Topological analysis of the gp41 MPER on lipid bilayers relevant to the metastable HIV-1 envelope prefusion state

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.

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Functional Optimization of Broadly Neutralizing HIV-1 Antibody 10E8 by Promotion of Membrane Interactions

Journal of Virology ◽

10.1128/jvi.02249-17 ◽

2018 ◽

Vol 92 (8) ◽

Cited By ~ 9

Author(s):

Edurne Rujas ◽

Daniel P. Leaman ◽

Sara Insausti ◽

Lei Ortigosa-Pascual ◽

Lei Zhang ◽

...

Keyword(s):

Lipid Bilayers ◽

Mechanism Of Action ◽

Neutralizing Antibodies ◽

Positive Charge ◽

Transmembrane Domain ◽

Membrane Surface ◽

Passive Immunization ◽

Viral Membrane ◽

Mutation Strategy ◽

Hiv 1

ABSTRACT The 10E8 antibody targets a helical epitope in the membrane-proximal external region (MPER) and transmembrane domain (TMD) of the envelope glycoprotein (Env) subunit gp41 and is among the broadest known neutralizing antibodies against HIV-1. Accordingly, this antibody and its mechanism of action valuably inform the design of effective vaccines and immunotherapies. 10E8 exhibits unusual adaptations to attain specific, high-affinity binding to the MPER at the viral membrane interface. Reversing the charge of the basic paratope surface (from net positive to net negative) reportedly lowered its neutralization potency. Here, we hypothesized that by increasing the net positive charge in similar polar surface patches, the neutralization potency of the antibody may be enhanced. We found that an increased positive charge at this paratope surface strengthened an electrostatic interaction between the antibody and lipid bilayers, enabling 10E8 to interact spontaneously with membranes. Notably, the modified 10E8 antibody did not gain any apparent polyreactivity and neutralized virus with a significantly greater potency. Binding analyses indicated that the optimized 10E8 antibody bound with a higher affinity to the epitope peptide anchored in lipid bilayers and to Env spikes on virions. Overall, our data provide a proof of principle for the rational optimization of 10E8 via manipulation of its interaction with the membrane element of its epitope. However, the observation that a similar mutation strategy did not affect the potency of the first-generation anti-MPER antibody 4E10 shows possible limitations of this principle. Altogether, our results emphasize the crucial role played by the viral membrane in the antigenicity of the MPER-TMD of HIV-1. IMPORTANCE The broadly neutralizing antibody 10E8 blocks infection by nearly all HIV-1 isolates, a capacity which vaccine design seeks to reproduce. Engineered versions of this antibody also represent a promising treatment for HIV infection by passive immunization. Understanding its mechanism of action is therefore important to help in developing effective vaccines and biologics to combat HIV/AIDS. 10E8 engages its helical MPER epitope where the base of the envelope spike submerges into the viral membrane. To enable this interaction, this antibody evolved an unusual property: the ability to interact with the membrane surface. Here, we provide evidence that 10E8 can be made more effective by enhancing its interactions with membranes. Our findings strengthen the idea that to elicit antibodies similar to 10E8, vaccines must reproduce the membrane environment where these antibodies perform their function.

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Neutralizing antibodies induced by first-generation gp41-stabilized HIV-1 envelope trimers and nanoparticles

10.1101/2020.12.02.408328 ◽

2020 ◽

Author(s):

Sonu Kumar ◽

Xiaohe Lin ◽

Timothy Ngo ◽

Benjamin Shapero ◽

Cindy Sou ◽

...

Keyword(s):

