P20A inhibits HIV-1 fusion through its electrostatic interaction with the distal region of the gp41 fusion core

We previously identified an HIV-1 fusion inhibitor P20A targeting HIV-1 gp41 6-HB fusion core. Using alanine scanning mutagenesis, we investigated the effect of 6-HB surface residue mutations on the binding affinity between P20A and 6-HB. Substitution of positively or negatively charged residues in the distal region of 6-HB with alanines resulted in significant decrease or increase of its binding affinity to P20A, respectively. The 6-HB with E630K, D632K, or E634K mutation exhibited enhanced binding affinity with P20A, suggesting that P20A blocks HIV-1 fusion through electrostatic interaction with the positively charged residues in the distal region of the gp41 fusion core.     

The HIV Env-mediated fusion reaction

The current general model of HIV viral entry involves the binding of the trimeric viral envelope glycoprotein gp120/gp41 to cell surface receptor CD4 and chemokine co-receptor CXCR4 or CCR5, which triggers conformational changes in the envelope proteins. Gp120 then dissociates from gp41, allowing for the fusion peptide to be inserted into the target membrane and the pre-hairpin configuration of the ectodomain to form. The C-terminal heptad repeat region and the leucine/isoleucine zipper region then form the thermostable six-helix coiled-coil, which drives the membrane merger and eventual fusion. This model needs updating, as there has been a wealth of data produced in the last few years concerning HIV entry, including target cell dependencies, fusion kinetic data, and conformational intermediates. A more complete model must include the involvement of membrane microdomains, actin polymerization, glycosphingolipids, and possibly CD4 and chemokine signaling in entry. In addition, kinetic experiments involving the addition of fusion inhibitors have revealed some of the rate-limiting steps in this process, adding a temporal component to the model. A review of these data that may require an updated version of the original model is presented here.     

Design,synthesis and activity evaluation of novel peptide fusion inhibitors targeting HIV-1 gp41

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes about 2 million people to death every year. Fusion inhibitors targeted the envelope protein (gp41) represent a novel and alternative approach for anti-AIDS therapy, which terminates the HIV-1 life cycle at an early stage. Using CP621-652 as a template, a series of peptides were designed, synthesized and evaluated in vitro assays. An interesting phenomenon was found that the substitution of hydrophobic residues at solvent accessible sites could increase the anti-HIV activity when the C-terminal sequence was extended with an enough numbers of amino acids. After the active peptides was synthesized and evaluated, peptide 8 showed the best anti-HIV-1 IIIB whole cell activity (MAGI IC₅₀ = 53.02 nM). Further study indicated that peptide 8 bound with the gp41 NHR helix, and then blocked the conformation of 6-helix, thus inhibited virus–cell membrane fusion. The results would be helpful for the design of peptide fusion inhibitors against HIV-1 infection.     

Structural and immunological characterisation of heteroclitic peptide analogues corresponding to the 600-612 region of the HIV envelope gp41 glycoprotein

The conformational and immunological properties of different analogues corresponding to the 600-612 disulfide loop of the human immunodeficiency virus (HIV) gp41 glycoprotein envelope were studied. Fourteen analogues were designed and synthesised; namely, a series of seven analogues in which the disulfide bond was replaced by a lactam bridge and a series of seven analogues in which one residue of each analogue at a time, was replaced by its corresponding homologised alpha-amino acid (beta(3)-amino acid). In the case of the lactam analogues, the influence of the two possible CO-NH and NH-CO orientations of the lactam bridge as well as the size of the lactam ring was explored. The analogues were tested in ELISA with monoclonal antibodies raised against the 600-612 cyclic parent peptide as well as with sera from HIV-1 infected patients. A structural analysis of the parent and analogue peptides was carried out in dimethyl sulfoxide (DMSO-d(6)) using two-dimensional NMR techniques and molecular dynamics simulations. Comparison of the own conformation of the cyclic analogues with their either strong or weak reactivity with the antibodies reveals structural features that may be correlated with the antibody reactivity. Thus, a close structural similarity, particularly a characteristic orientation of the side-chains of residues Lys606, Leu607 and Ile608 in the loop, was found in certain beta(3)-analogues that were better recognised than the parent peptide by anti-peptide mouse monoclonal antibodies and patients' antibodies.     

