Effect of L- and D-REKR amino acid-containing peptides on HIV and SIV envelope glycoprotein precursor maturation and viral replication (original) (raw)
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Inhibition of human immunodeficiency virus type 1 entry in cells expressing gp41-derived peptides
Journal of virology, 2004
As the limitations of antiretroviral drug therapy, such as toxicity and resistance, become evident, interest in alternative therapeutic approaches for human immunodeficiency virus (HIV) infection is growing. We developed the first gene therapeutic strategy targeting entry of a broad range of HIV type 1 (HIV-1) variants. Infection was inhibited at the level of membrane fusion by retroviral expression of a membrane-anchored peptide derived from the second heptad repeat of the HIV-1 gp41 transmembrane glycoprotein. To achieve maximal expression and antiviral activity, the peptide itself, the scaffold for presentation of the peptide on the cell surface, and the retroviral vector backbone were optimized. This optimized construct effectively inhibited virus replication in cell lines and primary blood lymphocytes. The membrane-anchored C-peptide was also shown to bind to free gp41 N peptides, suggesting that membrane-anchored antiviral C peptides have a mode of action similar to that of fr...
Virology, 2002
N36(L6)C34 is a recombinant protein that forms a six-helix bundle with high thermal stability. It consists of the N-terminal heptad-repeat region (N36 peptide) and the C-terminal heptad-repeat region (C34) of HIV-1 gp41, connected by six polar amino acids. The protein inhibits HIV-1 envelope-induced membrane fusion. Whether inhibition occurs while N36(L6)C34 is in its six-helix bundle configuration was investigated. Mutating a critical residue within the N36 region to promote dissociation of C34 from the grooves of the N36 coiled coil reduced bundle stability and increased the inhibition of fusion. In contrast, mutating a key residue within the C34 region to reduce bundle stability decreased inhibitory potency. The data provide strong evidence that the proteins inhibit fusion while they expose their C34 segments, rather than as six-helix bundles. Thus, despite high thermal stability of the bundle, the recombinants' less folded structures are present in sufficient concentration to inhibit fusion at physiological temperatures. © 2002 Elsevier Science (USA)
Biochemistry, 1995
The envelope glycoprotein gp41 from human immunodeficiency virus type 1 (HIV-1) is involved in membrane fusion and virus entry. It contains a functionally important leucine zipper-like heptad repeat region (residues 553-590). To investigate the solution structure and membrane-binding properties of this region, cysteine-substituted variants of a 38-residue peptide derived from the heptad repeat were synthesized and modified with nitroxide spin labels. Analytical equilibrium ultracentrifugation studies indicated it is primarily tetrameric in solution, in contrast to the protein gp160 which is a mixture of trimers and tetramers. Electron paramagnetic resonance (EPR) measurements indicated that the peptide was bound to vesicles containing 10 mol % negatively charged lipids. The peptides were bound parallel to the membrane surface, near the water-membrane interface, in a structure different from the solution structure, most likely as monomers. When Asp, Pro, or Ser was substituted for Ile at the core "a" position of the heptad repeat in the middle of the peptide, the coiled coil was destabilized. In addition, these peptides showed reduced membrane-binding affinities. Thus, mutations that destabilized coiled-coil formation also decreased membrane-binding propensity. These experimental results, taken with previous evidence, suggest two functions for the heptad repeat of gp41 after CD4 binding: (1) to form an extended coiled coil; (2) to provide a hydrophobic face that binds to the host-cell membrane, bringing the viral and cellular membranes closer and facilitating fusion. Retrovirus surfaces are coated with envelope (env) glycoproteins. These proteins are synthesized as a singlepolypeptide precursor that is subsequently cleaved into a surface attachment subunit (SU)] responsible for cell recognition and a fusion peptide containing a transmembrane domain (TM) responsible for viral-cell membrane fusion. After cleavage, the two domains are still attached. Specifically, for HIV-1, the env protein gp160 is cleaved into CD4 receptor-binding gp120 (SU) and gp41 (TM). They are noncovalently associated (
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...
