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.     

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.     

Inhibition of Human Immunodeficiency Virus Type 1 Infectivity by the gp41 Core: Role of a Conserved Hydrophobic Cavity in Membrane Fusion

The gp41 envelope protein of human immunodeficiency virus type 1 (HIV-1) contains an alpha-helical core structure responsible for mediating membrane fusion during viral entry. Recent studies suggest that a conserved hydrophobic cavity in the coiled coil of this core plays a distinctive structural role in maintaining the fusogenic conformation of the gp41 molecule. Here we investigated the importance of this cavity in determining the structure and biological activity of the gp41 core by using the N34(L6)C28 model. The high-resolution crystal structures of N34(L6)C28 of two HIV-1 gp41 fusion-defective mutants reveal that each mutant sequence is accommodated in the six-helix bundle structure by forming the cavity with different sets of atoms. Remarkably, the mutant N34(L6)C28 cores are highly effective inhibitors of HIV-1 infection, with 5- to 16-fold greater activity than the wild-type molecule. The enhanced inhibitory activity by fusion-defective mutations correlates with local structural perturbations close to the cavity that destabilize the six-helix bundle. Taken together, these results indicate that the conserved hydrophobic coiled-coil cavity in the gp41 core is critical for HIV-1 entry and its inhibition and provides a potential antiviral drug target.     

Buried polar interactions and conformational stability in the simian immunodeficiency virus (SIV) gp41 core

For human (HIV) and simian (SIV) immunodeficiency viruses, the gp41 envelope protein undergoes a receptor-activated conformational change from a labile native structure to an energetically more stable fusogenic conformation, which then mediates viral-cell membrane fusion. The core structure of fusion-active gp41 is a six-helix bundle in which three antiparallel carboxyl-terminal helices are packed against an amino-terminal trimeric coiled coil. Here we show that a recombinant model of the SIV gp41 core, designated N36(L6)C34, forms an alpha-helical trimer that exhibits a cooperative two-state folding-unfolding transition. We investigate the importance of buried polar interactions in determining the overall fold of the gp41 core. We have replaced each of four polar amino acids at the heptad a and d positions of the coiled coil in N36(L6)C34 with a representative hydrophobic amino acid, isoleucine. The Q565I, T582I, and T586I variants form six-helix bundle structures that are significantly more stable than that of the wild-type peptide, whereas the Q575I variant misfolds into an insoluble aggregate under physiological conditions. Thus, the buried polar residues within the amino-terminal heptad repeat are important determinants of the structural specificity and stability of the gp41 core. We suggest that these conserved buried polar interactions play a role in governing the conformational state of the gp41 molecule.     

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.     

Identification of a critical motif for the human immunodeficiency virus type 1 (HIV-1) gp41 core structure: implications for designing novel anti-HIV fusion inhibitors

Human immunodeficiency virus type 1 (HIV-1) entry into the host cell involves a cascade of events and currently represents one of most attractive targets in the search for new antiviral drugs. The fusion-active gp41 core structure is a stable six-helix bundle (6-HB) folded by its trimeric N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR). Peptides derived from the CHR region of HIV-1 gp41 are potent fusion inhibitors that target the NHR to block viral and cellular membrane fusion in a dominant negative fashion. However, all CHR peptides reported to date are derived primarily from residues 628 to 673 of gp41; little attention has been paid to the upstream sequence of the pocket binding domain (PBD) in the CHR. Here, we have identified a motif ((621)QIWNNMT(627)) located at the upstream region of the gp41 CHR, immediately adjacent to the PBD ((628)WMEWEREI(635)). Biophysical characterization demonstrated that this motif is critical for the stabilization of the gp41 6-HB core. The peptide CP621-652, containing the (621)QIWNNMT(627) motif, was able to interact with T21, a counterpart peptide derived from the NHR, to form a typical 6-HB structure with a high thermostability (thermal unfolding transition [T(m)] value of 82 degrees C). In contrast, the 6-HB formed by the peptides N36 and C34, which has been considered to be a core structure of the fusion-active gp41, had a T(m) of 64 degrees C. Different from T-20 (brand name Fuseon), which is the first and only HIV-1 fusion inhibitor approved for clinical use, CP621-652 could efficiently block 6-HB formation in a dose-dependent manner. Significantly, CP621-652 had potent inhibitory activity against HIV-1-mediated cell-cell fusion and infection, especially against T-20- and C34-resistant virus. Therefore, our works provide important information for understanding the core structure of the fusion-active gp41 and for designing novel anti-HIV peptides.     

