Structure-Related Roles for the Conservation of the HIV-1 Fusion Peptide Sequence Revealed by Nuclear Magnetic Resonance.

Despite extensive characterization of the human immunodeficiency virus type 1 (HIV-1) hydrophobic fusion peptide (FP), the structure-function relationships underlying its extraordinary degree of conservation remain poorly understood. Specifically, the fact that the tandem repeat of the FLGFLG tripeptide is absolutely conserved suggests that high hydrophobicity may not suffice to unleash FP function. Here, we have compared the nuclear magnetic resonance (NMR) structures adopted in nonpolar media by two FP surrogates, wtFP-tag and scrFP-tag, which had equal hydrophobicity but contained wild-type and scrambled core sequences LFLGFLG and FGLLGFL, respectively. In addition, these peptides were tagged at their C-termini with an epitope sequence that folded independently, thereby allowing Western blot detection without interfering with FP structure. We observed similar α-helical FP conformations for both specimens dissolved in the low-polarity medium 25% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), but important differences in contact with micelles of the membrane mimetic dodecylphosphocholine (DPC). Thus, whereas wtFP-tag preserved a helix displaying a Gly-rich ridge, the scrambled sequence lost in great part the helical structure upon being solubilized in DPC. Western blot analyses further revealed the capacity of wtFP-tag to assemble trimers in membranes, whereas membrane oligomers were not observed in the case of the scrFP-tag sequence. We conclude that, beyond hydrophobicity, preserving sequence order is an important feature for defining the secondary structures and oligomeric states adopted by the HIV FP in membranes.

Envelope glycoprotein mobility on HIV-1 particles depends on the virus maturation state.

Human immunodeficiency virus type 1 (HIV-1) assembles as immature particles, which require the proteolytic cleavage of structural polyprotein Gag and the clustering of envelope glycoprotein Env for infectivity. The details of mechanisms underlying Env clustering remain unknown. Here, we determine molecular dynamics of Env on the surface of individual HIV-1 particles using scanning fluorescence correlation spectroscopy on a super-resolution STED microscope. We find that Env undergoes a maturation-induced increase in mobility, highlighting diffusion as one cause for Env clustering. This mobility increase is dependent on Gag-interacting Env tail but not on changes in viral envelope lipid order. Diffusion of Env and other envelope incorporated proteins in mature HIV-1 is two orders of magnitude slower than in the plasma membrane, indicating that HIV-1 envelope is intrinsically a low mobility environment, mainly due to its general high lipid order. Our results provide insights into dynamic properties of proteins on the surface of individual virus particles.To become infectious, HIV-1 particles undergo a maturation process involving the clustering of envelope glycoprotein Env. Here, Chojnacki et al. employ super-resolution STED-FCS microscopy to study dynamics of Env molecules on HIV-1 particles and show that Env undergoes a maturation-induced increase in mobility.

Effects of HIV-1 gp41-Derived Virucidal Peptides on Virus-like Lipid Membranes.

Membrane fusion induced by the envelope glycoprotein enables the intracellular replication of HIV-1; hence, this process constitutes a major target for antiretroviral compounds. It has been proposed that peptides having propensity to interact with membrane interfaces might exert broad antiviral activity against enveloped viruses. To test this hypothesis, in this contribution we have analyzed the antiviral effects of peptides derived from the membrane-proximal external region and the transmembrane domain of the envelope glycoprotein subunit gp41, which display different degrees of interfacial hydrophobicity. Our data support the virucidal activity of a region that combines hydrophobic-at-interface membrane-proximal external region aromatics with hydrophobic residues of the transmembrane domain, and contains the absolutely conserved 679LWYIK/R683 sequence, proposed to embody a "cholesterol recognition/interaction amino acid consensus" motif. We further sought to correlate the antiviral activity of these peptides and their effects on membranes that mimic lipid composition and biophysical properties of the viral envelope. The data revealed that peptides endowed with virucidal activity were membrane active and induced permeabilization and fusion of virus-like lipid vesicles. In addition, they modulated lipid packing and miscibility of laterally segregated liquid domains, two properties that depend on the high cholesterol content of the viral membrane. Thus, the overall experimental evidence is consistent with a pattern of HIV inhibition that involves direct alteration of the physical chemistry of the virus membrane. Furthermore, the sequence-dependent effects observed might guide the development of new virucidal peptides.

Functional organization of the HIV lipid envelope.

