Gp41 Fusion Peptide Research Articles

Peptide Bispecifics Inhibiting HIV-1 Infection by an Orthogonal Chemical and Supramolecular Strategy.

Viral infections pose a significant threat to human health, and effective antiviral strategies are urgently needed. Antiviral peptides have emerged as a promising class of therapeutic agents due to their unique properties and mechanisms of action. While effective on their own, combining antiviral peptides may allow us to enhance their potency and to prevent viral resistance. Here, we developed an orthogonal chemical strategy to prepare a heterodimeric peptide conjugate assembled on a protein-based nanoplatform. Specifically, we combined the optimized version of two peptides inhibiting HIV-1 by distinct mechanisms. Virus-inhibitory peptide (VIRIP) is a 20 amino acid fragment of α1-antitrypsin that inhibits HIV-1 by targeting the gp41 fusion peptide. Endogenous peptide inhibitor of CXCR4 (EPI-X4) is a 16-residue fragment of human serum albumin that prevents HIV-1 entry by binding to the viral CXCR4 co-receptor. Optimized forms of both peptides are assembled on supramolecular nanoplatforms through the streptavidin-biotin interaction. We show that the construct consisting of the two different peptides (SAv-VIR-102C9-EPI-X4 JM#173-C) shows increased activity against CCR5- and CXCR4-tropic HIV-1 variants. Our results are a proof of concept that peptides with different modes of action can be assembled on nanoplatforms to enhance their antiviral activity.

Combination of Oleic Acid and the gp41 Fusion Peptide Switches the Phosphatidylethanolamine-Induced Membrane Fusion Mechanism from a Nonclassical to a Classical Stalk Model

Membrane fusion is considered to be one of the crucial processes for the existence of eukaryotes and the entry of enveloped viruses into host cells. The fusion mechanism depends on the lipid composition of the membrane as well as the properties of fusion proteins or peptides. The gp41 fusion peptide from the human immunodeficiency virus (HIV) is known to catalyze membrane fusion by altering the physical properties of the membrane. Earlier, we demonstrated that a membrane containing 30 mol % phosphatidylethanolamine (PE) circumvents the classical stalk model because of its intrinsic negative curvature. In this work, we demonstrated how the gp41 fusion peptide influences the fusion mechanism of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phos-pho¬ethanolamine (DOPE) (70/30 mol %) membranes. We further evaluated the effect of the same peptide on the mechanism of fusion for membranes containing 30 mol % PE and a fatty acid with an intrinsic positive curvature (oleic acid (OA)). Our results show that gp41 switches the fusion mechanism from a nonclassical to a classical stalk model when membranes contain OA, but fails to do so for DOPC/DOPE membranes. This could be due to the extreme influence of the intrinsic negative curvature of PE, which is partially downregulated in the presence of OA.

A small-angle neutron scattering study of the physical mechanism that drives the action of a viral fusion peptide

Viruses have evolved a variety of ways for delivering their genetic cargo to a target cell. One mechanism relies on a short sequence from a protein of the virus that is referred to as a fusion peptide. In some cases, the isolated fusion peptide is also capable of causing membranes to fuse. Infection by HIV-1 involves the 23 amino acid N-terminal sequence of its gp41 envelope protein, which is capable of causing membranes to fuse by itself, but the mechanism by which it does so is not fully understood. Here, a variant of the gp41 fusion peptide that does not strongly promote fusion was studied in the presence of vesicles composed of a mixture of unsaturated lipids and cholesterol by small-angle neutron scattering and circular dichroism spectroscopy to improve the understanding of the mechanism that drives vesicle fusion. The peptide concentration and cholesterol content govern both the peptide conformation and its impact on the bilayer structure. The results indicate that the mechanism that drives vesicle fusion by the peptide is a strong distortion of the bilayer structure by the peptide when it adopts the β-sheet conformation.