Cell Sorting ◽

Neutralizing Antibodies ◽

First Generation ◽

Heptad Repeat ◽

X Ray ◽

X Ray Crystallography ◽

Clade C ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing ◽

Hiv 1

ABSTRACTAntigen-specific B-cell sorting and next-generation sequencing (NGS) were combined to isolate HIV-1 neutralizing antibodies (NAbs) from mice and rabbits immunized with BG505 trimers and nanoparticles. Three mouse NAbs potently neutralize BG505.T332N and recognize a glycan epitope centered at the C3/V4 region, as revealed by electron microscopy (EM), x-ray crystallography, and epitope mapping. Three potent NAbs were sorted from rabbit B cells that target glycan holes on the BG505 envelope glycoprotein (Env) and account for a significant portion of autologous NAb response. We then determined a 3.4Å-resolution crystal structure for the clade C transmitted/founder Du172.17 Env with a redesigned heptad repeat 1 (HR1) bend. This clade C Env, as a soluble trimer and attached to a ferritin nanoparticle, along with a clade A Q482-d12 Env trimer, elicited distinct NAb responses in rabbits. Our study demonstrates that nanoparticles presenting gp41-stabilized trimers can induce potent NAb responses in mice and rabbits with Env-dependent breadth.TEASERMouse and rabbit NAbs elicited by gp41-stabilized trimers and nanoparticles neutralize autologous HIV-1 by targeting different epitopes

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Similarities and differences between native HIV-1 envelope glycoprotein trimers and stabilized soluble trimer mimetics

10.1101/500975 ◽

2018 ◽

Cited By ~ 1

Author(s):

Alba Torrents de la Peña ◽

Kimmo Rantalainen ◽

Christopher A. Cottrell ◽

Joel D. Allen ◽

Marit J. van Gils ◽

...

Keyword(s):

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

Transmembrane Domain ◽

Cytoplasmic Tail ◽

Full Length ◽

Broadly Neutralizing Antibodies ◽

Vaccine Research ◽

Similarities And Differences ◽

Membrane Tether ◽

Hiv 1

AbstractThe HIV-1 envelope glycoprotein (Env) trimer is located on the surface of the virus and is the target of broadly neutralizing antibodies (bNAbs). Recombinant native-like soluble Env trimer mimetics, such as SOSIP trimers, have taken a central role in HIV-1 vaccine research aimed at inducing bNAbs. We therefore performed a direct and thorough comparison of a full-length native Env trimer containing the transmembrane domain and the cytoplasmic tail, with the sequence matched soluble SOSIP trimer, both based on an early Env sequence (AMC011) from an HIV+ individual that developed bNAbs. The structures of the full-length AMC011 trimer bound to either bNAb PGT145 or PGT151 were very similar to the structures of SOSIP trimers. Antigenically, the full-length and SOSIP trimers were comparable, but in contrast to the full-length trimer, the SOSIP trimer did not bind at all to non-neutralizing antibodies, most likely as a consequence of the intrinsic stabilization of the SOSIP trimer. Furthermore, the glycan composition of full-length and SOSIP trimers was similar overall, but the SOSIP trimer possessed slightly less complex and less extensively processed glycans, which may relate to the intrinsic stabilization as well as the absence of the membrane tether. These data provide insights into how to best use and improve membrane-associated full-length and soluble SOSIP HIV-1 Env trimers as immunogens.

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Nanoparticle Vaccines for Inducing HIV-1 Neutralizing Antibodies

Vaccines ◽

10.3390/vaccines7030076 ◽

2019 ◽

Vol 7 (3) ◽

pp. 76 ◽

Cited By ~ 15

Author(s):

Mitch Brinkkemper ◽

Kwinten Sliepen

Keyword(s):

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

Critical Role ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Broadly Neutralizing Antibodies ◽

Viral Membrane ◽

Immunodeficiency Virus ◽

Hiv 1

The enormous sequence diversity between human immunodeficiency virus type 1 (HIV-1) strains poses a major roadblock for generating a broadly protective vaccine. Many experimental HIV-1 vaccine efforts are therefore aimed at eliciting broadly neutralizing antibodies (bNAbs) that are capable of neutralizing the majority of circulating HIV-1 strains. The envelope glycoprotein (Env) trimer on the viral membrane is the sole target of bNAbs and the key component of vaccination approaches aimed at eliciting bNAbs. Multimeric presentation of Env on nanoparticles often plays a critical role in these strategies. Here, we will discuss the different aspects of nanoparticles in Env vaccination, including recent insights in immunological processes underlying their perceived advantages, the different nanoparticle platforms and the various immunogenicity studies that employed nanoparticles to improve (neutralizing) antibody responses against Env.

