On the interaction between gp41 and membranes: the immunodominant loop stabilizes gp41 helical hairpin conformation

gp41 is the protein responsible for the process of membrane fusion that allows primate lentiviruses (HIV and SIV) to enter into their host cells. gp41 ectodomain contains an N-terminal and a C-terminal heptad repeat region (NHR and CHR) connected by an immunodominant loop. In the absence of membranes, the NHR and CHR segments fold into a protease-resistant core with a trimeric helical hairpin structure. However, when the immunodominant loop is not present (either in a complex formed by HIV-1 gp41-derived NHR and CHR peptides or by mild treatment with protease of recombinant constructs of HIV-1 gp41 ectodomain, which also lack the N-terminal fusion peptide and the C-terminal Trp-rich region) membrane binding induces a conformational change in the gp41 core structure. Here, we further investigated whether covalently linking the NHR and CHR segments by the immunodominant loop affects this conformational change. Specifically, we analyzed a construct corresponding to a fragment of SIVmac239 gp41ectodomain (residues 27-149, named e-gp41) by means of surface plasmon resonance, Trp and rhodamine fluorescence, ATR-FTIR spectroscopy, and differential scanning calorimetry. Our results suggest that the presence of the loop stabilizes the trimeric helical hairpin both when e-gp41 is in aqueous solution and when it is bound to the membrane surface. Bearing in mind possible differences between HIV-1 and SIV gp41, and considering that the gp41 ectodomain constructs analyzed to date lack the N-terminal fusion peptide and the C-terminal Trp-rich region, we discuss our observations in relation to the mechanism of virus-induced membrane fusion.     

Effects of amino acid changes in the extracellular domain of the human immunodeficiency virus type 1 gp41 envelope glycoprotein.

Changes were introduced into conserved amino acids within the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein. The effect of these changes on the structure and function of the HIV-1 envelope glycoproteins was examined. The gp41 glycoprotein contains an amino-terminal fusion peptide (residues 512 to 527) and a disulfide loop near the middle of the extracellular domain (residues 598 to 604). Mutations affecting the hydrophobic sequences between these two regions resulted in two phenotypes. Some changes in amino acids 528 to 562 resulted in a loss of the noncovalent association between gp41 and the gp120 exterior glycoprotein. Amino acid changes in other parts of the gp41 glycoprotein (residues 608 and 628) also resulted in subunit dissociation. Some changes affecting amino acids 568 to 596 resulted in envelope glycoproteins partially or completely defective in mediating membrane fusion. Syncytium formation was more sensitive than virus entry to these changes. Changes in several amino acids from 647 to 675 resulted in higher-than-wild-type syncytium-forming ability. One of these amino acid changes affecting tryptophan 666 resulted in escape from neutralization by an anti-gp41 human monoclonal antibody, 2F5. These results contribute to an understanding of the functional regions of the HIV-1 gp41 ectodomain.     

Asymmetric Deactivation of HIV-1 gp41 following Fusion Inhibitor Binding

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.     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.     

SBi4211阻断HIV-1 gp41促进新生隐球菌对人脑微血管内皮细胞的黏附作用

【背景】目前艾滋病和新型隐球菌性脑膜炎共病因素导致其高发病率和死亡率的机制尚不明确。【目的】探索S100B抑制剂SBi4211对HIV-1 gp41促进新生隐球菌黏附人脑微血管内皮细胞的影响和可能机制。【方法】黏附实验分析SBi4211是否能阻断HIV-1 gp41诱导下新生隐球菌黏附人脑微血管内皮细胞。使用免疫印迹方法进一步检测在此过程中SBi4211对脑微血管内皮细胞上新生隐球菌透明质酸受体CD44表达的影响。【结果】SBi4211可显著抑制HIV-1gp41对新生隐球菌黏附脑微血管内皮细胞的增强作用,且呈时间、剂量效应(P0.05);免疫印迹结果显示SBi4211可抑制新生隐球菌和/或HIV-1 gp41增加脑微血管内皮细胞上新生隐球菌透明质酸受体CD44的表达。【结论】SBi4211可通过下调受体CD44来阻断HIV-1 gp41对新生隐球菌黏附人脑微血管内皮细胞的增强效应,这为了解HIV-1与新生隐球菌共病机制及其防治策略提供了新思路。     

