Membrane-proximal Region Research Articles

Synaptic homeostasis requires the membrane-proximal carboxy tail of GluA2

Bidirectional scaling of synaptic transmission, expressed as a compensatory change in quantal size following chronic activity perturbation, is a critical effector mechanism underlying homeostatic plasticity in the brain. An emerging model posits that the GluA2 AMPA receptor (AMPAR) subunit may be important for the bidirectional scaling of excitatory transmission; however, whether this subunit plays an obligatory role in synaptic scaling, and the identity of the precise domain(s) involved, remain controversial. We set out to determine the specific AMPAR subunit required for scaling up in CA1 hippocampal pyramidal neurons, and found that the GluA2 subunit is both necessary and sufficient. In addition, our results point to a critical role for a single amino acid within the membrane-proximal region of the GluA2 cytoplasmic tail, and suggest a distinct model for the regulation of AMPAR trafficking in synaptic homeostasis.

The cytosolic domain of T-cell receptor ζ associates with membranes in a dynamic equilibrium and deeply penetrates the bilayer

Interactions between lipid bilayers and the membrane-proximal regions of membrane-associated proteins play important roles in regulating membrane protein structure and function. The T-cell antigen receptor is an assembly of eight single-pass membrane-spanning subunits on the surface of T lymphocytes that initiates cytosolic signaling cascades upon binding antigens presented by MHC-family proteins on antigen-presenting cells. Its ζ-subunit contains multiple cytosolic immunoreceptor tyrosine-based activation motifs involved in signal transduction, and this subunit by itself is sufficient to couple extracellular stimuli to intracellular signaling events. Interactions of the cytosolic domain of ζ (ζcyt) with acidic lipids have been implicated in the initiation and regulation of transmembrane signaling. ζcyt is unstructured in solution. Interaction with acidic phospholipids induces structure, but its disposition when bound to lipid bilayers is controversial. Here, using surface plasmon resonance and neutron reflection, we characterized the interaction of ζcyt with planar lipid bilayers containing mixtures of acidic and neutral lipids. We observed two binding modes of ζcyt to the bilayers in dynamic equilibrium: one in which ζcyt is peripherally associated with lipid headgroups and one in which it penetrates deeply into the bilayer. Such an equilibrium between the peripherally bound and embedded forms of ζcyt apparently controls accessibility of the immunoreceptor tyrosine-based activation signal transduction pathway. Our results reconcile conflicting findings of the ζ structure reported in previous studies and provide a framework for understanding how lipid interactions regulate motifs to tyrosine kinases and may regulate the T-cell antigen receptor biological activities for this cell-surface receptor system.

Immunologic Insights on the Membrane Proximal External Region: A Major Human Immunodeficiency Virus Type-1 Vaccine Target.

Broadly neutralizing antibodies (bNAbs) targeting conserved regions within the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein (Env) can be generated by the human immune system and their elicitation by vaccination will be a key point to protect against the wide range of viral diversity. The membrane proximal external region (MPER) is a highly conserved region within the Env gp41 subunit, plays a major role in membrane fusion and is targeted by naturally induced bNAbs. Therefore, the MPER is considered as an attractive vaccine target. However, despite many attempts to design MPER-based immunogens, further study is still needed to understand its structural complexity, its amphiphilic feature, and its limited accessibility by steric hindrance. These particular features compromise the development of MPER-specific neutralizing responses during natural infection and limit the number of bNAbs isolated against this region, as compared with other HIV-1 vulnerability sites, and represent additional hurdles for immunogen development. Nevertheless, the analysis of MPER humoral responses elicited during natural infection as well as the MPER bNAbs isolated to date highlight that the human immune system is capable of generating MPER protective antibodies. Here, we discuss the recent advances describing the immunologic and biochemical features that make the MPER a unique HIV-1 vulnerability site, the different strategies to generate MPER-neutralizing antibodies in immunization protocols and point the importance of extending our knowledge toward new MPER epitopes by the isolation of novel monoclonal antibodies. This will be crucial for the redesign of immunogens able to skip non-neutralizing MPER determinants.