In Vitro Evaluation of a Peptide-Mesoporous Silica Nanoparticle Drug Release System against HIV-1

It has been shown that the optimized VIR-576 derivative of the natural HIV-1 entry inhibitor targeting the viral gp41 fusion peptide is safe and effective in infected individuals. However, high doses of this peptide were required, and stability, as well as delivery, must be improved for clinical application. Here, we examined the loading and release of VIR-576 into/from mesoporous silica nanoparticles (MSNs) in vitro. We found that a moderately high peptide loading of 11.5 wt % could be achieved by adsorption from PBS buffer (pH 7.2), i.e., under mild, fully peptide-compatible conditions. The release rate of peptide into the same buffer was slow and the equilibrium concentration as indicated by the adsorption isotherm could not be reached even within 50 h at the particle concentrations studied. However, a faster release was observed at lower particle concentrations, indicating that partial particle dissolution had a positive influence on peptide release. To determine the antiviral activity of VIR-576-loaded MSNs, TZM-bl indicator cells were exposed to HIV-1 and the infection rates were followed as a function of time and VIR-576 concentration. The inhibitory activity observed for VIR-576 released from the MSNs was virtually identical to that of free VIR-576 at the 48 h time point, indicating that (a) VIR-576 was released in an active form from the MSNs, and (b) the release rate in the presence of serum proteins was clearly higher than that observed under protein-free conditions. These observations are discussed based on competitive peptide/protein adsorption, as well as potential influences of serum proteins on the dissolution-reprecipitation of silica under conditions where the total silica concentration is above the saturation level for dissolved silica. Our results highlight the need for studying drug release kinetics in the presence of serum proteins, in order to allow for a better extrapolation of in vitro data to in vivo conditions. Furthermore, due to the high peptide loadings that can be achieved using MSNs as carriers, such a formulation appears promising for local release applications. For systemic administration, however, peptides with a higher potency would be needed, due to their high molar masses limiting the drug loading in terms of moles per gram carrier.

2H nuclear magnetic resonance spectroscopy supports larger amplitude fast motion and interference with lipid chain ordering for membrane that contains β sheet human immunodeficiency virus gp41 fusion peptide or helical hairpin influenza virus hemagglutinin fusion peptide at fusogenic pH

Enveloped viruses are surrounded by a membrane which is obtained from an infected host cell during budding. Infection of a new cell requires joining (fusion) of the virus and cell membranes. This process is mediated by a monotopic viral fusion protein with a large ectodomain outside the virus. The ectodomains of class I enveloped viruses have a N-terminal “fusion peptide” (fp) domain that is critical for fusion and binds to the cell membrane. In this study, 2H NMR spectra are analyzed for deuterated membrane with fp from either HIV gp41 (GP) or influenza hemagglutinin (HA) fusion proteins. In addition, the HAfp samples are studied at more fusogenic pH 5 and less fusogenic pH 7. GPfp adopts intermolecular antiparallel β sheet structure whereas HAfp is a monomeric helical hairpin. The data are obtained for a set of temperatures between 35 and 0 °C using DMPC-d54 lipid with perdeuterated acyl chains. The DMPC has liquid-crystalline (Lα) phase with disordered chains at higher temperature and rippled gel (Pβ′) or gel phase (Lβ′) with ordered chains at lower temperature. At given temperature T, the no peptide and HAfp, pH 7 samples exhibit similar spectral lineshapes. Spectral broadening with reduced temperature correlates with the transition from Lα to Pβ′ and then Lβ′ phases. At given T, the lineshapes are narrower for HAfp, pH 5 vs. no peptide and HAfp, pH 7 samples, and even narrower for the GPfp sample. These data support larger-amplitude fast (>105 Hz) lipid acyl chain motion for samples with fusogenic peptides, and peptide interference with chain ordering. The NMR data of the present paper correlate with insertion of these peptides into the hydrocarbon core of the membrane and support a significant fusion contribution from the resultant lipid acyl chain disorder, perhaps because of reduced barriers between the different membrane topologies in the fusion pathway. Membrane insertion and lipid perturbation appear common to both β sheet and helical hairpin peptides.

Unsaturated Lipid Accelerates Formation of Oligomeric β-Sheet Structure of GP41 Fusion Peptide in Model Cell Membrane.