人类免疫缺陷病毒1型gp41结构与功能的研究进展

人类免疫缺陷病毒1型(HIV-1)通过其包膜糖蛋白(Env)介导侵入靶细胞.Env由受体特异性结合单位gp120和膜融合单位gp41组成.HIV-1的gp41分为3个功能区:膜外区、跨膜区和膜内区.膜外区是病毒感染时膜融合的主要结构基础;跨膜区通过疏水残基使Env锚定在脂质膜上;膜内区则表现多重功能,参与病毒的感染、复...     

Design, expression, and immunogenicity of a soluble HIV trimeric envelope fragment adopting a prefusion gp41 configuration

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) is comprised of non-covalently associated gp120/gp41 subunits that form trimeric spikes on the virion surface. Upon binding to host cells, Env undergoes a series of structural transitions, leading to gp41 rearrangement necessary for fusion of viral and host membranes. Until now, the prefusion state of gp41 ectodomain (e-gp41) has eluded molecular and structural analysis, and thus assessment of the potential of such an e-gp41 conformer to elicit neutralizing antibodies has not been possible. Considering the importance of gp120 amino (C1) and carboxyl (C5) segments in the association with e-gp41, we hypothesize that these regions are sufficient to maintain e-gp41 in a prefusion state. Based on the available gp120 atomic structure, we designed several truncated gp140 variants by including the C1 and C5 regions of gp120 in a gp41 ectodomain fragment. After iterative cycles of protein design, expression and characterization, we obtained a variant truncated at Lys(665) that stably folds as an elongated trimer under physiologic conditions. Several independent biochemical/biophysical analyses strongly suggest that this mini-Env adopts a prefusion e-gp41 configuration that is strikingly distinct from the postfusion trimer-of-hairpin structure. Interestingly, this prefusion mini-Env, lacking the fragment containing the 2F5/4E10 neutralizing monoclonal antibody binding sites, displays no detectable HIV-neutralizing epitopes when employed as an immunogen in rabbits. The result of this immunogenicity study has important implications for HIV-1 vaccine design efforts. Moreover, this engineered mini-Env protein should facilitate three-dimensional structural studies of the prefusion e-gp41 and serve to guide future attempts at pharmacologic and immunologic intervention of HIV-1.     

Identification of the HIV-1 gp41 core-binding motif--HXXNPF

The HIV-1 gp41 core, a six-helix bundle formed between the N- and C-terminal heptad repeats, plays a critical role in fusion between the viral and target cell membranes. Using N36(L8)C34 as a model of the gp41 core to screen phage display peptide libraries, we identified a common motif, HXXNPF (X is any of the 20 natural amino acid residues). A selected positive phage clone L7.8 specifically bound to N36(L8)C34 and this binding could be blocked by a gp41 core-specific monoclonal antibody (NC-1). JCH-4, a peptide containing HXXNPF motif, effectively inhibited HIV-1 envelope glycoprotein-mediated syncytium-formation. The epitope of JCH-4 was proven to be linear and might locate in the NHR regions of the gp41 core. These data suggest that HXXNPF motif may be a gp41 core-binding sequence and HXXNPF motif-containing molecules can be used as probes for studying the role of the HIV-1 gp41 core in membrane fusion process.     