The chemical composition of the human immunodeficiency virus type 1 (HIV-1) membrane is critical for fusion and entry into target cells, suggesting that preservation of a functional lipid bilayer organization may be required for efficient infection. HIV-1 acquires its envelope from the host cell plasma membrane at sites enriched in raft-type lipids. Furthermore, infectious particles display aminophospholipids on their surface, indicative of dissipation of the inter-leaflet lipid asymmetry metabolically generated at cellular membranes. By combining two-photon excited Laurdan fluorescence imaging and atomic force microscopy, we have obtained unprecedented insights into the phase state of membranes reconstituted from viral lipids (i.e., extracted from infectious HIV-1 particles), established the role played by the different specimens in the mixtures, and characterized the effects of membrane-active virucidal agents on membrane organization. In determining the molecular basis underlying lipid packing and lateral heterogeneity of the HIV-1 membrane, our results may help develop compounds with antiviral activity acting by perturbing the functional organization of the lipid envelope.

End-product diacylglycerol enhances the activity of PI-PLC through changes in membrane domain structure.

Diacylglycerol (DAG)-induced activation of phosphatidylinositol-phospholipase C (PI-PLC) was studied with vesicles containing PI, either pure or in mixtures with dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, sphingomyelin, or galactosylceramide, used as substrates. At 22°C, DAG at 33 mol % increased PI-PLC activity in all of the mixtures, but not in pure PI bilayers. DAG also caused an overall decrease in diphenylhexatriene fluorescence polarization (decreased molecular order) in all samples, and increased overall enzyme binding. Confocal fluorescence microscopy of giant unilamellar vesicles of all of the compositions under study, with or without DAG, and quantitative evaluation of the phase behavior using Laurdan generalized polarization, and of enzyme binding to the various domains, indicated that DAG activates PI-PLC whenever it can generate fluid domains to which the enzyme can bind with high affinity. In the specific case of PI/dimyristoyl phosphatidylcholine bilayers at 22°C, DAG induced/increased enzyme binding and activation, but no microscopic domain separation was observed. The presence of DAG-generated nanodomains, or of DAG-induced lipid packing defects, is proposed instead for this system. In PI/galactosylceramide mixtures, DAG may exert its activation role through the generation of small vesicles, which PI-PLC is known to degrade at higher rates. In general, our results indicate that global measurements obtained using fluorescent probes in vesicle suspensions in a cuvette are not sufficient to elucidate DAG effects that take place at the domain level. The above data reinforce the idea that DAG functions as an important physical agent in regulating membrane and cell properties.

Two-photon Laurdan studies of the ternary lipid mixture DOPC:SM:cholesterol reveal a single liquid phase at sphingomyelin:cholesterol ratios lower than 1.

The ternary lipid mixture DOPC:eggSM:cholesterol in excess water has been studied in the form of giant unilamellar vesicles using two-photon fluorescence microscopy. Previous publications based on single-photon fluorescence microscopy had reported heterogeneous phase behavior (phase coexistence) in the region of the triangular phase diagram corresponding to SM:cholesterol molar ratios <1. We have examined this region by two-photon microscopy of Laurdan-labeled mixtures and have found that, under our conditions, only a single liquid phase exists. We have shown that macroscopic phase separation in the above region can be artifactually induced by one-photon excitation of the fluorescent probes and ensuing photooxidation and is prevented using two-photon excitation. The main effect of increasing the concentration of cholesterol in mixtures containing 30 mol % SM was to increase the rigidity of the disordered domains. Increasing the concentration of SM in mixtures containing 20 mol % cholesterol gradually augmented the rigidity of the ordered domains, while the disordered domains reached minimal order at a SM:cholesterol 2.25:1 molar ratio, which then increased again. Moreover, the detailed measurement of Laurdan generalized polarization across the whole phase diagram allowed the representation, for both the single- and two-phase regions, of the gradual variation of membrane lateral packing along the diagram, which we found to be governed largely by SM:cholesterol interactions.

Cholesterol-dependent membrane fusion induced by the gp41 membrane-proximal external region-transmembrane domain connection suggests a mechanism for broad HIV-1 neutralization.