Membrane fusion of the viral and host cell membranes is the initial step of virus infection and is catalyzed by fusion peptides. Although the β-sheet structure of fusion peptides has been proposed to be the most important fusion-active conformation, it is still very challenging to experimentally identify different types of β-sheet structures at the cell membrane surface in situ and in real time. In this work, we demonstrate that the interface-sensitive amide II spectral signals of protein backbones, generated by the sum frequency generation vibrational spectroscopy, provide a sensitive probe for directly capturing the formation of oligomeric β-sheet structure of fusion peptides. Using human immunodeficiency virus (HIV) glycoprotein GP41 fusing peptide (FP23) as the model, we find that formation speed of oligomeric β-sheet structure depends on lipid unsaturation. The unsaturated lipid such as POPG can accelerate formation of oligomeric β-sheet structure of FP23. The β-sheet structure is more deeply inserted into the hydrophobic region of the POPG bilayer than the α-helical segment. This work will pave the way for future researches on capturing intermediate structures during membrane fusion processes and revealing the fusion mechanism.

Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody.

The HIV-1 envelope (Env) glycoprotein, a (gp120-gp41)3 trimer, mediates fusion of viral and host cell membranes after gp120 binding to host receptor CD4. Receptor binding triggers conformational changes allowing coreceptor (CCR5) recognition through CCR5’s tyrosine-sulfated N-terminus, release of the gp41 fusion peptide, and fusion. We present 3.3Å and 3.5Å cryo-EM structures of E51, a tyrosine-sulfated coreceptor-mimicking antibody, complexed with a CD4-bound open HIV-1 native-like Env trimer. Two classes of asymmetric Env interact with E51, revealing tyrosine-sulfated interactions with gp120 mimicking CCR5 interactions, and two conformations of gp120-gp41 protomers (A and B protomers in AAB and ABB trimers) that differ in their degree of CD4-induced trimer opening and induction of changes to the fusion peptide. By integrating the new structural information with previous closed and open envelope trimer structures, we modeled the order of conformational changes on the path to coreceptor binding site exposure and subsequent viral–host cell membrane fusion.

Strain-Dependent Activation and Inhibition of Human Immunodeficiency Virus Entry by a Specific PF-68742 Stereoisomer.

Human immunodeficiency virus (HIV-1) entry into cells is mediated by the viral envelope glycoprotein (Env) trimer, which consists of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. When gp120 binds sequentially to the receptors CD4 and CCR5 on the target cell, the metastable Env trimer is triggered to undergo entry-related conformational changes. PF-68742 is a small molecule that inhibits the infection of a subset of HIV-1 strains by interfering with an Env function other than receptor binding. Determinants of HIV-1 resistance to PF-68742 map to the disulfide loop and fusion peptide of gp41. Of the four possible PF-68742 stereoisomers, only one, MF275, inhibited the infection of CD4-positive CCR5-positive cells by some HIV-1 strains. MF275 inhibition of these HIV-1 strains occurred after CD4 binding but before the formation of the gp41 six-helix bundle. Unexpectedly, MF275 activated the infection of CD4-negative CCR5-positive cells by several HIV-1 strains resistant to the inhibitory effects of the compound in CD4-positive target cells. In contrast to CD4 complementation by CD4-mimetic compounds, activation of CD4-independent infection by MF275 did not depend upon the availability of the gp120 Phe 43 cavity. Sensitivity to inhibitors indicates that MF275-activated virus entry requires formation/exposure of the gp41 heptad repeat (HR1) as well as CCR5 binding. MF275 apparently activates a virus entry pathway parallel to that triggered by CD4 and CD4-mimetic compounds. Strain-dependent divergence in Env conformational transitions allows different outcomes, inhibition or activation, in response to MF275. Understanding the mechanisms of MF275 activity should assist efforts to optimize its utility.IMPORTANCE Envelope glycoprotein (Env) spikes on the surface of human immunodeficiency virus (HIV-1) bind target cell receptors, triggering changes in the shape of Env. We studied a small molecule, MF275, that also induced shape changes in Env. The consequences of MF275 interaction with Env depended on the HIV-1 strain, with infection by some viruses inhibited and infection by other viruses enhanced. These studies reveal the strain-dependent diversity of HIV-1 Envs as they undergo shape changes in proceeding down the entry pathway. Appreciation of this diversity will assist attempts to develop broadly active inhibitors of HIV-1 entry.