Helical interactions in the HIV-1 gp41 core reveal structural basis for the inhibitory activity of gp41 peptides

The HIV-1 gp41 envelope protein mediates membrane fusion that leads to virus entry into the cell. The core structure of fusion-active gp41 is a six-helix bundle in which an N-terminal three-stranded coiled coil is surrounded by a sheath of antiparallel C-terminal helices. A conserved glutamine (Gln 652) buried in this helical interface replaced by leucine increases HIV-1 infectivity. To define the basis for this enhanced membrane fusion activity, we investigate the role of the Gln 652 to Leu substitution on the conformation, stability, and biological activity of the N34(L6)C28 model of the gp41 ectodomain core. The 2.0 A resolution crystal structure of the mutant molecule shows that the Leu 652 side chains make prominent contacts with hydrophobic grooves on the surface of the central coiled coil. The Gln 652 to Leu mutation leads to a marginal stabilization of the six-helix bundle by -0.8 kcal/mol, evaluated from thermal unfolding experiments. Strikingly, the mutant N34(L6)C28 peptide is a potent inhibitor of HIV-1 infection, with 10-fold greater activity than the wild-type molecule. This inhibitory potency can be traced to the corresponding C-terminal mutant peptide that likely has greater potential to interact with the coiled-coil trimer. These results provide strong evidence that conserved interhelical packing interactions in the gp41 core are important determinants of HIV-1 entry and its inhibition. These interactions also offer a test-bed for the development of more potent analogues of gp41 peptide inhibitors.     

Two interaction modes of the gp41-derived peptides with gp41 and their correlation with antimembrane fusion activity

Peptides derived from gp41 effectively block the gp41-mediated cell fusion or HIV infection. A 36-mer (naDP178), 51-mer (C51) and 27-mer peptide (C27) from the membrane proximal region of gp41 have been examined their interaction modes with the coiled-coil motif of gp41 presented in thioredoxin (Trx-N) or the bacterially expressed ectodomain of gp41 (Ec-gp41ec). All of these peptides effectively inhibited the gp41-mediated membrane fusion, however, they showed distinct interaction modes with Ec-gp41ec or Trx-N. C51 peptide bound tightly to Trx-N, and it increased the solubility of Ec-gp41ec. naDP178 showed very weak binding affinity to Trx-N, however, it effectively solubilized Ec-gp41ec. In contrast, C27 peptide showed significant binding to Trx-N; however, it did not affect the solubility of Ec-gp41ec. These interaction modes of C-peptides were assumed to be related to their different inhibitory mechanism against gp41-mediated cell fusion.     

Determinants of Human Immunodeficiency Virus Type 1 Resistance to gp41-Derived Inhibitory Peptides

A synthetic peptide, DP178, containing amino acids 127 to 162 of the human immunodeficiency virus type 1 (HIV-1) gp41 Env glycoprotein, is a potent inhibitor of virus infection and virus mediated cell-to-cell fusion (C. Wild, T. Greenwell, and T. Matthews, AIDS Res. Hum. Retroviruses 9:1051–1053, 1993). In an effort to understand the mechanism of action of this peptide, we derived resistant variants of HIV-1_IIIB and NL4-3 by serial virus passage in the presence of increasing doses of the peptide. Sequence analysis of the resistant isolates suggested that a contiguous 3-amino-acid sequence within the amino-terminal heptad repeat motif of gp41 was associated with resistance. Site-directed mutagenesis studies confirmed this observation and indicated that changes in two of these three residues were necessary for development of the resistant phenotype. Direct binding of DP178 to recombinant protein and synthetic peptide analogs containing the wild-type and mutant heptad repeat sequences revealed a strong correlation between DP178 binding and the biological sensitivity of the corresponding virus isolates to DP178. The results are discussed from the standpoints of the mechanism of action of DP178 and recent crystallographic information for a core structure of the gp41 ectodomain.     

Subdomain folding and biological activity of the core structure from human immunodeficiency virus type 1 gp41: implications for viral membrane fusion

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of two subunits, gp120 and gp41. The extraviral portion (ectodomain) of gp41 contains an alpha-helical domain that likely represents the core of the fusion-active conformation of the molecule. Here we report the identification and characterization of a minimal, autonomous folding subdomain that retains key determinants in specifying the overall fold of the gp41 ectodomain core. This subdomain, designated N34(L6)C28, is formed by covalent attachment of peptides N-34 and C-28 by a short flexible linker in place of the normal disulfide-bonded loop sequence. N34(L6)C28 forms a highly thermostable, alpha-helical trimer. Point mutations within the envelope protein complex that abolish membrane fusion and HIV-1 infectivity also impede the formation of the N34(L6)C28 core. Moreover, N34(L6)C28 is capable of inhibiting HIV-1 envelope-mediated membrane fusion. Taken together, these results indicate that the N34(L6)C28 core plays a direct role in the membrane fusion step of HIV-1 infection and thus provides a molecular target for the development of antiviral pharmaceutical agents.     