Monomer-trimer equilibrium of the ectodomain of SIV gp41: insight into the mechanism of peptide inhibition of HIV infection.

The monomer-trimer equilibrium of the ectodomain of SIV gp41 (residues 27-149, e-gp41) has been characterized by analytical ultracentrifugation, circular dichroism (CD), and NMR spectroscopy. Based on analytical ultracentrifugation experiments performed at different rotor speeds and protein concentrations, the equilibrium association constant for the SIV e-gp41 trimer is 3.1 x 10(11) M(-2). The presence of intermolecular nuclear Overhauser effects in a mixture of 12C and 13C-labeled e-gp41 prepared under nondenaturing conditions unambiguously demonstrates that there is a dynamic equilibrium between the monomer and trimer. The CD spectra taken as a function of SIV e-gp41 concentration suggest that the helical content of the monomeric state does not change significantly relative to that of the trimeric state. The relevance of the monomer-trimer equilibrium is discussed with respect to gp41 function and the inhibitory properties of gp41 peptides.     

HIV—1 P24截短体与gp41截短体的融合蛋白在大肠杆菌中的表达、纯化和鉴定

用基因重组技术将截短的HIV－1 p24基因和gp41基因连接成嵌合基因,插入质粒pGEX-4T3,构建成重组表达质粒pGEX-F。将pGEX-F转化大肠杆菌BL21。经IPTG诱导表达,pGEX-F在大肠杆菌BL21中获得了高效表达。融合蛋白P24－gp41经Glutathione-Sepharose4B亲和层析纯化后,用间接ELISA和免疫印迹检测HIV抗体阳性血清和正常人血清,P24－gp41只与HIV抗体阳性血清反应,证明获得的融合蛋白P24－gp41有很强的抗原特异性和免疫反应性,具有较高的应用价值。     

Structural analysis of the epitope of the anti-HIV antibody 2F5 sheds light into its mechanism of neutralization and HIV fusion

Inhibition of human immunodeficiency virus (HIV) fusion with the host cell has emerged as a viable therapeutic strategy, and rational design of inhibitors and vaccines, interfering with this process, is a prime target for antiviral research. To advance our knowledge of the structural biology of HIV fusion, we have studied the membrane-proximal region of the fusogenic envelope subunit gp41, which includes the epitope ELDKWA of the broadly neutralizing human antibody 2F5. The structural evidence available for this region is contradictory, with some studies suggesting an overall helical conformation, while the X-ray structure of the ELDKWAS peptide bound to the antibody shows it folded in a type I beta turn. We used a two-step strategy: Firstly, by a competition binding assay, we identified the proper boundaries of the domain recognized by 2F5, which we found considerably larger than the ELDKWAS hexapeptide. Secondly, we studied the structure of the resulting 13 amino acid residue peptide by collecting NMR data and analyzing them by our previously developed statistical method (NAMFIS). Our study revealed that the increase in binding affinity goes in parallel with stabilization of specific local and global conformational propensities, absent from the shorter epitope. When compounded with the available biological evidence, our structural analysis allows us to propose a specific role for the membrane-proximal region during HIV fusion, in terms of a conformational transition between the turn and the helical structure. At the same time, our hypothesis offers a structural explanation for the mechanism of neutralization of mAb 2F5.     