Comparative Analysis of Membrane-Associated Fusion Peptide Secondary Structure and Lipid Mixing Function of HIV gp41 Constructs that Model the Early Pre-Hairpin Intermediate and Final Hairpin Conformations

Fusion between viral and host cell membranes is the initial step of human immunodeficiency virus infection and is mediated by the gp41 protein, which is embedded in the viral membrane. The ∼ 20-residue N-terminal fusion peptide (FP) region of gp41 binds to the host cell membrane and plays a critical role in fusion catalysis. Key gp41 fusion conformations include an early pre-hairpin intermediate (PHI) characterized by extended coiled-coil structure in the region C-terminal of the FP and a final hairpin state with compact six-helix bundle structure. The large “N70” (gp41 1-70) and “FP-Hairpin” constructs of the present study contained the FP and respectively modeled the PHI and hairpin conformations. Comparison was also made to the shorter “FP34” (gp41 1-34) fragment. Studies were done in membranes with physiologically relevant cholesterol content and in membranes without cholesterol. In either membrane type, there were large differences in fusion function among the constructs with little fusion induced by FP-Hairpin, moderate fusion for FP34, and very rapid fusion for N70. Overall, our findings support acceleration of gp41-induced membrane fusion by early PHI conformation and fusion arrest after folding to the final six-helix bundle structure. FP secondary structure at Leu7 of the membrane-associated constructs was probed by solid-state nuclear magnetic resonance and showed populations of molecules with either β-sheet or helical structure with greater β-sheet population observed for FP34 than for N70 or FP-Hairpin. The large differences in fusion function among the constructs were not obviously correlated with FP secondary structure. Observation of cholesterol-dependent FP structure for fusogenic FP34 and N70 and cholesterol-independent structure for non-fusogenic FP-Hairpin was consistent with membrane insertion of the FP for FP34 and N70 and with lack of insertion for FP-Hairpin. Membrane insertion of the FP may therefore be associated with the early PHI conformation and FP withdrawal with the final hairpin conformation.     

Conserved salt bridge between the N- and C-terminal heptad repeat regions of the human immunodeficiency virus type 1 gp41 core structure is critical for virus entry and inhibition

The fusogenic human immunodeficiency virus type 1 (HIV-1) gp41 core structure is a stable six-helix bundle formed by its N- and C-terminal heptad repeat sequences. Notably, the negatively charged residue Asp⁶³² located at the pocket-binding motif in the C-terminal heptad repeat interacts with the positively charged residue Lys⁵⁷⁴ in the pocket formation region of the N-terminal heptad repeat to form a salt bridge. We previously demonstrated that the residue Lys⁵⁷⁴ plays an essential role in six-helix bundle formation and virus infectivity and is a key determinant of the target for anti-HIV fusion inhibitors. In this study, the functionality of residue Asp⁶³² has been specifically characterized by mutational analysis and biophysical approaches. We show that Asp⁶³² substitutions with positively charged residues (D632K and D632R) or a hydrophobic residue (D632V) could completely abolish Env-mediated viral entry, while a protein with a conserved substitution (D632E) retained its activity. Similar to the Lys⁵⁷⁴ mutations, nonconserved substitutions of Asp⁶³² also severely impaired the α-helicity, stability, and conformation of six-helix bundles as shown by N36 and C34 peptides as a model system. Furthermore, nonconserved substitutions of Asp⁶³² significantly reduced the potency of C34 to sequestrate six-helix bundle formation and to inhibit HIV-1-mediated cell-cell fusion and infection, suggesting its importance for designing antiviral fusion inhibitors. Taken together, these data suggest that the salt bridge between the N- and C-terminal heptad repeat regions of the fusion-active HIV-1 gp41 core structure is critical for viral entry and inhibition.     

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.     

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.     