UNLABELLED: The HIV-1 glycoprotein 41 promotes fusion of the viral membrane with that of the target cell. Structural, biochemical, and biophysical studies suggest that its membrane-proximal external region (MPER) may interact with the HIV-1 membrane and induce its disruption and/or deformation during the process. However, the high cholesterol content of the envelope (ca. 40 to 50 mol%) imparts high rigidity, thereby acting against lipid bilayer restructuring. Here, based on the outcome of vesicle stability assays, all-atom molecular dynamics simulations, and atomic force microscopy observations, we propose that the conserved sequence connecting the MPER with the N-terminal residues of the transmembrane domain (TMD) is involved in HIV-1 fusion. This junction would function by inducing phospholipid protrusion and acyl-chain splay in the cholesterol-enriched rigid envelope. Supporting the functional relevance of such a mechanism, membrane fusion was inhibited by the broadly neutralizing 4E10 antibody but not by a nonneutralizing variant with the CDR-H3 loop deleted. We conclude that the MPER-TMD junction embodies an envelope-disrupting C-terminal fusion peptide that can be targeted by broadly neutralizing antibodies. IMPORTANCE: Fusion of the cholesterol-enriched viral envelope with the cell membrane marks the beginning of the infectious HIV-1 replicative cycle. Consequently, the Env glycoprotein-mediated fusion function constitutes an important clinical target for inhibitors and preventive vaccines. Antibodies 4E10 and 10E8 bind to one Env vulnerability site located at the gp41 membrane-proximal external region (MPER)-transmembrane domain (TMD) junction and block infection. These antibodies display broad viral neutralization, which underscores the conservation and functionality of the MPER-TMD region. In this work, we combined biochemical assays with molecular dynamics simulations and microscopy observations to characterize the unprecedented fusogenic activity of the MPER-TMD junction. The fact that such activity is dependent on cholesterol and inhibited by the broadly neutralizing 4E10 antibody emphasizes its physiological relevance. Discovery of this functional element adds to our understanding of the mechanisms underlying HIV-1 infection and its blocking by antibodies.

The three lives of viral fusion peptides.

Fusion peptides comprise conserved hydrophobic domains absolutely required for the fusogenic activity of glycoproteins from divergent virus families. After 30 years of intensive research efforts, the structures and functions underlying their high degree of sequence conservation are not fully elucidated. The long-hydrophobic viral fusion peptide (VFP) sequences are structurally constrained to access three successive states after biogenesis. Firstly, the VFP sequence must fulfill the set of native interactions required for (meta) stable folding within the globular ectodomains of glycoprotein complexes. Secondly, at the onset of the fusion process, they get transferred into the target cell membrane and adopt specific conformations therein. According to commonly accepted mechanistic models, membrane-bound states of the VFP might promote the lipid bilayer remodeling required for virus-cell membrane merger. Finally, at least in some instances, several VFPs co-assemble with transmembrane anchors into membrane integral helical bundles, following a locking movement hypothetically coupled to fusion-pore expansion. Here we review different aspects of the three major states of the VFPs, including the functional assistance by other membrane-transferring glycoprotein regions, and discuss briefly their potential as targets for clinical intervention.

Structure and immunogenicity of a peptide vaccine, including the complete HIV-1 gp41 2F5 epitope: implications for antibody recognition mechanism and immunogen design.

The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund's adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development.

Pore-forming activity of pestivirus p7 in a minimal model system supports genus-specific viroporin function.

Viroporins are small integral membrane proteins functional in viral assembly and egress by promoting permeabilization. Blocking of viroporin function therefore constitutes a target for antiviral development. Classical swine fever virus (CSFV) protein p7 has been recently regarded as a class II viroporin. Here, we sought to establish the determinants of the CSFV p7 permeabilizing activity in a minimal model system. Assessment of an overlapping peptide library mapped the porating domain to the C-terminal hydrophobic stretch (residues 39-67). Pore-opening dependence on pH or sensitivity to channel blockers observed for the full protein required the inclusion of a preceding polar sequence (residues 33-38). Effects of lipid composition and structural data further support that the resulting peptide (residues 33-67), may comprise a bona fide surrogate to assay p7 activity in model membranes. Our observations imply that CSFV p7 relies on genus-specific structures-mechanisms to perform its viroporin function.

Recognition of membrane-bound fusion-peptide/MPER complexes by the HIV-1 neutralizing 2F5 antibody: implications for anti-2F5 immunogenicity.

