trimeric coiled-coil core with three exterior helices packed in the grooves (core structure), has been proposed to be part of a fusion-active structure of Env (D. C. Chan, D. Fass, J. M. Berger, and P. S. Kim, Cell 89:263-273, 1997; W. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387:426-430, 1997; and K. Tan, J. Liu, J. Wang, S. Shen, and M. Lu, Proc. Natl. Acad. Sci. USA 94:12303, 1997). We analyzed the effects of amino acid substitutions of arginine or glutamic acid in residues in the coiled-coil (heptad repeat) domain that line the interface between the helices in the gp41 core structure. We found that mutations of leucine to arginine or glutamic acid in position 556 and of alanine to arginine in position 558 resulted in undetectable levels of Env expression. Seven other mutations in six positions completely abolished fusion activity despite incorporation of the mutant Env into virions and normal gp160 processing. Single-residue substitutions of glutamic acid at position 570 or 577 resulted in the only viable mutants among the 16 mutants studied, although both viable mutants exhibited impaired fusion activity compared to that of the wild type. The glutamic acid 577 mutant was more sensitive than the wild type to inhibition by a gp41 coiled-coil peptide (DP-107) but not to that by another peptide corresponding to the C helix in the gp41 core structure (DP-178). These results provide insight into the gp41 fusion mechanism and suggest that the DP-107 peptide may inhibit fusion by binding to the homologous region in gp41, probably by forming a peptide-gp41 coiled-coil structure.

Capture of an early fusion-active conformation of HIV-1 gp41.

Using an inhibitory synthetic peptide (DP-178) from HIV-1 gp41, we have trapped HIV-1 envelope glycoprotein (Env) undergoing conformational changes during virus entry. Our data show that DP-178 binds gp41 and inhibits Env-mediated membrane fusion after gp120 interacts with cellular receptors, indicating that conformational changes involving the coiled coil domain of gp41 are required for entry. Capture of this fusion-active conformation of Env provides insights into the early events leading to Env-mediated membrane fusion.

Studies of HIV-1 envelope glycoprotein-mediated fusion using a simple fluorescence assay.

OBJECTIVE: To study HIV envelope glycoprotein (Env)-mediated entry using a sensitive fusion assay. DESIGN AND METHODS: CD4+ lymphocytes or T-cell lines were labelled with fluorescent cytoplasm or membrane markers. Fusion with Env-expressing adherent cells was monitored by observing dye transfer from CD4+ cells to Env cells. RESULTS: Cell-cell fusion began 20-30 min after co-cultivation at 37 degrees C. Pre-binding at 4 degrees C was observed not to decrease the lag phase before fusion. Cells expressing envelope glycoproteins from non-syncytium-inducing (NSI) HIV strains showed dye transfer between two cells without progression to syncytia. A glycosylphosphatidylinositol anchored Env was found to be incapable of mediating membrane fusion, as measured either by lipid or cytoplasm contents mixing. Primary mouse cells expressing human CD4 and mouse 3T3 cells stably expressing both human CD4 and human CD26 did not support fusion with our Env-expressing cells. CONCLUSIONS: Env-mediated cell-cell fusion is a relatively slow process, probably reflecting a multi-step process occurring after CD4 binding and requiring the transmembrane domain of gp41. Env proteins are able to mediate cell-cell fusion at least under some experimental conditions, indicating that lack of a syncytia phenotype does not rule out the possibility of fusion occurring between only two or a few cells.

Characterization of stable Chinese hamster ovary cells expressing wild-type, secreted, and glycosylphosphatidylinositol-anchored human immunodeficiency virus type 1 envelope glycoprotein.

We generated Chinese hamster ovary cell lines that stably express wild-type, secreted, and glycosylphosphatidylinositol (GPI)-anchored envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). The cells expressing wild-type Env (WT cells) express both the precursor gp160 and the mature gp120/gp41 and readily form large syncytia when cocultivated with CD4+ human cells. The cells expressing secreted Env (SEC cells) release 140-kDa precursor and mature 120-kDa envelope glycoproteins into the supernatants. The cells expressing GPI-anchored Env (PI cells) express both 140-kDa precursor and mature gp120/gp41 envelope glycoproteins, which can be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Both the secreted and PI-PLC-released envelope glycoproteins form oligomers that can be detected on nonreducing sodium dodecyl sulfate-polyacrylamide gels. In contrast to the WT cells, the SEC and PI cells do not form syncytia when cocultivated with CD4+ human cells. The availability of cells producing water-soluble oligomers of HIV-1 Env should facilitate studies of envelope glycoprotein structure and function. The WT cells, which readily induce syncytia with CD4+ cells, provide a convenient system for assessing potential fusion inhibitors and for studying the fusion mechanism of the HIV Env glycoprotein.

Oligomeric organization of gp120 on infectious human immunodeficiency virus type 1 particles.

The oligomeric structure of the human immunodeficiency virus type 1 envelope glycoprotein (gp120) was examined by treating infectious virions with chemical cross-linking agents and subjecting the protein to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and velocity centrifugation. Immunoblots of cross-linked samples revealed three gp120 bands and an approximately threefold shift in gp120 sedimentation. Our finding of cross-linking solely between gp120 suggests that the gp120 subunits are closely associated in the native envelope structure.

Optimal conditions for recovery of the human immunodeficiency virus from peripheral blood mononuclear cells.

Optimal conditions for demonstrating the presence of infectious human immunodeficiency virus in peripheral blood mononuclear cells (PMCs) from seropositive individuals involved cocultivation of infected cells with phytohemagglutinin-stimulated PMCs from seronegative donors in the presence of 2 micrograms of Polybrene per ml. The size of the culture vessel also influenced the results; smaller numbers of infected cells were detected under conditions of increased cell density. In addition, an increased normal donor/patient PMC ratio was helpful. The cocultivation approach permitted identification of human immunodeficiency virus in over 90% of seropositive individuals with different clinical conditions. Moreover, reconstruction experiments indicated that this method allows detection of one productively infected CD4+ cell in a population of over 10(6) PMCs.