Mutations that destabilize the gp41 core are determinants for stabilizing the simian immunodeficiency virus-CPmac envelope glycoprotein complex

The human and simian immunodeficiency viruses (HIV and SIV) envelope glycoprotein consists of a trimer of two noncovalently and weakly associated subunits, gp120 and gp41. Upon binding of gp120 to cellular receptors, this labile native envelope complex undergoes conformational changes, resulting in a stable trimer-of-hairpins structure in gp41. Formation of the hairpin structure is thought to mediate membrane fusion by placing the viral and cellular membranes in close proximity. An in vitro-derived variant of SIVmac251, denoted CPmac, has acquired an unusually stable virion-associated gp120-gp41 complex. This unique phenotype is conferred by five amino acid substitutions in the gp41 ectodomain. Here we characterize the structural and physicochemical properties of the N40(L6)C38 model of the CPmac gp41 core. The 1.7-A resolution crystal structure of N40(L6)C38 is very similar to the six-helix bundle structure present in the parent SIVmac251 gp41. In both structures, three N40 peptides form a central three-stranded coiled coil, and three C38 peptides pack in an antiparallel orientation into hydrophobic grooves on the coiled-coil surface. Thermal unfolding studies show that the CPmac mutations destabilize the SIVmac251 six-helix bundle by 15 kJ/mol. Our results suggest that the formation of the gp41 trimer-of-hairpins structure is thermodynamically coupled to the conformational stability of the native envelope glycoprotein and raise the intriguing possibility that introduction of mutations to destabilize the six-helix bundle may lead to the stabilization of the trimeric gp120-gp41 complex. This study suggests a potential strategy for the production of stably folded envelope protein immunogens for HIV vaccine development.     

The stability of the intact envelope glycoproteins is a major determinant of sensitivity of HIV/SIV to peptidic fusion inhibitors

C-peptides derived from the HIV envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (C-HR) region are potent HIV fusion inhibitors. These peptides interact with the gp41 N-terminal heptad repeat (N-HR) region and block the gp41 six-helix bundle formation that is required for fusion. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by comparing the ability of C34, derived from HIV-1, HIV-2 and SIV gp41, to inhibit HIV-1, HIV-2 and SIV envelope-mediated fusion and the ability of these peptides to form stable six-helix bundles with N36 peptides derived from gp41 of these three viruses. The ability to form six-helix bundles was examined by circular dichroism spectroscopy, and HIV/SIV Env-mediated membrane fusion was monitored by a dye transfer assay. HIV-1 N36 formed stable helix bundles with HIV-1, HIV-2 and SIV C34, which all inhibited HIV-1 Env-mediated fusion at IC(50)<10nM. The three C34 peptides were poor inhibitors of HIV-2 and SIV fusion (IC(50)>100nM), although HIV-2 and SIV N36 formed stable helix bundles with SIV C34. Priming experiments with sCD4 indicate that, in contrast to HIV-1, HIV-2 and SIV Env do not expose their N-HR region to SIV C34 following CD4 binding, but rapidly proceed to co-receptor engagement and six-helix bundle formation resulting in fusion. Our results suggest that several factors, including six-helix bundle stability and the ability of CD4 to destabilize the envelope glycoprotein, serve as determinants of sensitivity to entry inhibitors.     

Rapid and automated fluorescence-linked immunosorbent assay for high-throughput screening of HIV-1 fusion inhibitors targeting gp41

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 plays an important role in the virus entry. During the process of fusion between the viral and target cell membranes, the N- and C-terminal heptad repeat (HR) regions of the gp41 extracellular domain associate to form a 6-helical bundle, corresponding to the fusion-active gp41 core. Any compound that blocks the gp41 6-helix bundle formation between the N- and C-peptides, which are derived from the N- and C-terminal HR regions, respectively, may inhibit HIV-1 mediated membrane fusion. Based on this principle, we previously established a sandwich enzyme-linked immunosorbent assay (ELISA) for drug screening by using the N-peptide N36 and the C-peptide C34 and a monoclonal antibody (NC-1) which specifically recognizes the gp41 6-helix bundle. In the present study, a fluorescence-linked immunosorbent assay (FLISA) was developed by using fluorescein isothiocyanate (FITC)-conjugated C34 to replace C34 and by directly detecting fluorescence intensity instead of more complicated enzymatic reaction. Compared with the sandwich ELISA, this FLISA has similar sensitivity and specificity, but it is much more rapid, economic and convenient. Using an Integrated Robotic Sample Processing System, this assay has been applied for high-throughput screening of organic compounds on a large scale for HIV-1 fusion inhibitors targeting gp41.     