The membrane proximal external region (MPER) of the fusogenic HIV-1 glycoprotein-41 harbors the epitope sequence recognized by 2F5, a broadly neutralizing antibody isolated from an infected individual. Structural mimicry of the conserved MPER 2F5 epitope constitutes a pursued goal in the field of anti-HIV vaccine development. It has been proposed that 2F5 epitope folding into its native state is attained in the vicinity of the membrane interface and might involve interactions with other viral structures. Here we present results indicating that oligomeric complexes established between MPER and the conserved amino-terminal fusion peptide (FP) can partition into lipid vesicles and be specifically bound by the 2F5 antibody at their surfaces. Cryo-transmission electron microscopy of liposomes doped with MPER:FP peptide mixtures provided the structural grounds for complex recognition by antibody at lipid bilayer surfaces. Supporting the immunogenicity of the membrane-bound complex, these MPER:FP peptide-vesicle formulations could trigger cross-reactive anti-MPER antibodies in rabbits. Thus, our observations suggest that contacts with N-terminal regions of gp41 may stabilize the 2F5 epitope as a membrane-surface antigen.

Membrane-transferring regions of gp41 as targets for HIV-1 fusion inhibition and viral neutralization.

The fusogenic function of HIV-1 gp41 transmembrane Env subunit relies on two different kinds of structural elements: i) a collapsible ectodomain structure (the hairpin or six-helix bundle) that opens and closes, and ii) two membrane- transferring regions (MTRs), the fusion peptide (FP) and the membrane-proximal external region (MPER), which ensure coupling of hairpin closure to apposition and fusion of cell and viral membranes. The isolation of naturally produced short peptides and neutralizing IgG-s, that interact with FP and MPER, respectively, and block viral infection, suggests that these conserved regions might represent useful targets for clinical intervention. Furthermore, MTR-derived peptides have been shown to be membrane-active. Here, it is discussed the potential use of these molecules and how the analysis of their membrane activity in vitro could contribute to the development of HIV fusion inhibitors and effective immunogens.

A new paradigm in molecular recognition? Specific antibody binding to membrane-inserted HIV-1 epitopes.

The conserved membrane proximal external region (MPER), adjacent to the transmembrane domain (TMD) of human immunodeficiency virus type-1 (HIV-1) gp41 glycoprotein subunit, is accessible to the broadly neutralizing 4E10 and 2F5 monoclonal antibodies (mAbs) and, therefore, constitutes a potential target for vaccine design. This gp41 domain is postulated to be functional during the Env glycoprotein-mediated fusion reaction by destabilizing the highly rigid viral envelope. To perform this task, the aromatic-rich MPER is believed to insert into the interfacial region of the viral membrane external monolayer, thereby inducing the restructuring of the lipid bilayer required for fusion-pore opening. This model predicts that: (i) 2F5 and 4E10 mAbs are capable of binding epitopes inserted into the membrane interface; (ii) in-membrane binding will result in effective blocking of MPER membrane activity; and (iii) both processes, in-membrane recognition and blocking of membrane activity, can be modulated by altering both the lipid composition and the MPER amino acid sequence. We review here recently reported experimental data consistent with those predictions, and further speculate on their relevance for prospective anti-HIV vaccine development.

Interaction of anti-HIV type 1 antibody 2F5 with phospholipid bilayers and its relevance for the mechanism of virus neutralization.

Broadly neutralizing monoclonal antibody (MAb) 2F5 targets a linear epitope within the highly conserved membrane proximal external region (MPER) of the HIV-1 envelope protein gp41 integral subunit. Prospective vaccine developments warrant efforts currently underway to unveil the mechanistic and structural basis of its mode of action. One open question relates to the putative role that membrane phospholipids might play in the neutralization process. In this work, we establish experimental conditions that allow monitoring 2F5 insertion into lipid bilayers. Then, we compare the abilities of 2F5-based MAb, Fabs, and 2F5-specific antibodies recovered from immunized rabbits to directly penetrate into lipid bilayers and block the lytic activity of MPER-derived peptides. Antibody insertion induced membrane perturbation, which was blocked on interacting with the peptide epitope, thereby suggesting that such phenomenon was primarily mediated by the epitope-binding site. The long, hydrophobic complementarity-determining region (CDR)-H3 loop contributed little to this effect. In contrast, the CDR-H3 loop was required for blocking the lytic activity of MPER-based peptides and viral neutralization. Thus, our results suggest that core epitope binding plus association with lipid bilayers are not in conjunction sufficient to support viral neutralization by 2F5. Moreover, they support a role for the CDR-H3 loop in establishing secondary interactions with lipids and/or gp41 that would block the membrane-perturbing activity of MPER during fusion.