Designing a Soluble Near Full-length HIV-1 gp41 Trimer

The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates virus entry by binding to host receptors, whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has been extensively studied, the detailed mechanism is still poorly understood. Design of gp41 recombinants that mimic key intermediates is essential to elucidate the mechanism as well as to develop potent therapeutics and vaccines. Here, using molecular genetics and biochemical approaches, a series of hypotheses was tested to overcome the extreme hydrophobicity of HIV-1 gp41 and design a soluble near full-length gp41 trimer. The two long heptad repeat helices HR1 and HR2 of gp41 ectodomain were mutated to disrupt intramolecular HR1-HR2 interactions but not intermolecular HR1-HR1 interactions. This resulted in reduced aggregation and improved solubility. Attachment of a 27-amino acid foldon at the C terminus and slow refolding channeled gp41 into trimers. The trimers appear to be stabilized in a prehairpin-like structure, as evident from binding of a HR2 peptide to exposed HR1 grooves, lack of binding to hexa-helical bundle-specific NC-1 mAb, and inhibition of virus neutralization by broadly neutralizing antibodies 2F5 and 4E10. Fusion to T4 small outer capsid protein, Soc, allowed display of gp41 trimers on the phage nanoparticle. These approaches for the first time led to the design of a soluble gp41 trimer containing both the fusion peptide and the cytoplasmic domain, providing insights into the mechanism of entry and development of gp41-based HIV-1 vaccines.     

Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein interacts with the viral receptor (CD4) and with the gp41 transmembrane envelope glycoprotein. To study the interaction of the gp120 and gp41 envelope glycoproteins, we compared the abilities of anti-gp120 monoclonal antibodies to bind soluble gp120 and a soluble glycoprotein, sgp140, that contains gp120 and gp41 exterior domains. The occlusion or alteration of a subset of gp120 epitopes on the latter molecule allowed the definition of a gp41 "footprint" on the gp120 antibody competition map. The occlusion of these epitopes on the sgp140 glycoprotein was decreased by the binding of soluble CD4. The gp120 epitopes implicated in the interaction with the gp41 ectodomain were disrupted by deletions of the first (C1) and fifth (C5) conserved gp120 regions. These deletions did not affect the integrity of the discontinuous binding sites for CD4 and neutralizing monoclonal antibodies. Thus, the gp41 interface on the HIV-1 gp120 glycoprotein, which elicits nonneutralizing antibodies, can be removed while retaining immunologically desirable gp120 structures.     

Target cell-specific determinants of membrane fusion within the human immunodeficiency virus type 1 gp120 third variable region and gp41 amino terminus.

The entry of human immunodeficiency virus type 1 (HIV-1) into target cells involves binding to the viral receptor (CD4) and membrane fusion events, the latter influenced by target cell factors other than CD4. The third variable (V3) region of the HIV-1 gp120 exterior envelope glycoprotein and the amino terminus of the HIV-1 gp41 transmembrane envelope glycoprotein have been shown to be important for the membrane fusion process. Here we demonstrate that some HIV-1 envelope glycoproteins containing an altered V3 region or gp41 amino terminus exhibit qualitatively different abilities to mediate syncytium formation and virus entry when different target cells are used. These results demonstrate that the structure of these HIV-1 envelope glycoprotein regions determines the efficiency of membrane fusion in a target cell-specific manner and support a model in which the gp41 amino terminus interacts directly or indirectly with the target cell during virus entry.     