Journal of Biological Chemistry, 2007
The binding of CD4 and chemokine receptors to the gp120 attachment glycoprotein of human immunodeficiency virus triggers refolding of the associated gp41 fusion glycoprotein into a trimer of hairpins with a 6-helix bundle (6HB) core. These events lead to membrane fusion and viral entry. Here, we examined the functions of the fusion peptide-proximal polar segment and membrane-proximal Trp-rich region (MPR), which are exterior to the 6HB. Alanine substitution of Trp 666 , Trp 672 , Phe 673 , and Ile 675 in the MPR reduced entry by up to 120-fold without affecting gp120-gp41 association or cell-cell fusion. The L537A polar segment mutation led to the loss of gp120 from the gp120-gp41 complex, reduced entry by ϳ10-fold, but did not affect cell-cell fusion. Simultaneous Ala substitution of Leu 537 with Trp 666 , Trp 672 , Phe 673 , or Ile 675 abolished entry with 50 -80% reductions in cell-cell fusion. gp120-gp41 complexes of fusion-defective double mutants were resistant to soluble CD4-induced shedding of gp120, suggesting that their ability to undergo receptor-induced conformational changes was compromised. Consistent with this idea, a representative mutation, L537A/W666A, led to an ϳ80% reduction in lipophilic fluorescent dye transfer between gp120-gp41-expressing cells and receptor-expressing targets, indicating a block prior to the lipid-mixing phase. The L537A/W666A double mutation increased the chymotrypsin sensitivity of the polar segment in a trimer of hairpins model, comprising the 6HB core, the polar segment, and MPR linked N-terminally to maltose-binding protein. The data indicate that the polar segment and MPR of gp41 act synergistically in forming a fusion-competent gp120-gp41 complex and in stabilizing the membrane-interactive end of the trimer of hairpins. The abbreviations used are: Env, envelope glycoprotein; HIV-1, human immunodeficiency virus type 1; N-helix, N-terminal coiled coil forming ␣-helix of gp41; C-helix, C-terminal ␣-helix of gp41; MPR, membrane proximal region; TMD, transmembrane domain; mAb, monoclonal antibody; sCD4, soluble CD4; DiO, 3,3Ј-dioctadecyloxacarbocyanine perchlorate; DiI, 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine perchlorate; PBS, phosphate-buffered saline; MBP, maltose-binding protein; MALDI, matrixassisted laser desorption ionization; BHK, baby hamster kidney; gp, glycoprotein; 6HB, 6-helix bundle; SIV, simian immunodeficiency virus.
J Mol Biol, 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.
Journal of Virology, 2005
cytoplasmic tail (CT) can modulate the fusogenicity of the envelope glycoprotein (Env) on infected cells and virions. However, the CT domains involved and the underlying mechanism responsible for this "inside-out" regulation of Env function are unknown. HIV and SIV CTs are remarkably long and contain amphipathic alpha-helical domains (LLP1, LLP2, and LLP3) that likely interact with cellular membranes. Using a cell-cell fusion assay and a panel of HIV Envs with stop codons at various positions in the CT, we show that truncations of gp41 proximal to the most N-terminal alpha helix, LLP2, increase fusion efficiency and expose CD4-induced epitopes in the Env ectodomain. These effects were not seen with a truncation distal to this domain and before LLP1. Using a dye transfer assay to quantitate fusion kinetics, we found that these truncations produced a twoto fourfold increase in the rate of fusion. These results were observed for X4-, R5-, and dual-tropic Envs on CXCR4-and CCR5-expressing target cells and could not be explained by differences in Env surface expression. These findings suggest that distal to the membrane-spanning domain, an interaction of the gp41 LLP2 domain with the cell membrane restricts Env fusogenicity during Env processing. As with murine leukemia viruses, where cleavage of a membrane-interactive R peptide at the C terminus is required for Env to become fusogenic, this restriction of Env function may serve to protect virus-producing cells from the membrane-disruptive effects of the Env ectodomain.
Journal of Biological Chemistry, 2002
The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36 Mut(e,g) and N36 Mut(a,d) , were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546-581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36 Mut(e,g) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil. N36 Mut(a,d) contains nine substitutions designed to block formation of the trimeric coiled-coil but retains residues that interact with the C-terminal region of gp41. N36 Mut(a,d) is monomeric, is largely random coil, does not interact with the C34 peptide derived from the C-terminal region of gp41 (residues 628-661), and does not inhibit fusion. The trimeric coiled-coil structure is therefore a prerequisite for interaction with the C-terminal region of gp41. N36 Mut(e,g) forms a monodisperse, helical trimer in solution, does not interact with C34, and yet inhibits fusion about 50-fold more effectively than the parent N36 peptide (IC 50 ϳ 308 nM versus ϳ16 M). These results indicate that N36 Mut(e,g) acts by disrupting the homotrimeric coiled-coil of N-terminal helices in the prehairpin intermediate to form heterotrimers. Thus N36 Mut(e,g) represents a novel third class of gp41-targeted HIV fusion inhibitor. A quantitative model describing the interaction of N36 Mut(e,g) with the pre-hairpin intermediate is presented.
Journal of virology, 1995
An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusio...