Structural and Functional Analysis of the HIV gp41 Core Containing an Ile573 to Thr Substitution:  Implications for Membrane Fusion^,

Abstract

The envelope glycoprotein of HIV-1 consists of the surface subunit gp120 and the transmembrane subunit gp41. Binding of gp120 to target cell receptors induces a conformational change in gp41, which then mediates the fusion of viral and cellular membranes. A buried isoleucine (Ile573) in a central trimeric coiled coil within the fusion-active gp41 ectodomain core is thought to favor this conformational activation. The role of Ile573 in determining the structure and function of the gp120-gp41 complex was investigated by mutating this residue to threonine, a nonconservative substitution in HIV-1 that occurs naturally in SIV. While the introduction of Thr573 markedly destabilized the gp41 core, the three-dimensional structure of the mutant trimer of hairpins was very similar to that of the wild-type molecule. A new hydrogen-bonding interaction between the buried Thr573 and Thr569 residues appears to allow formation of the trimer-of-hairpins structure at physiological temperature. The mutant envelope glycoprotein expressed in 293T cells and incorporated within pseudotyped virions displayed only a moderate reduction in syncytium-inducing capacity and virus infectivity, respectively. Our results demonstrate that the proper folding of the gp41 core underlies the membrane fusion properties of the gp120-gp41 complex. An understanding of the gp41 activation process may suggest novel strategies for vaccine and antiviral drug development.

Human Immunodeficiency Virus (HIV) gp41 Escape Mutants: Cross-Resistance to Peptide Inhibitors of HIV Fusion and Altered Receptor Activation of gp120

“…Such destabilizing mutations would also likely reduce infectivity of the mutant virus. A correlation between decreased 6HB stability and reduced infectivity has been reported for other mutations in gp41 (16,22). Yet T20-resistant viruses can acquire resistance by mutating residues that directly impair peptide binding (2,31,36).…”

Structural and Functional Analysis of Interhelical Interactions in the Human Immunodeficiency Virus Type 1 gp41 Envelope Glycoprotein by Alanine-Scanning Mutagenesis

“…Biophysical and structural characterization directly demonstrates that this substitution strengthens the interhelix interaction in the fusogenic hairpin structure by providing additional hydrophobic packing forces (63). Taken together, these results have been interpreted to indicate that the receptor-triggered conformational changes of the HIV-1 envelope glycoprotein are thermodynamically controlled and that the process of membrane apposition and lipid bilayer fusion is driven by the currency of energy released from the formation of the fusogenic gp41 core (33,49,51,63,69).…”

“…The ability of the wild-type and mutant envelope glycoproteins to mediate cell-cell fusion was determined by coculturing envelope glycoprotein-expressing COS-7 cells with U87 cells expressing CD4 and CXCR4 coreceptor (31,45,49). Transfected COS-7 cells were resuspended using PBS with 0.5 mM EDTA, and 5 ϫ 10 3 cells were added to 96-well microcultures containing 5 ϫ 10 3 U87-CD4-CXCR4 cells.…”

Stabilization of the Soluble, Cleaved, Trimeric Form of the Envelope Glycoprotein Complex of Human Immunodeficiency Virus Type 1

“…However, no structural or other data are available on the likely points of contact between the gp41 moieties in the native, prefusion form of the envelope glycoprotein trimer. We therefore adopted a more theoretical approach, based on the premise that destabilization of the postfusion state of gp41 might stabilize its prefusion configuration, by shifting the conformational equilibrium in favor of the prefusion state (40,41,(49)(50)(51). A plethora of structural and genetic data on the postfusion, six-helix bundle structure of gp41 was available to guide our mutagenesis strategy (3,10,13,16,20,21,39,49,51,54,74,89,(94)(95)(96) The trimeric stability of gp41 in the six-helix bundle form of the protein is determined by the residues at positions a and d of the N-terminal heptad repeat region (40,41,52) (Fig.…”

“…An emphasis of our mutagenesis approach was to alter the Ile-559 and Ile-573 residues, because core isoleucines are known to confer the greatest stability to trimeric coiled coils (also known as isoleucine zippers) (38). In most simian immunodeficiency virus (SIV) strains, Val and Thr residues are found, respectively, at these positions, where they may serve to destabilize the postfusion, coiled-coil structure (40,49,50). Of note is that SIV gp140 UNC has a greater tendency to be trimeric than the corresponding HIV-1 protein (15,19).…”