HIV-12 cellular invasion proceeds through fusion of viral and cellular membranes, a process mediated by the viral surface glycoprotein Env in response to binding cellular receptors (1, 2). Functional Env is composed of two noncovalently linked subunits, gp120 and gp41, arranged as a trimer of heterodimers on the virion surface. The interaction of gp120 with cellular CD4 and a chemokine receptor promotes structural rearrangements that ultimately lead gp41 to insert its N-terminal fusion peptide segment into the target cell membrane (Fig. 1A). Eventually, gp41 collapses into a stable, compact trimer-of-hairpins conformation essential for efficient membrane fusion (3).The core of the gp41 trimer-of-hairpins is a bundle of six ␣-helices formed by two hydrophobic heptad repeat sequences (HR1 and HR2) within each of the three gp41 ectodomains (4 -7). The N-terminal HR1 segments form a central, trimeric coiled coil. The C-terminal HR2 segments pack in an antiparallel manner into hydrophobic grooves on the exterior of the HR1 coiled coil. The antiparallel packing of HR1 and HR2 segments ensures that the fusion peptide and transmembrane region of gp41 are brought into close proximity (Fig. 1A). Since the fusion peptide is inserted within the target cell membrane and the transmembrane region is embedded within the viral membrane, formation of the gp41 trimer-of-hairpins promotes the juxtaposition of the two membranes (6). The similarity of the gp41 trimer-of-hairpins to the fusion-active conformations of glycoproteins from distantly related enveloped viruses suggests that this structure plays a critical role in promoting viral membrane fusion (see Ref. 3 and references therein).Peptides derived from the HR2 region of the gp41 ectodomain can disrupt formation of the gp41 trimer-of-hairpins, thereby inhibiting HIV-1 entry (4, 8, 9). Denoted as C-peptides, they work in a dominant negative fashion by binding the gp41 HR1 regions exposed in the extended, prehairpin intermediate conformation (4, 10 -13). Once bound, they prevent association of the HR1 and HR2 segments, thus blocking viral membrane fusion. That C-peptides bind an intermediate state in the fusion process is supported by the following observations: 1) C-peptides can precipitate gp41 only after Env binds cellular receptors (12, 13); 2) the extent of inhibition depends on C-peptide concentration present during membrane fusion and not on preincubation levels (12); 3) conditions that alter the rate of gp41-mediated membrane fusion influence the inhibitory potency of C-peptides (14 -16); and 4) at intermediate concentrations that block fusion pore formation, C-peptides can lock gp41 in a state that still permits lipid mixing (17). Despite the transient exposure of their binding site, some C-peptides possess potent (low nanomolar) inhibitory activity in vitro. One C-peptide, T20 (enfuvirtide), also demonstrates antiviral activity in vivo and has been approved by the FDA for treatment of HIV-1 infected individuals (18,19).In a complementary manner, agents that specific...

Both equilibrium and nonequilibrium factors influence the efficacy of pharmaceutical agents that target intermediate states of biochemical reactions. We explored the intermediate state inhibition of gp41, part of the HIV-1 envelope glycoprotein complex (Env) that promotes viral entry through membrane fusion. This process involves a series of gp41 conformational changes coordinated by Env interactions with cellular CD4 and a chemokine receptor. In a kinetic window between CD4 binding and membrane fusion, the N- and C-terminal regions of the gp41 ectodomain become transiently susceptible to inhibitors that disrupt Env structural transitions. In this study, we sought to identify kinetic parameters that influence the antiviral potency of two such gp41 inhibitors, C37 and 5-Helix. Employing a series of C37 and 5-Helix variants, we investigated the physical properties of gp41 inhibition, including the ability of inhibitor-bound gp41 to recover its fusion activity once inhibitor was removed from solution. Our results indicated that antiviral activity critically depended upon irreversible deactivation of inhibitor-bound gp41. For C37, which targets the N-terminal region of the gp41 ectodomain, deactivation was a slow process that depended on chemokine receptor binding to Env. For 5-Helix, which targets the C-terminal region of the gp41 ectodomain, deactivation occurred rapidly following inhibitor binding and was independent of chemokine receptor levels. Due to this kinetic disparity, C37 inhibition was largely reversible, while 5-Helix inhibition was functionally irreversible. The fundamental difference in deactivation mechanism points to an unappreciated asymmetry in gp41 following inhibitor binding and impacts the development of improved fusion inhibitors and HIV-1 vaccines. The results also demonstrate how the activities of intermediate state inhibitors critically depend upon the final disposition of inhibitor-bound states.

The recent success of the fusion inhibitor T-20 (enfuvirtide) in clinical studies has ushered in a new chapter in the development of anti-HIV-1 therapeutics. T-20 is the first FDA-approved drug that targets the viral transmembrane protein gp41. This protein, along with gp120, promotes viral entry through a coordinated cascade of conformational transitions that lead to the fusion of the HIV-1 and target cell membranes. The interaction of gp120 with CD4 and a chemokine receptor stimulates gp41 to extend and bridge the space between the virus and cell. Subsequently, gp41 collapses into a trimer-of-hairpins structure that brings the viral and cellular membranes into close proximity necessary for fusion. Enfuvirtide targets the gp41 amino-terminal region exposed in the transient extended state, blocking the ultimate collapse into the trimer-of hairpins and inhibiting membrane fusion. The vulnerability of this transient extended state has stimulated the development of new agents, ranging from small molecules to large proteins, that bind to gp41 and inhibit its structural transformations. The discovery and characterization of these inhibitors have not only led to new antiviral strategies, but have also shed light on the accessibility of gp41 epitopes that might play a role in HIV-1 vaccine development.