Confocal microscopy of giant vesicles supports the absence of HIV-1 neutralizing 2F5 antibody reactivity to plasma membrane phospholipids.

The broadly neutralizing anti-HIV-1 2F5 monoclonal antibody recognizes a gp41 epitope proximal to the viral membrane. Potential phospholipid autoreactivity at cell surfaces has raised concerns about the use of this antibody for development of vaccines or immunotherapy. In this study, confocal microscopy of giant unilamellar vesicles (GUVs) was used to assess 2F5 reactivity with phospholipids assembled into bilayers with surface charge and curvature stress approximating those of the eukaryotic plasma membranes. Antibody partitioning into lipid bilayers required the specific recognition of membrane-inserted epitope, indicating that 2F5 was unable to directly react with GUV phospholipids, even under fluid phase segregation conditions. Our results thus support the feasibility of raising 2F5-like neutralizing responses through vaccination, and the medical safety of mAb infusions.

Ablation of the complementarity-determining region H3 apex of the anti-HIV-1 broadly neutralizing antibody 2F5 abrogates neutralizing capacity without affecting core epitope binding.

The identification and characterization of broadly neutralizing antibodies (bnAbs) against HIV-1 has formed a major research focus, with the ultimate goal to help in the design of an effective AIDS vaccine. One of these bnAbs, 2F5, has been extensively characterized, and residues at the apex of its unusually long complementarity-determining region (CDR) H3 loop have been shown to be crucial for neutralization. Structural studies, however, have revealed that the (100)TLFGVPI(100F) apex residues of the CDR H3 loop do not interact directly with residues of its core gp41 epitope. In an attempt to gain better insight into the functional role of this element, we have recombinantly expressed native 2F5 Fab and two mutants in which either the apical Phe100B(H) residue was changed to an alanine or the CDR H3 residues (100)TLFGVPI(100F) were replaced by a Ser-Gly dipeptide linker. Isothermal titration calorimetry (ITC) and competitive-binding enzyme-linked immunosorbent assays (ELISAs) rendered strikingly similar affinity constants (K(d) [dissociation constant] of approximately 20 nM) for linear peptide epitope binding by 2F5 Fabs, independent of the presence or absence of the apex residues. Ablation of the CDR H3 apex residues, however, abolished the cell-cell fusion inhibition and pseudovirus neutralization capacities of 2F5 Fab. We report competitive ELISA data that suggest a role of 2F5 CDR H3 apex residues in mediating weak hydrophobic interactions with residues located at the C terminus of the gp41 membrane proximal external region and/or membrane components in the context of core epitope binding. The present data therefore imply an extended 2F5 paratope that includes weak secondary interactions that are crucial for neutralization of Env-mediated fusion.

Structural constraints imposed by the conserved fusion peptide on the HIV-1 gp41 epitope recognized by the broadly neutralizing antibody 2F5.

The HIV-1 gp41 epitope recognized by the broadly neutralizing 2F5 antibody has focused much attention as a suitable target in the design of peptide immunogens. Peptides mimicking the linear 2F5 epitope (2F5ep) are however intrinsically disordered, while the structural constraints existing in the cognate gp41 native structure recognized by the antibody are presently unknown. In recent reports, we have shown that core residues of the amino-terminal fusion peptide (FP) increase MAb2F5 affinity. Here, we have inferred the sequence-specific structural constraints imposed by the FP residues on the 2F5 epitope from the comparison of two hybrid peptides: HybK3, which connects through a flexible tether residues derived from 2F5ep and FP sequences, and scrHybK3, combining 2F5ep and an FP sequence with the conserved core scrambled. Circular dichroism, conventional and two-dimensional correlation infrared spectroscopy, and X-ray diffraction studies revealed specific structural features that were dependent on the exact FP sequence, namely, (i) the production with moderate low polarity of an intermediate folded structure enriched in beta-turns and alpha-helix; (ii) the existence in this intermediate of a thermotropic conformational transition taking place at ca. 18-20 degrees C, consistent with the conversion of 3(10)-helices into beta-turn conformers; and (iii) the presence of a C-terminal alpha-helix in crystals of Fab'-peptide complexes. Those features support the existence of native-like tertiary interactions between FP and 2F5 epitope residues, which might be important to recreate when developing an effective AIDS peptide vaccine.