Cholesterol Modulates Membrane Properties and the Interaction of gp41 Fusion Peptide To Promote Membrane Fusion.

An envelope glycoprotein, gp41, is crucial for the entry of human immunodeficiency virus (HIV) into the host cell. The 20-23 N-terminal amino acid sequence of gp41 plays an important role in promoting fusion between viral and host cells. Interestingly, the structure and function of the fusion peptide are extremely sensitive to the characteristics of the lipid environment. In this present work, we have extensively utilized steady-state and time-resolved fluorescence spectroscopy in tandem with molecular dynamics simulation to elucidate peptide binding and peptide-induced perturbation to the membrane. We have used two depth-dependent fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and its trimethylammonium derivative (TMA-DPH), to monitor the effect of peptide binding along the bilayer normal and have reconciled the experimental observation with the insights from the simulated molecular events. We have further monitored the effect of membrane cholesterol on peptide-induced membrane perturbation. The molecular dynamics simulation data show that the peptide alters the membrane properties in the vicinity of the peptide and it penetrates to a larger extent into the bilayer when the membrane contains cholesterol. Our results clearly elucidate that cholesterol alters the membrane physical properties in favor of membrane fusion and interaction pattern of the fusion peptide with the membrane in a concentration-dependent fashion. The role of cholesterol is specifically important as the host eukaryotic cells contain a decent amount of cholesterol that might be critical for the entry of HIV into the host cells.

Exploration of HIV-1 fusion peptide-antibody VRC34.01 binding reveals fundamental neutralization sites.

Antibody binding to a vulnerable site of HIV envelope glycoprotein (Env), the eight N-terminal residues of the gp41 fusion peptide, renders robust HIV neutralization. Here, we theoretically investigate HIV-1 fusion peptide binding to the neutralizing antibody N123-VRC34.01. We explore numerous fusion peptide mutations using all-atom molecular dynamics simulation with explicit-solvent models. Simulation results show that the hydrophobic interaction between Ile515 in the HIV-1 fusion peptide and the antibody VRC34.01 Fab plays an important role in antibody binding. Furthermore, we verify by free energy perturbation (FEP) calculations that two point mutations of Ile515Thr or Ile515Ala can dramatically weaken the binding affinity. Our findings provide new insights into fusion peptide-VRC34.01 binding, which can ultimately be utilized to design effective HIV vaccines.

Gating Charge Calculations by Computational Electrophysiology Simulations

Electrical cell signaling requires adjustment of ion channel, receptor, or transporter function in response to changes in membrane potential. For the majority of such membrane proteins, the molecular details of voltage sensing remain insufficiently understood. Here, we present a molecular dynamics simulation-based method to determine the underlying charge movement across the membrane—the gating charge—by measuring electrical capacitor properties of membrane-embedded proteins. We illustrate the approach by calculating the charge transfer upon membrane insertion of the HIV gp41 fusion peptide, and validate the method on two prototypical voltage-dependent proteins, the Kv1.2 K+ channel and the voltage sensor of the Ciona intestinalis voltage-sensitive phosphatase, against experimental data. We then use the gating charge analysis to study how the T1 domain modifies voltage sensing in Kv1.2 channels and to investigate the voltage dependence of the initial binding of two Na+ ions in Na+-coupled glutamate transporters. Our simulation approach quantifies various mechanisms of voltage sensing, enables direct comparison with experiments, and supports mechanistic interpretation of voltage sensitivity by fractional amino acid contributions.