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Conformational Engineering of HIV-1 Env Based on Mutational Tolerance in the CD4 and PG16 Bound States

Journal of Virology ◽

10.1128/jvi.00219-19 ◽

2019 ◽

Vol 93 (11) ◽

Cited By ~ 3

Author(s):

Jeremiah D. Heredia ◽

Jihye Park ◽

Hannah Choi ◽

Kevin S. Gill ◽

Erik Procko

Keyword(s):

Virus Replication ◽

Conformational Changes ◽

Neutralizing Antibodies ◽

Bound States ◽

Structural Changes ◽

Electrostatic Repulsion ◽

Closed Conformation ◽

Open Conformation ◽

Outer Domain ◽

Hiv 1

ABSTRACTHIV-1 infection is initiated by viral Env engaging the host receptor CD4, triggering Env to transition from a “closed” to “open” conformation during the early events of virus-cell membrane fusion. To understand how Env sequence accommodates this conformational change, mutational landscapes decoupled from virus replication were determined for Env from BaL (clade B) and DU422 (clade C) isolates interacting with CD4 or antibody PG16 that preferentially recognizes closed trimers. Sequence features uniquely important to each bound state were identified, including glycosylation and binding sites. Notably, the Env apical domain and trimerization interface are under selective pressure for PG16 binding. Based on this key observation, mutations were found that increase presentation of quaternary epitopes associated with properly conformed trimers when Env is expressed at the plasma membrane. Many mutations reduce electrostatic repulsion at the Env apex and increase PG16 recognition of Env sequences from clades A and B. Other mutations increase hydrophobic packing at the gp120 inner-outer domain interface and were broadly applicable for engineering Env from diverse strains spanning tiers 1, 2, and 3 across clades A, B, C, and BC recombinants. Core mutations predicted to introduce steric strain in the open state show markedly reduced CD4 interactions. Finally, we demonstrate how our methodology can be adapted to interrogate interactions between membrane-associated Env and the matrix domain of Gag. These findings and methods may assist vaccine design.IMPORTANCEHIV-1 Env is dynamic and undergoes large conformational changes that drive fusion of virus and host cell membranes. Three Env proteins in a trimer contact each other at their apical tips to form a closed conformation that presents epitopes recognized by broadly neutralizing antibodies. The apical tips separate, among other changes, to form an open conformation that binds tightly to host receptors. Understanding how Env sequence facilitates these structural changes can inform the biophysical mechanism and aid immunogen design. Using deep mutational scans decoupled from virus replication, we report mutational landscapes for Env from two strains interacting with conformation-dependent binding proteins. Residues in the Env trimer interface and apical domains are preferentially conserved in the closed conformation, and conformational diversity is facilitated by electrostatic repulsion and an underpacked core between domains. Specific mutations are described that enhance presentation of the trimeric closed conformation across diverse HIV-1 strains.

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Neutralizing Antibodies Targeting HIV-1 gp41

Viruses ◽

10.3390/v12111210 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1210

Author(s):

Christophe Caillat ◽

Delphine Guilligay ◽

Guidenn Sulbaran ◽

Winfried Weissenhorn

Keyword(s):

Conformational Changes ◽

Neutralizing Antibodies ◽

Somatic Mutations ◽

Heptad Repeat ◽

Broadly Neutralizing Antibodies ◽

Vaccine Research ◽

Membrane Components ◽

And Function ◽

Hiv 1

HIV-1 vaccine research has obtained an enormous boost since the discovery of many broadly neutralizing antibodies (bnAbs) targeting all accessible sites on the HIV-1 envelope glycoprotein (Env). This in turn facilitated high-resolution structures of the Env glycoprotein in complex with bnAbs. Here we focus on gp41, its highly conserved heptad repeat region 1 (HR1), the fusion peptide (FP) and the membrane-proximal external region (MPER). Notably, the broadest neutralizing antibodies target MPER. Both gp41 HR1 and MPER are only fully accessible once receptor-induced conformational changes have taken place, although some studies suggest access to MPER in the close to native Env conformation. We summarize the data on the structure and function of neutralizing antibodies targeting gp41 HR1, FP and MPER and we review their access to Env and their complex formation with gp41 HR1, MPER peptides and FP within native Env. We further discuss MPER bnAb binding to lipids and the role of somatic mutations in recognizing a bipartite epitope composed of the conserved MPER sequence and membrane components. The problematic of gp41 HR1 access and MPER bnAb auto- and polyreactivity is developed in the light of inducing such antibodies by vaccination.