Interhelical interactions in the gp41 core: implications for activation of HIV-1 membrane fusion

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex (gp120-gp41) promotes viral entry by mediating the fusion of viral and cellular membranes. Formation of a stable trimer-of-hairpins structure in the gp41 ectodomain brings the two membranes into proximity, leading to membrane fusion. The core of this hairpin structure is a six-helix bundle in which three carboxyl-terminal outer helices pack against an inner trimeric coiled coil. Here we investigate the role of these conserved interhelical interactions on the structure and function of both the envelope glycoprotein and the gp41 core. We have replaced each of the eight amino acids at the buried face of the carboxyl-terminal helix with a representative amino acid, alanine. Structural and physicochemical characterization of the alanine mutants shows that hydrophobic interactions are a dominant factor in the stabilization of the six-helix bundle. Alanine substitutions at the Trp628, Trp631, Ile635, and Ile642 residues also affected envelope processing and/or gp120-gp41 association and abrogated the ability of the envelope glycoprotein to mediate cell-cell fusion. These results suggest that the amino-terminal region of the gp41 outer-layer alpha-helix plays a key role in the sequence of events associated with HIV-1 entry and have implications for the development of antibodies and small-molecule inhibitors of this conserved element.     

Functional and structural characterization of HIV-1 gp41 ectodomain regions in phospholipid membranes suggests that the fusion-active conformation is extended

HIV-1 entry into its host cell involves a sequential interaction whereby gp41 is in direct contact with the plasma membrane. Understanding the effect of membrane composition on the fusion mechanism can shed light on the unsolved phases of this complex mechanism. Here, we studied N36, a peptide derived from the N-heptad-repeat (NHR) of the gp41 ectodomain, its six helix bundle (SHB) forming counterpart C34, together with the N-terminal 70-mer wild-type peptide (N70), and additional gp41 ectodomain-derived peptides in the presence of two membranes, modeling inner and outer leaflets of the plasma membrane. Information on the structure of these peptides, their affinity towards phospholipids and their ability to induce vesicle fusion was gathered by a variety of fluorescence, spectroscopic and microscopy methods. We found that N36, having strong affinity towards phospholipids, prominently shifts conformation from alpha-helix in an outer leaflet-like zwitterionic membrane to beta-sheet in a membrane mimicking the negatively charged inner leaflet environment, leading to pronounced fusion-activity. Real-time atomic force microscopy (AFM) was used to study the peptides' effect on the membrane morphology, revealing severe bilayer perturbation and extensive pore formation.We also found, that the N36/C34 core is destabilized by electronegative, but not zwitterionic phospholipids. Taken together, our data suggest that the fusion-active pore forming conformation of gp41 is extended, upstream of the SHB. In this manner, folding of the ectodomain into a SHB might also serve as a negative regulator of fusion by impeding gp41 fusion-active surfaces, thus preventing irreversible damage to the cell membrane. This assumption is supported by the finding that pre-incubation of large unilamellar vesicles (LUV) with C-heptad repeat (CHR)-derived fusion inhibitors reduces the fusogenic activity of N-terminal peptides in a dose-dependant manner, and suggests that CHR-derived fusion inhibitors inhibit HIV entry in an analogous mechanism.     

A Conformation-Specific Monoclonal Antibody Reacting with Fusion-Active gp41 from the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein plays a major role in the membrane fusion step of viral infection. The ectodomain of gp41 contains a six-helix structural domain that likely represents the core of the fusion-active conformation of the molecule. A monoclonal antibody (MAb), designated NC-1, was generated and cloned from a mouse immunized with the model polypeptide N36(L6)C34, which folds into a stable six-helix bundle. NC-1 binds specifically to both the α-helical core domain and the oligomeric forms of gp41. This conformation-dependent reactivity is dramatically reduced by point mutations within the N-terminal coiled-coil region of gp41 which impede formation of the gp41 core. NC-1 binds to the surfaces of HIV-1-infected cells only in the presence of soluble CD4. These results indicate that NC-1 is capable of reacting with fusion-active gp41 in a conformation-specific manner and can be used as a valuable biological reagent for studying the receptor-induced conformational changes in gp41 required for membrane fusion and HIV-1 infection.