Changes in lipid bilayer structure caused by the helix-to-sheet transition of an HIV-1 gp41 fusion peptide derivative

HIV-1, like other enveloped viruses, undergoes fusion with the cell membrane to infect it. Viral coat proteins are thought to bind the virus to the membrane and actively fuse the viral and cellular membranes together. The actual molecular mechanism of fusion is challenging to visualize, resulting in the use of model systems. Here, the bilayer curvature modifying properties of a synthetic variant of the HIV-1 gp41 fusion peptide with lipid bilayer vesicles composed of a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) were studied. In 7:3 DMPC:DMPS vesicles made with deuterium-labeled DMPC, the peptide was observed to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel β-sheet. Through the use of small-angle neutron scattering (SANS) and selective deuterium labeling, it was revealed that conformational transition of the peptide is also accompanied by a transition in the structure of the lipid bilayer. In addition to changes in the distribution of the lipid between the leaflets of the vesicle, the SANS data are consistent with two regions having different thicknesses. Of the two different bilayer structures, the one corresponding to the smaller area fraction, being ∼8% of the vesicle area, is much thicker than the remainder of the vesicle, which suggests that there are regions of localized negative curvature similar to what takes place at the point of contact between two membranes immediately preceding fusion.

An HIV-1 antibody from an elite neutralizer implicates the fusion peptide as a site of vulnerability.

The induction by vaccination of broadly neutralizing antibodies (bNAbs) capable of neutralizing various HIV-1 viral strains is challenging, but understanding how a subset of HIV-infected individuals develops bNAbs may guide immunization strategies. Here, we describe the isolation and characterization of the bNAb ACS202 from an elite neutralizer that recognizes a new, trimer-specific and cleavage-dependent epitope at the gp120-gp41 interface of the envelope glycoprotein (Env), involving the glycan N88 and the gp41 fusion peptide. In addition, an Env trimer, AMC011 SOSIP.v4.2, based on early virus isolates from the same elite neutralizer, was constructed, and its structure by cryo-electron microscopy at 6.2 Å resolution reveals a closed, pre-fusion conformation similar to that of the BG505 SOSIP.664 trimer. The availability of a native-like Env trimer and a bNAb from the same elite neutralizer provides the opportunity to design vaccination strategies aimed at generating similar bNAbs against a key functional site on HIV-1.

HIV-1 Escape from a Peptidic Anchor Inhibitor through Stabilization of the Envelope Glycoprotein Spike

Because of the rapid development of HIV-1 drug resistance, new drug targets need to be explored continuously. The fusion peptide of the envelope glycoprotein can be targeted by anchor inhibitors. Here we describe virus escape from the anchor inhibitor VIR165. Interestingly, some escape viruses became dependent on the inhibitor for cell entry. We show that the identified escape mutations stabilize the ground state of the envelope glycoprotein and should thus be useful in the design of stabilized envelope-based HIV vaccines.

A novel bispecific peptide HIV-1 fusion inhibitor targeting the N-terminal heptad repeat and fusion peptide domains in gp41.

HIV-1 fusion with the target cell is initiated by the insertion of the gp41 fusion peptide (FP) into the target cell membrane and the interaction between the gp41N- and C-terminal heptad repeats (NHR and CHR), followed by the formation of the six-helix bundle (6-HB) fusion core. Therefore, both FP and NHR are important targets for HIV-1 fusion inhibitors. Here, we designed and synthesized a dual-target peptidic HIV-1 fusion inhibitor, 4HR-LBD-VIRIP, in which 4HR-LBD is able to bind to the gp41 NHR domain, while VIRIP is able to interact with gp41 FP. We found that 4HR-LBD-VIRIP is about tenfold more potent than 4HR-LBD and VIRIP in inhibiting HIV-1IIIB infection and HIV-1 envelope glycoprotein (Env)-mediated cell-cell fusion, suggesting that this dual-target HIV-1 fusion inhibitor possesses a strong synergistic antiviral effect. A biophysical analysis indicates that 4HR-LBD-VIRIP can interact with N70 peptide that contains the gp41 NHR and FP domains and binds with lipid membrane. This study provides a new approach for designing novel viral fusion inhibitors against HIV and other enveloped viruses with class I membrane fusion proteins.

Single-Chain Soluble BG505.SOSIP gp140 Trimers as Structural and Antigenic Mimics of Mature Closed HIV-1 Env.

