Inhibition of HIV-1 entry before gp41 folds into its fusion-active conformation 1 1 Edited by J. Karn (original) (raw)
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Inhibition of HIV-1 entry before gp41 folds into its fusion-active conformation
Journal of Molecular Biology, 2000
HIV-1 entry into its host cell is modulated by its transmembrane envelope glycoprotein (gp41). The core of the activated conformation of gp41 consists of a trimer of heterodimers comprising a leucine/isoleucine zipper sequence (represented here by the synthetic peptide N36 or by the longer N51 peptide) and a C-terminal highly conserved region (represented here by C34). A correlation was found between the action of DP178, which is a potent inhibitor of HIV-1 entry into its host cell, and its ability to interact with the leucine/isoleucine zipper sequence. This correlation was further tested and con®rmed by circular dichroism spectroscopy. We found that whereas DP178 perturbs the partial a-helix nature of peptides corresponding to the leucine/isoleucine zipper sequence (N36 or N51), it cannot perturb the trimer of heterodimers conformation, modeled by the complex of N36 or N51 with C34. Therefore, we suggest that the already formed trimer of heterodimers is not the target of inhibition by DP178. Our results are consistent with a model in which DP178 acquires its inhibitory activity by binding to an earlier intermediate of gp41, in which the N and C peptide regions are not yet associated, thus allowing DP178 to bind to the leucine/isoleucine zipper sequence and consequently to inhibit transition to the fusion-active conformation.
Mode of Action of an Antiviral Peptide from HIV-1
Journal of Biological Chemistry, 2001
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 * This work was supported by the National Institutes of Health intramural AIDS targeted antiviral program.
Mode of Action of an Antiviral Peptide from HIV-1. INHIBITION AT A POST-LIPID MIXING STAGE
Journal of Biological Chemistry, 2001
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 * This work was supported by the National Institutes of Health intramural AIDS targeted antiviral program.
Biochemistry, 2005
HIV-1 envelope glycoprotein-mediated fusion is driven by the concerted coalescence of the HIV-1 gp41 N-and C-helical regions, which results in the formation of 6-helix bundles. These two regions are considered prime targets for peptides and antibodies that inhibit HIV-1 entry. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by monitoring the temporal sequence of conformational states of HIV-1 gp41 during the course of HIV-1-mediated cell-cell fusion by quantitative video microscopy using reagents that bind to N-and C-helical regions, respectively. Envexpressing cells were primed by incubation with target cells at different times at 37°C followed by washing. The reactivity of triggered gp41 to the NC-1 monoclonal antibody, which we demonstrate here to bind to N-helical gp41 trimers, increased rapidly to a maximal level in the primed state but decreased once stable fusion junctions had formed. In contrast, reactivity with 5-helix, which binds to the C-helical region of gp41, increased continuously as a function of time following the priming. The peptide N36 Mut(e,g) reduced NC-1 monoclonal antibody binding and enhanced 5-helix binding, consistent with the notion that this molecule promotes dissociation of gp41 trimers. This inactivation pathway may be important for the design of entry inhibitors and vaccine candidates.
Cell, 1999
1995; Lu et al., 1995) as seen in the X-ray crystal structure of a protease-resistant core of gp41 (Chan et al.The trimer-of-hairpins structure is a common feature Massachusetts Institute of Technology of diverse viral membrane fusion proteins (Singh et al., Nine Cambridge Center 1999, and references therein). In gp41, a central three-Cambridge, Massachusetts 02142 stranded coiled coil (formed by the N-terminal regions of gp41) is surrounded by helices derived from the C-terminal end of the gp41 ectodomains, packed in an Summary antiparallel manner around the outside of the coiled coil (Figure 1, inset). Peptides corresponding to these re-The HIV-1 gp41 protein promotes viral entry by mediatgions of gp41 are referred to as N-peptides and C-peping the fusion of viral and cellular membranes. A promitides, respectively. nent pocket on the surface of a central trimeric coiled Synthetic C-peptides are potent inhibitors of HIV-1 coil within gp41 was previously identified as a potential infection. These inhibitors, such as C34 or DP178 (see target for drugs that inhibit HIV-1 entry. We designed Figure 1 legend), inhibit HIV-1 infection and syncytia a peptide, IQN17, which properly presents this pocket. formation at nanomolar concentrations in cell culture Utilizing IQN17 and mirror-image phage display, we experiments (Jiang et al., 1993; Wild et al., 1994; Lu et identified cyclic, D-peptide inhibitors of HIV-1 infection al., 1995; Chan et al., 1998; Rimsky et al., 1998).