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DNA Duplexes with Hydrophobic Modifications Inhibit Fusion between HIV-1 and Cell Membranes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00758-13 ◽

2013 ◽

Vol 57 (10) ◽

pp. 4963-4970 ◽

Cited By ~ 8

Author(s):

Liang Xu ◽

Lifeng Cai ◽

Xueliang Chen ◽

Xifeng Jiang ◽

Huihui Chong ◽

...

Keyword(s):

Lipid Bilayers ◽

Cell Membranes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

New Drugs ◽

Heptad Repeat ◽

Dna Duplexes ◽

Dna Duplex ◽

Modified Dna ◽

Hiv 1

ABSTRACTDiscovery of new drugs for the treatment of AIDS typically possessing unique structures associated with novel mechanisms of action has been of great importance due to the quick drug-resistant mutations of HIV-1 strains. The work presented in this report describes a novel class of DNA duplex-based HIV-1 fusion inhibitors. Hydrophobic groups were introduced into a DNA duplex skeleton either at one end, at both ends, or in the middle. These modified DNA duplexes inhibited fusion between HIV-1 and human cell membranes at micro- or submicromolar concentrations. Respective inhibitors adopted an aptamer pattern instead of a base-pairing interaction pattern. Structure-activity relationship studies of the respective DNA duplexes showed that the rigid and negatively charged DNA skeletons, in addition to the presence of hydrophobic groups, were crucial to the anti-HIV-1 activity of these compounds. A fluorescent resonance energy transfer (FRET)-based inhibitory assay showed that these duplex inhibitors interacted with the primary pocket in the gp41 N-terminal heptad repeat (NHR) instead of interacting with the lipid bilayers.

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Viral Entry Inhibitors Targeted to the Membrane Site of Action

Journal of Virology ◽

10.1128/jvi.00135-10 ◽

2010 ◽

Vol 84 (13) ◽

pp. 6760-6768 ◽

Cited By ~ 62

Author(s):

Matteo Porotto ◽

Christine C. Yokoyama ◽

Laura M. Palermo ◽

Bruce Mungall ◽

Mohamad Aljofan ◽

...

Keyword(s):

Conformational Changes ◽

Viral Entry ◽

Target Cell ◽

Fusion Proteins ◽

Transmembrane Domain ◽

Hendra Virus ◽

Heptad Repeat ◽

Membrane Microdomains ◽

Site Of Action ◽

Hiv 1

ABSTRACT The fusion of enveloped viruses with the host cell is driven by specialized fusion proteins to initiate infection. The “class I” fusion proteins harbor two regions, typically two heptad repeat (HR) domains, which are central to the complex conformational changes leading to fusion: the first heptad repeat (HRN) is adjacent to the fusion peptide, while the second (HRC) immediately precedes the transmembrane domain. Peptides derived from the HR regions can inhibit fusion, and one HR peptide, T20 (enfuvirtide), is in clinical use for HIV-1. For paramyxoviruses, the activities of two membrane proteins, the receptor-binding protein (hemagglutinin-neuraminidase [HN] or G) and the fusion protein (F), initiate viral entry. The binding of HN or G to its receptor on a target cell triggers the activation of F, which then inserts into the target cell and mediates the membrane fusion that initiates infection. We have shown that for paramyxoviruses, the inhibitory efficacy of HR peptides is inversely proportional to the rate of F activation. For HIV-1, the antiviral potency of an HRC-derived peptide can be dramatically increased by targeting it to the membrane microdomains where fusion occurs, via the addition of a cholesterol group. We report here that for three paramyxoviruses—human parainfluenza virus type 3 (HPIV3), a major cause of lower respiratory tract diseases in infants, and the emerging zoonotic viruses Hendra virus (HeV) and Nipah virus (NiV), which cause lethal central nervous system diseases—the addition of cholesterol to a paramyxovirus HRC-derived peptide increased antiviral potency by 2 log units. Our data suggest that this enhanced activity is indeed the result of the targeting of the peptide to the plasma membrane, where fusion occurs. The cholesterol-tagged peptides on the cell surface create a protective antiviral shield, target the F protein directly at its site of action, and expand the potential utility of inhibitory peptides for paramyxoviruses.