Structure of the Ebola virus envelope protein MPER/TM domain and its interaction with the fusion loop explains their fusion activity

Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM-FL interaction in EBOV entry and fusion.

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.

Mutational Replacements at the "Glycine Hinge" of the Escherichia coli Chemoreceptor Tsr Support a Signaling Role for the C-Helix Residue.

Bacterial chemoreceptors are dimeric membrane proteins that transmit signals from a periplasmic ligand-binding domain to the interior of the cells. The highly conserved cytoplasmic domain consists of a long hairpin that in the dimer forms a four-helix coiled-coil bundle. The central region of the bundle couples changes in helix packing that occur in the membrane proximal region to the signaling tip, controlling the activity of an associated histidine kinase. This subdomain contains certain glycine residues that are postulated to form a hinge in chemoreceptors from enteric bacteria and have been largely postulated to play a role in the coupling mechanism, and/or in the formation of higher-order chemoreceptor assemblies. In this work, we directly assessed the importance of the "glycine hinge" by obtaining nonfunctional replacements at each of its positions in the Escherichia coli serine receptor Tsr and characterizing them. Our results indicate that, rather than being essential for proper receptor-receptor interaction, the "glycine hinge" residues are involved in the ability of the receptor to switch between different signaling states. Mainly, the C-helix residue G439 has a key role in shifting the equilibrium toward a kinase-activating conformation. However, we found second-site mutations that restore the chemotactic proficiency of some of the "glycine hinge" mutants, suggesting that a complete hinge is not strictly essential. Rather, glycine residues seem to favor the coupling activity that relies on some other structural features of the central subdomain.

Adaptation of HIV-1 to cells with low expression of the CCR5 coreceptor

The binding of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ((gp120/gp41)3) to the receptors CD4 and CCR5 triggers virus entry into host cells. To identify Env regions that respond to CCR5 binding, HIV-1 was serially passaged on a CD4-positive canine cell line expressing progressively lower levels of CCR5. HIV-1 replication was observed in cells expressing ~1300 CCR5 molecules/cell. Env changes that conferred this low-CCR5 replication phenotype were located outside of the known CCR5-binding region of the gp120 Env subunit and did not apparently increase CCR5 binding affinity. The adaptation-associated changes, located in the gp120 α1 helix and in the gp41 HR1 heptad repeat and membrane-proximal external region (MPER), enhanced HIV-1 replication in cells at all levels of CCR5 expression. The adapted Envs exhibited a greater propensity to undergo conformational changes, as evidenced by increased exposure of conserved regions near the CD4- and CCR5-binding sites.

Antigp41 membrane proximal external region antibodies and the art of using the membrane for neutralization.

We summarize the latest research on the progress to understand the neutralizing epitopes present within the membrane proximal external region (MPER) of the HIV-1 fusion protein subunit gp41. The HIV-1 fusion protein subunit gp41 contains a highly conserved sequence that is essential for membrane fusion and targeted by broadly neutralizing antibodies such as 2F5, 4E10, Z13e1, and 10E8. These antibodies recognize a linear gp41 epitope with high affinity, but require additional hydrophobic sequences present in their heavy chain CDR3 for neutralization. Recent structural studies on mAbs 4E10 and 10E8 provide molecular details for specific interactions with lipids and implicate part of the transmembrane region as the relevant 10E8 epitope. Although many different approaches have been applied to engineer gp41 immunogens that can induce broadly neutralizing antibodies directed toward MPER, only modest success has yet been reported. The new structural details on the complex gp41-lipidic epitope will spur new approaches to design gp41-MPER immunogens that might induce broadly neutralizing antibody responses.

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1.

The Membrane-Proximal Region of C-C Chemokine Receptor Type 5 Participates in the Infection of HIV-1.

The initial infection and transmission of HIV-1 requires C–C chemokine receptor type 5 (CCR5). Here, we report that the membrane-proximal region (MPR, aa 22–38) of CCR5 participates in the infection of HIV-1. First, MPR-specific antibodies elicited in mice dose-dependently inhibited the infection of CCR5-tropic HIV-1. Second, substituting MPR with the same region from other co-receptors significantly impaired HIV-1 infection, while the key residues identified by alanine scanning mutagenesis formed an exposed leucine zipper-like structure. Moreover, a peptide derived from MPR could block the infection of a number of HIV-1 strains only before the formation of gp41 six-helix bundle, coincide with the early interaction between CCR5 and the gp120 protein during HIV-1 infection. These promising results ensured the potential of this previously uncharacterized domain as a starting point for the development of antiviral drugs, blocking antibodies, and HIV vaccines.

Broad and potent cross clade neutralizing antibodies with multiple specificities in the plasma of HIV-1 subtype C infected individuals

Broadly Cross clade Neutralizing (BCN) antibodies are recognized as potential therapeutic tools and leads for the design of a vaccine that can protect human beings against various clades of Human Immunodeficiency Virus (HIV). In the present study, we screened plasma of 88 HIV-1 infected ART naïve individuals for their neutralization potential using a standard panel of 18 pseudoviruses belonging to different subtypes and different levels of neutralization. We identified 12 samples with good breadth of neutralization (neutralized >90% of the viruses). Four of these samples neutralized even the difficult-to-neutralize tier-3 pseudoviruses with great potency (GMT > 600). Analysis of neutralization specificities indicated that four samples had antibodies with multiple epitope binding specificities, viz. CD4-binding site (CD4BS), glycans in the V1/V2 and V3 regions and membrane proximal external region (MPER). Our findings indicate the strong possibility of identifying highly potent bNAbs with known or novel specificities from HIV-1 subtype C infected individuals from India that can be exploited as therapeutic tools or lead molecules for the identification of potential epitopes for design of a protective HIV-1 vaccine.

Development of a DNA vaccine expressing a secreted HIV-1 gp41 ectodomain that includes the membrane-proximal external region

A limited number of sites on the HIV-1 Envelope protein are vulnerable to broadly neutralizing antibodies (bnAbs). One of these sites, the membrane proximal external region (MPER), is located at the C-terminus of the gp41 ectodomain (gp41ecto). This highly conserved sequence is bound by several well-characterized bnAbs. Efforts to produce a gp41 immunogen are in part hampered by the MPER’s hydrophobicity and propensity to induce aggregation. We sought to produce a DNA vaccine expressing a gp41ecto that is both secreted from mammalian cells and maintains binding by bnAbs to the MPER. Through in silico analysis, we predicted regions of gp41ecto that could induce aggregation and possibly hinder secretion. We generated deletion mutants of gp41ecto and tested their ability to be secreted by mammalian cells. Upon deletion of regions in either the fusion peptide (FP) or MPER, secretion of the gp41ecto increased. In an effort to both augment secretion and maintain binding by bnAbs, we developed constructs with the FP deletion and introduced point mutations in the MPER. Two constructs (gp41 ΔFP and gp41 ΔFP+I682E) maintained binding by gp41 MPER-specific bnAbs (4E10, Z13e1 and 10E8). These were evaluated as DNA vaccines in a mouse model. Both vaccines proved to be immunogenic and appeared to elicit some MPER-specific antibodies that bound gp41 ectodomain-derived proteins but not short peptides spanning the MPER. No neutralizing capacity was detected against a clade C virus containing a homologous MPER.

Identification and Preparation of a Novel Chemokine Receptor-Binding Domain in the Cytoplasmic Regulator FROUNT

FROUNT is a cytoplasmic protein that binds to the membrane-proximal C-terminal regions (Pro-Cs) of chemokine receptors, CCR2 and CCR5. The FROUNT-chemokine receptor interactions play a pivotal role in the migration of inflammatory immune cells, indicating the potential of FROUNT as a drug target for inflammatory diseases. To provide the foundation for drug development, structural information of the Pro-C binding region of FROUNT is desired. Here, we defined the novel structural domain (FNT-CB), which mediates the interaction with the chemokine receptors. A recombinant GST-tag-fused FNT-CB protein expression system was constructed. The protein was purified by affinity chromatography and then subjected to in-gel protease digestion of the GST-tag. The released FNT-CB was further purified by anion-exchange and size-exclusion chromatography. Purified FNT-CB adopts a helical structure, as indicated by CD. NMR line-broadening indicated that weak aggregation occurred at sub-millimolar concentrations, but the line-broadening was mitigated by using a deuterated sample in concert with transverse relaxation-optimized spectroscopy. The specific binding of FNT-CB to CCR2 Pro-C was confirmed by the fluorescence-based assay. The improved NMR spectral quality and the retained functional activity of FNT-CB support the feasibility of further structural and functional studies targeted at the anti-inflammatory drug development.

Functional Contacts between MPER and the Anti-HIV-1 Broadly Neutralizing Antibody 4E10 Extend into the Core of the Membrane

The exceptional breadth of broadly neutralizing antibodies (bNAbs) against the membrane-proximal external region (MPER) of the transmembrane protein gp41 makes this class of antibodies an ideal model to design HIV vaccines. From a practical point of view, however, the preparation of vaccines eliciting bNAbs is still a major roadblock that limits their clinical application. Fresh mechanistic insights are necessary to develop more effective strategies. In particular, the function of the unusually long complementarity-determining region three of the heavy chain (CDRH3) of 4E10, an anti-MPER bNAb, is an open question that fascinates researchers in the field. Residues comprising the apex region are dispensable for engagement of the epitope in solution; still, their single mutation profoundly impairs the neutralization capabilities of the antibody. Since this region is very hydrophobic, it has been proposed that the apex is essential for anchoring the antibody to the viral membrane where MPER resides. Herein, we have critically examined this idea using structural, biophysical, biochemical, and cell-based approaches. Our results demonstrate that the apex region is not just a “greasy” spot merely increasing the affinity of the antibody for the membrane. We demonstrate the three-dimensional engagement of the apex region of the CDRH3 with the conglomerate of gp41 epitope and membrane lipids as a means of effective binding and neutralization of the virus. This mechanism of recognition suggests a standard route of antibody ontogeny. Therefore, we need to focus our efforts on recreating a more realistic MPER/lipid immunogen in order to generate more effective anti-HIV-1 vaccines.

Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1.

The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus—1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1 immunopathogenesis through modulation of the cytokine release and active inhibition of immune responses.

Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers.

ABSTRACTBroadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches.

Evaluation of a novel multi-immunogen vaccine strategy for targeting 4E10/10E8 neutralizing epitopes on HIV-1 gp41 membrane proximal external region

The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by broadly neutralizing antibodies (bnAbs) 4E10 and 10E8. In this proof-of-concept study, we evaluated a novel multi-immunogen vaccine strategy referred to as Incremental, Phased Antigenic Stimulation for Rapid Antibody Maturation (IPAS-RAM) to induce 4E10/10E8-like bnAbs. Rabbits were immunized sequentially, but in a phased manner, with three immunogens that are progressively more native (gp41-28×3, gp41-54CT, and rVV-gp160DH12). Although nAbs were not induced, epitope-mapping analyses indicated that IPAS-RAM vaccination was better able to target antibodies towards the 4E10/10E8 epitopes than homologous prime-boost immunization using gp41-28×3 alone. MPER-specific rabbit monoclonal antibodies were generated, including 9F6. Although it lacked neutralizing activity, the target epitope profile of 9F6 closely resembled those of 4E10 and 10E8 (671NWFDITNWLWYIK683). B-cell repertoire analyses suggested the importance of co-immunizations for maturation of 9F6, which warrants further evaluation of our IPAS-RAM vaccine strategy using an improved priming immunogen.

Lipid interactions and angle of approach to the HIV-1 viral membrane of broadly neutralizing antibody 10E8: Insights for vaccine and therapeutic design.

Among broadly neutralizing antibodies to HIV, 10E8 exhibits greater neutralizing breadth than most. Consequently, this antibody is the focus of prophylactic/therapeutic development. The 10E8 epitope has been identified as the conserved membrane proximal external region (MPER) of gp41 subunit of the envelope (Env) viral glycoprotein and is a major vaccine target. However, the MPER is proximal to the viral membrane and may be laterally inserted into the membrane in the Env prefusion form. Nevertheless, 10E8 has not been reported to have significant lipid-binding reactivity. Here we report x-ray structures of lipid complexes with 10E8 and a scaffolded MPER construct and mutagenesis studies that provide evidence that the 10E8 epitope is composed of both MPER and lipid. 10E8 engages lipids through a specific lipid head group interaction site and a basic and polar surface on the light chain. In the model that we constructed, the MPER would then be essentially perpendicular to the virion membrane during 10E8 neutralization of HIV-1. As the viral membrane likely also plays a role in selecting for the germline antibody as well as size and residue composition of MPER antibody complementarity determining regions, the identification of lipid interaction sites and the MPER orientation with regard to the viral membrane surface during 10E8 engagement can be of great utility for immunogen and therapeutic design.

Antigenic characterization of the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor incorporated into nanodiscs.

The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the viral envelope glycoproteins (Envs), which are derived by the proteolytic cleavage of a trimeric gp160 Env precursor. The mature Env trimer is a major target for entry inhibitors and vaccine-induced neutralizing antibodies. Env interstrain variability, conformational flexibility and heavy glycosylation contribute to evasion of the host immune response, and create challenges for structural characterization and vaccine development. Here we investigate variables associated with reconstitution of the HIV-1 Env precursor into nanodiscs, nanoscale lipid bilayer discs enclosed by membrane scaffolding proteins. We identified detergents, as well as lipids similar in composition to the viral lipidome, that allowed efficient formation of Env-nanodiscs (Env-NDs). Env-NDs were created with the full-length Env precursor and with an Env precursor with the majority of the cytoplasmic tail intact. The self-association of Env-NDs was decreased by glutaraldehyde crosslinking. The Env-NDs exhibited an antigenic profile expected for the HIV-1 Env precursor. Env-NDs were recognized by broadly neutralizing antibodies. Of note, neutralizing antibody epitopes in the gp41 membrane-proximal external region and in the gp120:gp41 interface were well exposed on Env-NDs compared with Env expressed on cell surfaces. Most Env epitopes recognized by non-neutralizing antibodies were masked on the Env-NDs. This antigenic profile was stable for several days, exhibiting a considerably longer half-life than that of Env solubilized in detergents. Negative selection with weak neutralizing antibodies could be used to improve the antigenic profile of the Env-NDs. Finally, we show that lipid adjuvants can be incorporated into Env-NDs. These results indicate that Env-NDs represent a potentially useful platform for investigating the structural, functional and antigenic properties of the HIV-1 Env trimer in a membrane context.

Fusion on a Pedestal: The Structure of the Full-Length HSV-1 Fusogen gB

All enveloped viruses must catalyze the fusion of their surrounding membrane with that of their target cell. In herpesviruses, fusion is powered by a large structural rearrangement of the trimeric fusogen gB, with help from viral glycoproteins gD and gH/gL. Although the structure of the postfusion gB ectodomain has been solved, the architecture of the membrane-interacting portion of gB - its membrane proximal region (MPR), transmembrane domain (TMD), and cytoplasmic domain (CTD) - is unknown. These domains both facilitate and regulate fusion, but the lack of structural information on them has hindered reconstruction of the gB refolding process. To fill this gap, we have crystallized full-length, postfusion gB in a detergent-amphipol environment. The resulting structure is the first to encompass the membrane-spanning portion of a viral fusogen in its postfusion conformation. In each protomer of the gB trimer, the MPR is a lipid embedded helix that forms a platform beneath the ectodomain. The TMD helices traverse the membrane at an angle, creating a flexible conduit that could transmit the status of the regulatory CTD below. In the CTD, the three protomers form a shallow basket bounded by a long helix that angles upward toward the membrane. While the remainder of the CTD is unstructured in these crystals, electron spin resonance (ESR) spectroscopy data indicate that many of these distal residues form an amphipathic helix embedded in the outer bilayer. Protein/protein and protein/membrane contacts may enable the CTD to restrain fusion by acting as a clamp that braces against the bilayer. These results thus present a possible mechanism for regulation of gB mediated fusion and a strategy for capturing another critical gB form - the elusive prefusion conformation.