An Inducible ESCRT-III Inhibition Tool to Control HIV-1 Budding.

HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated autocleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization, and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins. Notably, upon drug administration, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted no effect but synergized with CHMP2A-NS3. Localization studies demonstrated the relocalization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes.

An inducible ESCRT-III inhibition tool to control HIV-1 budding.

HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated auto-cleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins with variable modification of Gag VLP budding upon drug administration. Notably, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted a minor effect and synergized with CHMP2A-NS3. Localization studies demonstrated the re-localization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes.

SARS-CoV-2 S Glycoprotein Stabilization Strategies.

The SARS-CoV-2 pandemic has again shown that structural biology plays an important role in understanding biological mechanisms and exploiting structural data for therapeutic interventions. Notably, previous work on SARS-related glycoproteins has paved the way for the rapid structural determination of the SARS-CoV-2 S glycoprotein, which is the main target for neutralizing antibodies. Therefore, all vaccine approaches aimed to employ S as an immunogen to induce neutralizing antibodies. Like all enveloped virus glycoproteins, SARS-CoV-2 S native prefusion trimers are in a metastable conformation, which primes the glycoprotein for the entry process via membrane fusion. S-mediated entry is associated with major conformational changes in S, which can expose many off-target epitopes that deviate vaccination approaches from the major aim of inducing neutralizing antibodies, which mainly target the native prefusion trimer conformation. Here, we review the viral glycoprotein stabilization methods developed prior to SARS-CoV-2, and applied to SARS-CoV-2 S, in order to stabilize S in the prefusion conformation. The importance of structure-based approaches is highlighted by the benefits of employing stabilized S trimers versus non-stabilized S in vaccines with respect to their protective efficacy.

Structural basis of CHMP2A-CHMP3 ESCRT-III polymer assembly and membrane cleavage.

The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo-EM structures of membrane-coated CHMP2A-CHMP3 filaments from Homo sapiens of two different diameters at 3.3 and 3.6 Å resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence toward the tube interior. Inter-filament interactions are electrostatic, which may facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high-speed atomic force microscopy imaging corroborate that VPS4 can constrict and cleave CHMP2A-CHMP3 membrane tubes. We therefore conclude that CHMP2A-CHMP3-VPS4 act as a minimal membrane fission machinery.

Immunization with synthetic SARS-CoV-2 S glycoprotein virus-like particles protects macaques from infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused an ongoing global health crisis. Here, we present as a vaccine candidate synthetic SARS-CoV-2 spike (S) glycoprotein-coated lipid vesicles that resemble virus-like particles. Soluble S glycoprotein trimer stabilization by formaldehyde cross-linking introduces two major inter-protomer cross-links that keep all receptor-binding domains in the "down" conformation. Immunization of cynomolgus macaques with S coated onto lipid vesicles (S-LVs) induces high antibody titers with potent neutralizing activity against the vaccine strain, Alpha, Beta, and Gamma variants as well as T helper (Th)1 CD4+-biased T cell responses. Although anti-receptor-binding domain (RBD)-specific antibody responses are initially predominant, the third immunization boosts significant non-RBD antibody titers. Challenging vaccinated animals with SARS-CoV-2 shows a complete protection through sterilizing immunity, which correlates with the presence of nasopharyngeal anti-S immunoglobulin G (IgG) and IgA titers. Thus, the S-LV approach is an efficient and safe vaccine candidate based on a proven classical approach for further development and clinical testing.

The archaeal division protein CdvB1 assembles into polymers that are depolymerized by CdvC.

The Cdv proteins constitute the cell division system of the Crenarchaea, a machinery closely related to the ESCRT system of eukaryotes. Using a combination of TEM imaging and biochemical assays, we here present an in vitro study of Metallosphaera sedula CdvB1, the Cdv protein that is believed to play a major role in the constricting ring that drives cell division in the Crenarchaea. We show that CdvB1 self-assembles into filaments that are depolymerized by the Vps4-homolog ATPase CdvC. Furthermore, we find that CdvB1 binds to negatively charged lipid membranes and can be detached from the membrane by the action of CdvC. Our findings provide novel insight into one of the main components of the archaeal cell division machinery.