HIV-1 variants with a single-point mutation in the gp41 pocket region exhibiting different susceptibility to HIV fusion inhibitors with pocket- or membrane-binding domain

Enfuvirtide (T20), the first FDA-approved peptide HIV fusion/entry inhibitor derived from the HIV-1 gp41 C-terminal heptad-repeat (CHR) domain, is believed to share a target with C34, another well-characterized CHR-peptide, by interacting with the gp41 N-terminal heptad-repeat (NHR) to form six-helix bundle core. However, our previous studies showed that T20 mainly interacts with the N-terminal region of the NHR (N-NHR) and lipid membranes, while C34 mainly binds to the NHR C-terminal pocket region. But so far, no one has shown that C34 can induce drug-resistance mutation in the gp41 pocket region. In this study, we constructed pseudoviruses in which the Ala at the position of 67 in the gp41 pocket region was substituted with Asp, Gly or Ser, respectively, and found that these mutations rendered the viruses highly resistant to C34, but sensitive to T20. The NHR-peptide N36 with mutations of A67 exhibited reduced anti-HIV-1 activity and decreased α-helicity. The stability of six-helix bundle formed by C34 and N36 with A67 mutations was significantly lower than that formed by C34 and N36 with wild-type sequence. The combination of C34 and T20 resulted in potent synergistic anti-HIV-1 effect against the viruses with mutations in either N- or C-terminal region in NHR. These results suggest that C34 with a pocket-binding domain and T20 containing the N-NHR- and membrane-binding domains inhibit HIV-1 fusion by interacting with different target sites and the combinatorial use of C34 and T20 is expected to be effective against HIV-1 variants resistant to HIV fusion inhibitors.     

Determination of the HIV-1 gp41 fusogenic core conformation modeled by synthetic peptides: applicable for identification of HIV-1 fusion inhibitors

Triggered by receptor binding of gp120, the human immunodeficiency virus type 1 (HIV-1) gp41 changes its conformation to a fusogenic six-helix bundle structure. In the present study, this core conformation modeled by the peptides derived from the gp41 N- and C-terminal heptad repeat regions was determined by fluorescence native polyacrylamide gel electrophoresis and size exclusion high-performance liquid chromatography (HPLC). Two previously described small molecule HIV-1 fusion inhibitors significantly blocked the six-helix bundle formation. It suggests that these biophysical techniques can be used in a novel way to study the conformational change of gp41 during virus entry into cells and to identify HIV-1 fusion inhibitors.     

Conserved residue Lys574 in the cavity of HIV-1 Gp41 coiled-coil domain is critical for six-helix bundle stability and virus entry

The fusion-active HIV-1 gp41 core structure is a stable six-helix bundle (6-HB) formed by its N- and C-terminal heptad-repeat sequences (NHR and CHR). A highly conserved, deep hydrophobic cavity on the surface of the N-helical trimer is important for stability of the 6-HB and serves as an ideal target for developing anti-human immunodeficiency virus (HIV) fusion inhibitors. We have recently identified several small molecule HIV-1 fusion inhibitors that bind to the gp41 cavity through hydrophobic and ionic interactions and block the gp41 6-HB formation. Molecular docking analysis reveals that these small molecules fit inside the hydrophobic cavity and interact with positively charged residue Lys574 to form a conserved salt bridge. In this study, the functionality of Lys574 has been finely characterized by mutational analysis and biophysical approaches. We found that substitutions of Lys574 with non-conserved residues (K574D, K574E, and K574V) could completely abolish virus infectivity. With a set of wild-type and mutant N36 peptides derived from the NHR sequence as a model, we demonstrated that non-conservative Lys574 substitutions severely impaired the stability and conformation of 6-HBs as detected by circular dichroism spectroscopy, native polyacrylamide gel electrophoresis, and enzyme-linked immunosorbent assay. The binding affinity of N36 mutants bearing non-conservative Lys574 substitutions to the peptide C34 derived from the CHR sequence dramatically decreased as measured by isothermal titration calorimetry. These substitutions also significantly reduced the potency of N-peptides to inhibit HIV-1 infection. Collectively, these data suggest that conserved Lys574 plays a critical role in 6-HB formation and HIV-1 infectivity, and may serve as an important target for designing anti-HIV drugs.     