Small molecule fusion inhibitors: Design,synthesis and biological evaluation of (Z)-3-(5-(3-benzyl-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrroles and related derivatives targeting HIV-1 gp41

By a scaffold elongation strategy, a series of (Z)-3-(5-(3-benzyl-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrroles and related derivatives with a linear multi-aromatic-ring skeleton were designed, synthesized, and evaluated in HIV-1 gp41 and cellular assays. Among them, the most active compounds, 12e, 12g, and 12k with a one-carbon linker (n = 1) between the rhodanine (C) and phenyl (D) rings, exhibited very promising inhibitory potency with IC₅₀ values of 1.8–2.6 μM and EC₅₀ values of 0.3–1.5 μM against gp41 6-HB formation and HIV-1 replication in MT-2 cells, respectively. Additionally, they were almost equally effective against both T20-sensitive and resistant strains. The related SAR studies and molecular modeling results provided potential for further developing a new class of non-peptide small molecular fusion inhibitors targeting the HIV-1 gp41.     

Structural and functional roles of HIV-1 gp41 pretransmembrane sequence segmentation

The membrane-proximal segment connecting the helical core with the transmembrane anchor of human immunodeficiency virus type 1 gp41 is accessible to broadly neutralizing antibodies and plays a crucial role in fusion activity. New predictive approaches including computation of interfacial affinity and the corresponding hydrophobic moments suggest that this region is functionally segmented into two consecutive subdomains: one amphipathic at the N-terminal side and one fully interfacial at the C-terminus. The N-terminal subdomain would extend alpha-helices from the preceding carboxy-terminal heptad repeat and provide, at the same time, a hydrophobic-at-interface surface. Experiments were performed to compare a wild-type representing pretransmembrane peptide with a nonamphipathic defective sequence, which otherwise conserved interfacial hydrophobicity at the carboxy-subdomain. Results confirmed that both penetrated equally well into lipid monolayers and both were able to partition into membrane interfaces. However only the functional sequence: 1), adopted helical structures in solution and in membranes; 2), formed homo-oligomers in solution and membranes; and 3), inhibited gp41-induced cell-cell fusion. These data support two roles for gp41 aromatic-rich pretransmembrane sequence: 1), oligomerization of gp41; and 2), immersion into the viral membrane interface. Accessibility to membrane interfaces and subsequent adoption of the low-energy structure may augment helical bundle formation and perhaps be related to a concomitant loss of immunoreactivity. These results may have implications in the development of HIV-1 fusion inhibitors and vaccines.     

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Monoclonal anti‐HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L ‐Phe/β‐Ala bi‐substituted 1,3,5‐triazine (Trz) scaffold (β‐Ala‐Trz‐L ‐Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10‐binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K_D) of 0.41 ± 0.05 µM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60–80%). Analysis of the antibody preparation by SDS‐PAGE, enzyme‐linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids. Copyright © 2009 John Wiley & Sons, Ltd.     

The membranotropic regions of the endo and ecto domains of HIV gp41 envelope glycoprotein

We have identified the membranotropic regions of the full sequence of the HIV gp41 envelope glycoprotein by performing an exhaustive study of membrane rupture, phospholipid-mixing and fusion induced by two 15-mer gp41-derived peptide libraries from HIV strains HIV_MN and HIV_consensus_B on model membranes having different phospholipid compositions. The data obtained for the two strains and its comparison have led us to identify different gp41 membranotropic segments in both ecto- and endodomains which might be implicated in viral membrane fusion and/or membrane interaction. The membranotropic segments corresponding to the gp41 ectodomain were the fusion domain, a stretch located on the N-heptad repeat region adjacent to the fusion domain, part of the immunodominant loop, the pre-transmembrane domain and the transmembrane domain. The membranotropic segments corresponding to the gp41 endodomain were mainly located at some specific parts of the previously described lentivirus lytic sequences. Significantly, the C-heptad repeat region and the Kennedy sequence located in the ectodomain and in the endodomain, respectively, presented no membranotropic activity in any model membrane assayed. The identification of these gp41 segments as well as their membranotropic propensity sustain the notion that different segments of gp41 provide the driving force for the merging of the viral and target cell membranes as well as they help us to define those segments as attractive targets for further development of new anti-viral compounds.     

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26-42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).