Lipid modulation of membrane-bound epitope recognition and blocking by HIV-1 neutralizing antibodies.

The conserved, aromatic-rich membrane-proximal external region (MPER) of gp41 is functional in human immunodeficiency virus (HIV)-cell fusion by perturbing membrane integrity. Broadly-neutralizing 2F5 and 4E10 monoclonal antibodies (MAb-s) recognize amino- and carboxy-terminal epitope sequences within this domain, respectively. An MPER peptide overlapping 2F5 and 4E10 epitope sequences was capable of breaching the permeability barrier of lipid vesicles. Cholesterol and sphingomyelin raft-lipids, present at high quantities in the HIV-1 envelope, promoted exposure or occlusion of 4E10 epitope, respectively. Conversely, 2F5 epitope accessibility was affected to a lesser extent by these envelope lipids. These observations support the idea that MPER epitopes on membranes are segmented in terms of how they are affected by envelope lipids, which may have implications for MPER-based vaccine development.

Functional and structural characterization of 2B viroporin membranolytic domains.

Nonstructural 2B viroporin is an intracellularly produced pore-forming protein required for effective enteroviral and rhinoviral replication. The sequence of 2B displays two putative interconnected transmembrane domains, which are predicted to insert into the negatively charged membranes of target organelles forming an integral hairpin. The use of an overlapping peptide library that spanned the complete 2B sequence has recently allowed the mapping of the cell plasma membrane porating activity to the partially amphipathic, amino-terminal transmembrane domain (TM1, residues 35-55). We describe here that although the TM1 peptide was effective in permeabilizing uncharged membranes, it induced marginal lysis of anionic bilayers. In fact, only the peptide representing the highly conserved carboxy-terminal transmembrane domain (TM2, residues 59-82) reproduced the capacity of the full 2B protein to efficiently permeabilize bilayers made of anionic phospholipids. Insertion into lipid monolayers and circular dichroism determinations were, however, consistent with penetration of the TM1 helix into both anionic and zwitterionic membranes, while TM2 interacting with membranes assumed a mixture of conformations. Moreover, addition of TM1 strongly stimulated TM2-induced permeabilization of the anionic membranes. In combination, TM1 and TM2 formed a complex that had structural properties, including a high proportion of extended nonhelical secondary structure, that were distinct from those of the individual peptides. Finally, a comparison of antimicrobial and hemolytic activities further underscored the TM1 domain's cytolytic character. Overall, our data support the idea that the cytolytic activity of TM1 in the negatively charged cell endomembranes targeted by 2B viroporin requires the cooperation of both transmembrane domains.

The broadly neutralizing anti-human immunodeficiency virus type 1 4E10 monoclonal antibody is better adapted to membrane-bound epitope recognition and blocking than 2F5.

The broadly neutralizing 2F5 and 4E10 monoclonal antibodies (MAbs) recognize epitopes within the membrane-proximal external region (MPER) that connects the human immunodeficiency virus type 1 (HIV-1) envelope gp41 ectodomain with the transmembrane anchor. By adopting different conformations that stably insert into the virion external membrane interface, such as helical structures, a conserved aromatic-rich sequence within the MPER is thought to participate in HIV-1-cell fusion. Recent experimental evidence suggests that the neutralizing activity of 2F5 and 4E10 might correlate with the MAbs' capacity to recognize epitopes inserted into the viral membrane, thereby impairing MPER fusogenic activity. To gain new insights into the molecular mechanism underlying viral neutralization by these antibodies, we have compared the capacities of 2F5 and 4E10 to block the membrane-disorganizing activity of MPER peptides inserted into the surface bilayer of solution-diffusing unilamellar vesicles. Both MAbs inhibited leakage of vesicular aqueous contents (membrane permeabilization) and intervesicular lipid mixing (membrane fusion) promoted by MPER-derived peptides. Thus, our data support the idea that antibody binding to a membrane-inserted epitope may interfere with the function of the MPER during gp41-induced fusion. Antibody insertion into a cholesterol-containing, uncharged virion-like membrane is mediated by specific epitope recognition, and moreover, partitioning-coupled folding into a helix reduces the efficiency of 2F5 MAb binding to its epitope in the membrane. We conclude that the capacity to interfere with the membrane activity of conserved MPER sequences is best correlated with the broad neutralization of the 4E10 MAb.