Theaflavin derivatives in black tea and catechin derivatives in green tea inhibit HIV-1 entry by targeting gp41

Theaflavin derivatives and catechin derivatives are the major polyphenols in black tea and green tea, respectively. Several tea polyphenols, especially those with galloyl moiety, can inhibit HIV-1 replication with multiple mechanisms of action. Here we showed that the theaflavin derivatives had more potent anti-HIV-1 activity than catechin derivatives. These tea polyphenols could inhibit HIV-1 entry into target cells by blocking HIV-1 envelope glycoprotein-mediated membrane fusion. The fusion inhibitory activity of the tea polyphenols was correlated with their ability to block the formation of the gp41 six-helix bundle, a fusion-active core conformation. Computer-aided molecular docking analyses indicate that these tea polyphenols, theaflavin-3,3'-digallate (TF3) as an example, may bind to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled coil formed by the N-terminal heptad repeats of gp41. These results indicate that tea, especially black tea, may be used as a source of anti-HIV agents and theaflavin derivatives may be applied as lead compounds for developing HIV-1 entry inhibitors targeting gp41.     

The membrane proximal external region of the HIV-1 envelope glycoprotein gp41 contributes to the stabilization of the six-helix bundle formed with a matching N' peptide

The HIV-1 envelope glycoprotein gp41 undergoes a sequence of extensive conformational changes while participating in the fusion of the virus with the host cell. Since the discovery of its postfusion conformation, the structure and function of the protease-resistant six-helix bundle (6-HB) have been the subject of extensive investigation. In this work, we describe additional determinants (S528-Q540 and W666-N677) in the fusion peptide proximal region (FP-PR) and the membrane proximal external region (MPER) that stabilize the six-helix bundle and are involved in the interaction of T-20 (FUZEON, an anti-HIV-1 fusion inhibitor drug) with the gp41 FP-PR. Circular dichroism and sedimentation equilibrium measurements indicate that the 1:1 mixture of N' and C' peptides comprising residues A541-T569 and I635-K665 from the gp41 first and second helical repeats, HR1 and HR2, respectively, fail to form a stable six-helix bundle. Triglutamic acid and triarginine tags were added to these N' and C' peptides, respectively, at the termini distant from the FP-PR and the MPER to alter their pI and increase their solubility at pH 3.5. The tagged HR1 and HR2 peptides were elongated by addition of residues S528-Q540 from the FP-PR and residues W666-N677 from the MPER, respectively. A 1:1 complex of the elongated peptides formed a stable six-helix bundle which melted at 60 degrees C. These results underscore the importance of a detailed high-resolution characterization of MPER interactions, the results of which may improve our understanding of the structure-function relationship of gp41 and its role in HIV-1 fusion.     

Identification of the HIV-1 gp41 core-binding motif in the scaffolding domain of caveolin-1

The human immunodeficiency virus, type 1 (HIV-1) gp41 core plays an important role in fusion between viral and target cell membranes. A single chain polypeptide, N36(L8)C34, which forms a six-helix bundle in physiological solution, can be used as a model of gp41 core. Here we identified from a 12-mer phage peptide library a positive phage clone displaying a peptide sequence with high binding activity to the HIV-1 gp41 core. The peptide sequence contains a putative gp41-binding motif, PhiXXXXPhiXPhi (X is any amino acid residue, and Phi is any one of the aromatic amino acid residues Trp, Phe, or Tyr). This motif also exists in the scaffolding domain of caveolin-1 (Cav-1), a known gp41-binding protein. Cav-1-(61-101) and Cav-1-(82-101), two recombinant fusion proteins containing the Cav-1 scaffolding domain, bound significantly to the gp41 expressed in mammalian cells and interacted with the polypeptide N36(L8)C34. These results suggest that the scaffolding domain of Cav-1 may bind to the gp41 core via the motif. This interaction may be essential for formation of fusion pore or endocytosis of HIV-1 and affect the pathogenesis of HIV-1 infection. Further characterization of the gp41 core-binding motifs may shed light on the alternative mechanism by which HIV-1 enters into the target cell.