Similar to other type I fusion machines, the HIV-1 envelope glycoprotein (Env) requires proteolytic activation; specifically, cleavage of a gp160 precursor into gp120 and gp41 subunits creates an N-terminal gp41 fusion peptide and permits folding from an immature uncleaved state to a mature closed state. While the atomic-level consequences of cleavage for HIV-1 Env are still being determined, the uncleaved state is antigenically distinct from the mature closed state, and cleavage has been reported to be essential for mimicry of the mature viral spike by soluble versions of Env. Here we report the redesign of a current state-of-the-art soluble Env mimic, BG505.SOSIP, to make it cleavage independent. Specifically, we replaced the furin cleavage site between gp120 and gp41 with Gly-Ser linkers of various lengths. The resultant linked gp120-gp41 constructs, termed single-chain gp140 (sc-gp140), exhibited different levels of structural and antigenic mimicry of the parent cleaved BG505.SOSIP. When constructs were subjected to negative selection to remove subspecies recognized by poorly neutralizing antibodies, trimers of high antigenic mimicry of BG505.SOSIP could be obtained; negative-stain electron microscopy indicated these to resemble the mature closed state. Higher proportions of BG505.SOSIP-trimer mimicry were observed in sc-gp140s with linkers of 6 or more residues, with a linker length of 15 residues exhibiting especially promising traits. Overall, flexible linkages between gp120 and gp41 in BG505.SOSIP can thus substitute for cleavage, and sc-gp140s that closely mimicked the vaccine-preferred mature closed state of Env could be obtained. The trimeric HIV-1 envelope glycoprotein (Env) is the sole target of virus-directed neutralizing antibody responses and a primary focus of vaccine design. Soluble mimics of Env have proven challenging to obtain and have been thought to require proteolytic cleavage into two-component subunits, gp120 and gp41, to achieve structural and antigenic mimicry of mature Env spikes on virions. Here we show that replacement of the cleavage site between gp120 and gp41 in a lead soluble gp140 construct, BG505.SOSIP, with flexible linkers can result in molecules that do not require cleavage to fold efficiently into the mature closed state. Our results provide insights into the impact of cleavage on HIV-1 Env folding. In some contexts such as genetic immunization, optimized cleavage-independent soluble gp140 constructs may have utility over the parental BG505.SOSIP, as they would not require furin cleavage to achieve mimicry of mature Env spikes on virions.

HIV gp41 Fusion Peptide Increases Membrane Ordering in a Cholesterol-Dependent Fashion

Fusion between viral envelopes and host cell membranes, which is mediated by special glycoproteins anchored on the viral membrane, is required for HIV viral entry and infection. The HIV gp41 fusion peptide (FP), which initiates membrane fusion, adopts either an α-helical or β-sheeted structure depending on the cholesterol concentration. We used phosphocholine spin labels on the lipid headgroup and different positions on the acyl chain to detect its perturbation on lipid bilayers containing different cholesterol concentrations by electron-spin resonance. Our findings were as follows. 1), gp41 FP affects the lipid order in the same manner as previously shown for influenza hemagglutinin FP, i.e., it has a cooperative effect versus the peptide/lipid ratio, supporting our hypothesis that membrane ordering is a common prerequisite for viral membrane fusion. 2), gp41 FP induces membrane ordering in all lipid compositions studied, whereas a nonfusion mutant FP perturbs lipid order to a significantly smaller extent. 3), In high-cholesterol-containing lipid bilayers, where gp41 FP is in the β-aggregation conformation, its effect on the lipid ordering reaches deeper into the bilayer. The different extent to which the two conformers perturb is correlated with their fusogenicity. The possible role of the two conformers in membrane fusion is discussed.