Stepwise Conformational Stabilization of a HIV-1 Clade C Consensus Envelope Trimer Immunogen Impacts the Profile of Vaccine-Induced Antibody Responses.

Stabilization of the HIV-1 Envelope glycoprotein trimer (Env) in its native pre-fusion closed conformation is regarded as one of several requirements for the induction of neutralizing antibody (nAb) responses, which, in turn, will most likely be a prerequisite for the development of an efficacious preventive vaccine. Here, we systematically analyzed how the stepwise stabilization of a clade C consensus (ConC) Env immunogen impacts biochemical and biophysical protein traits such as antigenicity, thermal stability, structural integrity, and particle size distribution. The increasing degree of conformational rigidification positively correlates with favorable protein characteristics, leading to optimized homogeneity of the protein preparations, increased thermal stability, and an overall favorable binding profile of structure-dependent broadly neutralizing antibodies (bnAbs) and non-neutralizing antibodies (non-nAbs). We confirmed that increasing the structural integrity and stability of the Env trimers positively correlates with the quality of induced antibody responses by the immunogens. These and other data contribute to the selection of ConCv5 KIKO as novel Env immunogens for use within the European Union's H2020 Research Consortium EHVA (European HIV Alliance) for further preclinical analysis and phase 1 clinical development.

Structure of HIV-1 gp41 with its membrane anchors targeted by neutralizing antibodies.

The HIV-1 gp120/gp41 trimer undergoes a series of conformational changes in order to catalyze gp41-induced fusion of viral and cellular membranes. Here, we present the crystal structure of gp41 locked in a fusion intermediate state by an MPER-specific neutralizing antibody. The structure illustrates the conformational plasticity of the six membrane anchors arranged asymmetrically with the fusion peptides and the transmembrane regions pointing into different directions. Hinge regions located adjacent to the fusion peptide and the transmembrane region facilitate the conformational flexibility that allows high-affinity binding of broadly neutralizing anti-MPER antibodies. Molecular dynamics simulation of the MPER Ab-stabilized gp41 conformation reveals a possible transition pathway into the final post-fusion conformation with the central fusion peptides forming a hydrophobic core with flanking transmembrane regions. This suggests that MPER-specific broadly neutralizing antibodies can block final steps of refolding of the fusion peptide and the transmembrane region, which is required for completing membrane fusion.

The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization.

BACKGROUND: ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, ESCRT-III CHMP2 exists as two isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet. RESULTS: Here, we use a combination of techniques on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Thus, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect. CONCLUSIONS: We therefore conclude that CHMP2B and CHMP2A exhibit different mechanical properties and might thus contribute differently to the diverse ESCRT-III-catalyzed membrane remodeling processes.

Neutralizing Antibodies Targeting HIV-1 gp41.

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.

Author Correction: Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation.

Endosomal sorting complexes for transport-III (ESCRT-III) assemble in vivo onto membranes with negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and how ESCRT-III shapes membranes is yet unclear. Human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 are used to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron tomography and AFM. We show that CHMP4B filaments preferentially bind to flat membranes or to tubes with positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes. Combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes and reshape vesicles into helical "corkscrew-like" membrane tubes. Sub-tomogram averaging reveals that the ESCRT-III filaments assemble parallel and locally perpendicular to the tube axis, highlighting the mechanical stresses imposed by ESCRT-III. Our results underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature required for cellular membrane remodeling processes.

Structural Basis for Broad HIV-1 Neutralization by the MPER-Specific Human Broadly Neutralizing Antibody LN01.

