Mode of Action of an Antiviral Peptide from HIV-1

Abstract

DP178, a synthetic peptide corresponding to a segment of the transmembrane envelope glycoprotein (gp41) of human immunodeficiency virus, type 1 (HIV-1), is a potent inhibitor of viral infection and virus-mediated cell-cell fusion. Nevertheless, DP178 does not contain gp41 coiled-coil cavity binding residues postulated to be essential for inhibiting HIV-1 entry. We find that DP178 inhibits phospholipid redistribution mediated by the HIV-1 envelope glycoprotein at a concentration 8 times greater than that of solute redistribution (the IC 50 values are 43 and 335 nM, respectively). In contrast, C34, a synthetic peptide which overlaps with DP178 but contains the cavity binding residues, did not show this phenomenon (11 and 25 nM, respectively). The ability of DP178 to inhibit membrane fusion at a post-lipid mixing stage correlates with its ability to bind and oligomerize on the surface of membranes. Furthermore, our results are consistent with a model in which DP178 inhibits the formation of gp41 viral hairpin structure at low affinity, whereas C34 inhibits its formation at high affinity: the failure to form the viral hairpin prevents both lipid and solute from redistributing between cells. However, our data also suggest an additional membrane-bound inhibitory site for DP178 in the ectodomain of gp41 within a region immediately adjacent to the membrane-spanning domain. By binding to this higher affinity site, DP178 inhibits the recruitment of several gp41-membrane complexes, thus inhibiting fusion pore formation.The first step in HIV-1 1 infection involves the binding of the viral envelope glycoproteins gp120-gp41 to CD4 (1-3) and subsequently to a co-receptor (4 -8) (for recent review, see Refs. 9 -11). Consequently, gp41 undergoes conformational changes that mediate the fusion between the viral and the cellular membranes or between infected and healthy cells (12, 13). Gallaher and co-workers (14, 15) postulated a model of gp41, identifying a fusion peptide followed by a leucine/isoleucine zipper-like sequence (N-helix) and an amphipathic helical segment (C-helix) in the viral glycoprotein. The indispensability of the fusion peptide for viral infection was confirmed by sitedirected mutagenesis (16,17). Furthermore, gp41 was found to contain a protease-resistant core consisting of the postulated N-and C-helices (18). Specifically, peptides corresponding to these sequences co-crystallized as a six-helix bundle in which the N-and C-helices are arranged in a three-hairpin structure (19 -21). Three N peptides form a coiled-coil, and the C peptides are packed in an antiparallel manner into highly conserved, hydrophobic grooves on the surface of the coiled-coil. Recently, the solution and crystal structures of the ectodomain of the Simian immunodeficiency virus gp41, consisting of those two helices as well as the loop connecting them, confirmed the interplay of the N-and C-helices (22, 23). Remarkably, the coiled coil is a common motif found in many diverse viral membrane fusion proteins (24), as well as in proteins invol...

C-terminal Octylation Rescues an Inactive T20 Mutant

“…Second, Nieva and co-workers (38,39) have shown that HIV gp41-derived peptides corresponding to a Trp-rich region that partially overlaps the C terminus of T20 destabilize model membranes, suggesting that the equivalent region in gp41 might participate in the actual process of membrane fusion. Third, HIV gp41-derived T20 has been shown to bind to phospholipid membranes (23). Fourth, as already mentioned, membrane anchoring by fusion to a transmembrane domain restores the inhibitory potency of the ANAA mutant of HIV gp41-derived T20 (24).…”

“…However, the understanding of its detailed mechanism of inhibition has been hampered by the following. (i) T20 lacks the residues that bind to the coiled coil C-terminal cavity; (ii) the known crystal and solution structures of fragments of HIV or SIV gp41 ectodomains do not fully include the regions corresponding either to T20 or to its putative binding site at the N terminus of the coiled coil; and (iii) in addition to a primary target site within the N-terminal heptad repeat (21), T20 has been postulated to bind to a second site in gp41, presumably a non-specified region at the C terminus of the ectodomain close to the viral membrane (22,23). Indeed, that T20 acts in close proximity to the membrane is supported by the studies of von Laer and co-workers (24).…”

Enhancement of α-Helicity in the HIV-1 Inhibitory Peptide DP178 Leads to an Increased Affinity for Human Monoclonal Antibody 2F5 but Does Not Elicit Neutralizing Responses in Vitro

“…1A. act at the water-lipid membrane interface so that this region of GP41 would lie close to the bilayer surface with the 2F5 epitope bending outward in a loop to join the CHR region NH 2 -terminal to it. DP178 also overlaps a portion of the tryptophan-rich region (residues 665-673) and has been recently implicated in membrane interactions (54). Although we and others have shown that the isolated peptide is largely disordered in solution, it is entirely plausible that it exists as a more highly helical structure in the membrane-bound state of native GP41.…”

Role of the Envelope Genetic Context in the Development of Enfuvirtide Resistance in Human Immunodeficiency Virus Type 1-Infected Patients

“…ENF binding to HR1 is thought to compete with the folding of the HR2 domain onto HR1, thus preventing Env-mediated membrane fusion (1,5,17). Accordingly, viral variants selected both in vitro and in vivo in the presence of ENF carry resistance mutations in the HR1 domain (35,42).…”