Dynamics of Bilayer Interactions at the Air-Water Interface

A novel supported bilayer system at the air-water interface has been developed for use in synchrotron X-ray scattering experiments in order to drastically increase signal to noise ratio. This system gives a significant resolution improvement comparing to more traditional solid-supported bilayer system and is better suited for specular X-Ray reflectivity and grazing incidence X-ray off-specular scattering. Using this system we have successfully characterized anionic bilayers mimetic of Gram-positive bacterial membranes interacting with engineered biomimetic oligomers (peptoids), zwitterionic bilayers interacting with human islet amyloid polypeptide (IAPP), and insertion differences between zwitterionic membranes with and without cholesterol and the glycoprotein-41 subunit fragment of the viral envelope glycoprotein-160. Results from anionic and net neutral bilayers correspond to previous studies on monolayer insertion studies, where peptoid ML 2-6 acts by associating throughout the lipophilic regions and zwitterionic bilayers without cholesterol were completely penetrated by the GP-41 fusion peptide. Resolution of the dynamic formation of a zwitterionic lipid bilayer system in minute scale time resolution via Grazing Incidence X-Ray Off-Specular Scattering (GIXOS) was also observed, and found to be in agreement with the proposed mechanisms associated with vesicle fusion on a solid support.

GP41 Ectodomain Dissociates and Forms a Stable Monomer on Phospholipid Vesicles and Detergent Micelles: Implication for the HIV-1 Env-Mediated Membrane Fusion

The first step of HIV infection involves the fusion of the viral and target cell membranes, a process mediated by the viral envelope glycoproteins, gp120 and gp41. The binding of gp120 to the cell surface receptors CD4 triggers a cascade of conformational changes that disrupt the gp41-gp120 interactions and allows the insertion of the N-terminal fusion peptide of gp41 into the host cell membrane. The gp41 trimer then rapidly folds into a 6-helix bundle that pulls together the fusion peptide, inserted in the host cell membrane and the transmembrane domain, located in the viral membrane. Very little structural information is known about the pre-fusion state of gp41 notwithstanding its critical importance for the design of fusion inhibitors. To investigate the dynamics and structural properties of such metastable states, we designed a set of protein constructs mimicking the extracellular ectodomain of gp41. We found that the secondary structure of gp41 ectodomain is pronouncedly impacted by the presence of phospholipid vesicles or detergents containing phosphatidyl choline head groups. Our NMR and multi-angle light scattering data attribute these changes to the transition between a trimer in the absence of detergent to a monomer in the presence of a membrane-mimicking environment. The structure of gp41 in a monomeric state was determined by state-of-the-art NMR techniques, including measurements of NOE distance restraints and residual dipolar couplings in weakly aligned solutions. 15N relaxation data provided a detailed view at the backbone dynamics of both the trimeric and monomeric conformations. This stable monomeric state may represent a crucial structural intermediate facilitating both the gp41 conformational change during fusion and the local apposition of the viral and cellular membranes.

Solid-State NMR Structural Measurements and Models of the HIV and Influenza Fusion Proteins in Membranes

Solid-state NMR (SSNMR) has been applied to probe the structures and membrane locations of domains of the HIV gp41 and the influenza virus HA2 membrane fusion proteins. Advantages of SSNMR include: (1) use of membrane bilayers without detergent and with lipid and cholesterol composition similar to that of host cells; and (2) preparation of samples under conditions very similar to those used for functional vesicle fusion assays. The N-terminal ∼20-residue HA2 fusion peptide (IFP) domain has helix-turn-helix structure and in detergent there are reports of: (1) an open interhelical topology with a hydrophobic interhelical pocket and inverted V membrane insertion; and (2) a closed topology with tightly-packed antiparallel helices and membrane contact with a hydrophobic protein surface. SSNMR measurements in membranes lacking cholesterol show that the closed structure is predominant. Both the IFP and the corresponding N-terminal gp41 fusion peptide (HFP) have predominant oligomeric β sheet structure in membranes with 30 mole% cholesterol which is typical for viral and host cell membranes. SSNMR measurements for HFP show that there is antiparallel arrangement of adjacent stands with a broad distribution of antiparallel registries. The central regions of the antiparallel sheets are deeply inserted in the membrane and contact the methyl termini of the lipid acyl chains. This antiparallel distribution is also observed for a much larger segment of gp41 that includes the fusion peptide. A very different registry distribution is detected for the non-functional V2E mutant and suggests that the amino acid content of the antiparallel sheet is correlated to membrane fusion.