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Identification of HIV-1 Envelope Mutations that Enhance Entry Using Macaque CD4 and CCR5

Viruses ◽

10.3390/v12020241 ◽

2020 ◽

Vol 12 (2) ◽

pp. 241

Author(s):

Jeremy I. Roop ◽

Noah A. Cassidy ◽

Adam S. Dingens ◽

Jesse D. Bloom ◽

Julie Overbaugh

Keyword(s):

Viral Entry ◽

Neutralizing Antibodies ◽

Bound States ◽

Vaccine Development ◽

Rhesus Macaques ◽

Heptad Repeat ◽

Design Strategies ◽

Model Studies ◽

Macaque Model ◽

Hiv 1

Although Rhesus macaques are an important animal model for HIV-1 vaccine development research, most transmitted HIV-1 strains replicate poorly in macaque cells. A major genetic determinant of this species-specific restriction is a non-synonymous mutation in macaque CD4 that results in reduced HIV-1 Envelope (Env)-mediated viral entry compared to human CD4. Recent research efforts employing either laboratory evolution or structure-guided design strategies have uncovered several mutations in Env’s gp120 subunit that enhance binding of macaque CD4 by transmitted/founder HIV-1 viruses. In order to identify additional Env mutations that promote infection of macaque cells, we utilized deep mutational scanning to screen thousands of Env point mutants for those that enhance HIV-1 entry via macaque receptors. We identified many uncharacterized amino acid mutations in the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions of gp41 that increased entry into cells bearing macaque receptors up to 9-fold. Many of these mutations also modestly increased infection of cells bearing human CD4 and CCR5 (up to 1.5-fold). NHR/CHR mutations identified by deep mutational scanning that enhanced entry also increased sensitivity to neutralizing antibodies targeting the MPER epitope, and to inactivation by cold-incubation, suggesting that they promote sampling of an intermediate trimer conformation between closed and receptor bound states. Identification of this set of mutations can inform future macaque model studies, and also further our understanding of the relationship between Env structure and function.

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Immunogenicity Study of Glycoprotein-Deficient Rabies Virus Expressing Simian/Human Immunodeficiency Virus SHIV89.6P Envelope in a Rhesus Macaque

Journal of Virology ◽

10.1128/jvi.78.24.13455-13459.2004 ◽

2004 ◽

Vol 78 (24) ◽

pp. 13455-13459 ◽

Cited By ~ 12

Author(s):

Philip M. McKenna ◽

Pyone Pyone Aye ◽

Bernhard Dietzschold ◽

David C. Montefiori ◽

Louis N. Martin ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Rhesus Macaque ◽

Rabies Virus ◽

Neutralizing Antibodies ◽

Transmembrane Domain ◽

Cell Loss ◽

Glycoprotein G ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Rabies virus (RV) has recently been developed as a novel vaccine candidate for human immunodeficiency virus type 1 (HIV-1). The RV glycoprotein (G) can be functionally replaced by HIV-1 envelope glycoprotein (Env) if the gp160 cytoplasmic domain (CD) of HIV-1 Env is replaced by that of RV G. Here, we describe a pilot study of the in vivo replication and immunogenicity of an RV with a deletion of G (ΔG) expressing a simian/human immunodeficiency virus SHIV89.6P Env ectodomain and transmembrane domain fused to the RV G CD (ΔG-89.6P-RVG) in a rhesus macaque. An animal vaccinated with ΔG-89.6P-RVG developed SHIV89.6P virus-neutralizing antibodies and SHIV89.6P-specific cellular immune responses after challenge with SHIV89.6P. There was no evidence of CD4+ T-cell loss, and plasma viremia was controlled to undetectable levels by 6 weeks postchallenge and has remained suppressed out to 22 weeks postchallenge.

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