Potent and broadly neutralizing antibodies (bnAbs) are the hallmark of HIV-1 protection by vaccination. The membrane-proximal external region (MPER) of the HIV-1 gp41 fusion protein is targeted by the most broadly reactive HIV-1 neutralizing antibodies. Here, we examine the structural and molecular mechansims of neutralization by anti-MPER bnAb, LN01, which was isolated from lymph-node-derived germinal center B cells of an elite controller and exhibits broad neutralization breadth. LN01 engages both MPER and the transmembrane (TM) region, which together form a continuous helix in complex with LN01. The tilted TM orientation allows LN01 to interact simultaneously with the peptidic component of the MPER epitope and membrane via two specific lipid binding sites of the antibody paratope. Although LN01 carries a high load of somatic mutations, most key residues interacting with the MPER epitope and lipids are germline encoded, lending support for the LN01 epitope as a candidate for lineage-based vaccine development.

Super Potent Bispecific Llama VHH Antibodies Neutralize HIV via a Combination of gp41 and gp120 Epitopes.

Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown of the gp120 V3 loop, occluded in native Env. We present biophysical and structural details on the 2E7 interaction with gp41. In order to further increase breadth and potency, we constructed bi-specific VHH. The combination of CD4bs VHH (J3/3E3) with 2E7 group VHH enhanced strain-specific neutralization with potencies up to 1400-fold higher than the mixture of the individual VHHs. Thus, these new bivalent VHH are potent new tools to develop therapeutic approaches or microbicide intervention.

Activation of the ILT7 receptor and plasmacytoid dendritic cell responses are governed by structurally-distinct BST2 determinants.

Type I interferons (IFN-I) are key innate immune effectors predominantly produced by activated plasmacytoid dendritic cells (pDCs). By modulating immune responses at their foundation, IFNs can widely reshape immunity to control infectious diseases and malignancies. Nevertheless, their biological activities can also be detrimental to surrounding healthy cells, as prolonged IFN-I signaling is associated with excessive inflammation and immune dysfunction. The interaction of the human pDC receptor immunoglobulin-like transcript 7 (ILT7) with its IFN-I-regulated ligand, bone marrow stromal cell antigen 2 (BST2) plays a key role in controlling the IFN-I amounts produced by pDCs in response to Toll-like receptor (TLR) activation. However, the structural determinants and molecular features of BST2 that govern ILT7 engagement and activation are largely undefined. Using two functional assays to measure BST2-stimulated ILT7 activation as well as biophysical studies, here we identified two structurally-distinct regions of the BST2 ectodomain that play divergent roles during ILT7 activation. We found that although the coiled-coil region contains a newly defined ILT7-binding surface, the N-terminal region appears to suppress ILT7 activation. We further show that a stable BST2 homodimer binds to ILT7, but post-binding events associated with the unique BST2 coiled-coil plasticity are required to trigger receptor signaling. Hence, BST2 with an unstable or a rigid coiled-coil fails to activate ILT7, whereas substitutions in its N-terminal region enhance activation. Importantly, the biological relevance of these newly defined domains of BST2 is underscored by the identification of substitutions having opposing potentials to activate ILT7 in pathological malignant conditions.

Tumor suppression of novel anti-PD-1 antibodies mediated through CD28 costimulatory pathway.

Classical antagonistic antibodies (Abs) targeting PD-1, such as pembrolizumab and nivolumab, act through blockade of the PD-1-PDL-1 interaction. Here, we have identified novel antagonistic anti-PD-1 Abs not blocking the PD-1-PDL-1 interaction. The nonblocking Abs recognize epitopes on PD-1 located on the opposing face of the PDL-1 interaction and overlap with a newly identified evolutionarily conserved patch. These nonblocking Abs act predominantly through the CD28 coreceptor. Importantly, a combination of blocking and nonblocking Abs synergize in the functional recovery of antigen-specific exhausted CD8 T cells. Interestingly, nonblocking anti-PD-1 Abs have equivalent antitumor activity compared with blocker Abs in two mouse tumor models, and combination therapy using both classes of Abs enhanced tumor suppression in the mouse immunogenic tumor model. The identification of the novel nonblocker anti-PD-1 Abs and their synergy with classical blocker Abs may be instrumental in potentiating immunotherapy strategies and antitumor activity.

VPS4 triggers constriction and cleavage of ESCRT-III helical filaments.