Journal of Virology, 2006
Soluble peptides derived from the C-terminal heptad repeat domain of human immunodeficiency virus type 1 (HIV-1) gp41 are potent inhibitors of HIV-1 entry and gp41-induced fusion. Target membrane-anchored variants of these peptides have been shown to retain inhibitory activity. Both soluble and membrane-anchored C peptides (MACs) are thought to block fusion by binding to the N-terminal coiled coil domain of gp41 and preventing formation of the final six-helix bundle structure. However, interactions of target MACs with gp41 must be restricted to a subset of trimers that have their hydrophobic fusion peptides inserted into the target membrane. This unique feature of MACs was used to identify the intermediate step of fusion at which gp41 engaged the target membrane. Fusion between HIV envelope-expressing effector cells and target cells was measured by fluorescence microscopy. Expression of MACs in target cells led to less than twofold reduction in the extent of fusion. However, when re...
ChemBioChem, 2003
The selective proteolytic activation of the HIV-1 envelope glycoprotein gp160 by furin and other precursor convertases (PCs) occurs at the carboxyl side of the sequence Arg508-Glu-Lys-Arg511 (site 1), in spite of the presence of another consensus sequence: Lys500-Ala-Lys-Arg503 (site 2). We report on the solution structural analysis of a 19residue synthetic peptide, p498, which spans the two gp160-processing sites 1 and 2, and is properly digested by furin at site 1. A molecular model is obtained for p498, by means of molecular dynamics simulations, from NMR data collected in trifluoroethanol/water. The peptide N-terminal side presents a 9-residue helical segment, enclosing the processing site 2; the C-terminal segment can be described as a loop exposing the processing site 1. A hypothesis for the docking of p498 onto the catalytic domain of human furin, modeled by homology and fitting previous site-directed mutagenesis studies, is also presented. p498 site 1 is shown to have easy access to the furin catalytic site, unlike the nonphysiological site 2. Finally, on the basis of available data, we suggest a possible structural motif required for the gp160 ± PCs recognition.
Journal of Biological Chemistry, 1999
A peptide of 51 amino acids corresponding to the NH 2terminal region (5-55) of the glycoprotein gp41 of human immunodeficiency virus type 1 was synthesized to study its conformation and assembly. Nuclear magnetic resonance experiments indicated the sequence NH 2-terminal to the leucine zipper-like domain of gp41 was induced into helix in the micellar solution, in agreement with circular dichroism data. Light scattering experiment showed that the peptide molecules self-assembled in water into trimeric structure on average. That the peptide molecules oligomerize in aqueous solution was supported by gel filtration and diffusion coefficient experiments. Molecular dynamics simulation based on the NMR data revealed a flexible region adjacent to the hydrophobic NH 2 terminus of gp41. The biological significance of the present findings on the conformational flexibility and the propensity of oligomerization of the peptide may be envisioned by a proposed model for the interaction of gp41 with membranes during fusion process.
Membrane interactions of the synthetic N-terminal peptide of HIV-1 gp41 and its structural analogs
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1999
Structural and functional studies assessed the membrane actions of the N terminus of HIV-1 glycoprotein 41 000 (gp41). Earlier site-directed mutagenesis has shown that key amino acid changes in this gp41 domain inhibit viral infection and syncytia formation. Here, a synthetic peptide corresponding to the N terminus of gp41 (FP; 23 residues, 519^541), and also FP analogs (FP520V/E with ValCGlu at residue 520; FP527L/R with LeuCArg at 527; FP529F/Y with PheCTyr at 529; and FPCLP1 with FP truncated at 525) incorporating these modifications were prepared. When added to human erythrocytes at physiologic pH, the lytic and aggregating activities of the FP analogs were much reduced over those with the wild-type FP. With resealed human erythrocyte ghosts, the lipid-mixing activities of the FP analogs were also substantially depressed over that with the wild-type FP. Combined with results from earlier studies, theoretical calculations using hydrophobic moment plot analysis and physical experiments using circular dichroism and Fourier transform infrared spectroscopy indicate that the diminished lysis and fusion noted for FP analogs may be due to altered peptide-membrane lipid interactions. These data confirm that the N-terminal gp41 domain plays critical roles in the cytolysis and fusion underlying HIV-cell infection.