Many cellular processes such as endosomal vesicle budding, virus budding, and cytokinesis require extensive membrane remodeling by the endosomal sorting complex required for transport III (ESCRT-III). ESCRT-III protein family members form spirals with variable diameters in vitro and in vivo inside tubular membrane structures, which need to be constricted to proceed to membrane fission. Here, we show, using high-speed atomic force microscopy and electron microscopy, that the AAA-type adenosine triphosphatase VPS4 constricts and cleaves ESCRT-III CHMP2A-CHMP3 helical filaments in vitro. Constriction starts asymmetrically and progressively decreases the diameter of CHMP2A-CHMP3 tubular structure, thereby coiling up the CHMP2A-CHMP3 filaments into dome-like end caps. Our results demonstrate that VPS4 actively constricts ESCRT-III filaments and cleaves them before their complete disassembly. We propose that the formation of ESCRT-III dome-like end caps by VPS4 within a membrane neck structure constricts the membrane to set the stage for membrane fission.

The role of VPS4 in ESCRT-III polymer remodeling.

The endosomal sorting complex required for transport-III (ESCRT-III) and VPS4 catalyze a variety of membrane-remodeling processes in eukaryotes and archaea. Common to these processes is the dynamic recruitment of ESCRT-III proteins from the cytosol to the inner face of a membrane neck structure, their activation and filament formation inside or at the membrane neck and the subsequent or concomitant recruitment of the AAA-type ATPase VPS4. The dynamic assembly of ESCRT-III filaments and VPS4 on cellular membranes induces constriction of membrane necks with large diameters such as the cytokinetic midbody and necks with small diameters such as those of intraluminal vesicles or enveloped viruses. The two processes seem to use different sets of ESCRT-III filaments. Constriction is then thought to set the stage for membrane fission. Here, we review recent progress in understanding the structural transitions of ESCRT-III proteins required for filament formation, the functional role of VPS4 in dynamic ESCRT-III assembly and its active role in filament constriction. The recent data will be discussed in the context of different mechanistic models for inside-out membrane fission.

Induction of Tier 1 HIV Neutralizing Antibodies by Envelope Trimers Incorporated into a Replication Competent Vesicular Stomatitis Virus Vector.

A chimeric vesicular stomatitis virus with the glycoprotein of the lymphocytic choriomeningitis virus, VSV-GP, is a potent viral vaccine vector that overcomes several of the limitations of wild-type VSV. Here, we evaluated the potential of VSV-GP as an HIV vaccine vector. We introduced genes for different variants of the HIV-1 envelope protein Env, i.e., secreted or membrane-anchored, intact or mutated furin cleavage site or different C-termini, into the genome of VSV-GP. We found that the addition of the Env antigen did not attenuate VSV-GP replication. All HIV-1 Env variants were expressed in VSV-GP infected cells and some were incorporated very efficiently into VSV-GP particles. Crucial epitopes for binding of broadly neutralizing antibodies against HIV-1 such as MPER (membrane-proximal external region), CD4 binding site, V1V2 and V3 loop were present on the surface of VSV-GP-Env particles. Binding of quaternary antibodies indicated a trimeric structure of VSV-GP incorporated Env. We detected high HIV-1 antibody titers in mice and showed that vectors expressing membrane-anchored Env elicited higher antibody titers than vectors that secreted Envs. In rabbits, Tier 1A HIV-1 neutralizing antibodies were detectable after prime immunization and titers further increased after boosting with a second immunization. Taken together, VSV-GP-Env is a promising vector vaccine against HIV-1 infection since this vector permits incorporation of native monomeric and/or trimeric HIV-1 Env into a viral membrane.

Dynamic and Sequential Protein Reconstitution on Negatively Curved Membranes by Giant Vesicles Fusion.

In vitro investigation of the interaction between proteins and positively curved membranes can be performed using a classic nanotube pulling method. However, characterizing protein interaction with negatively curved membranes still represents a formidable challenge. Here, we describe our recently developed approach based on laser-triggered Giant Unilamellar Vesicles (GUVs) fusion. Our protocol allows sequential addition of proteins to a negatively curved membrane, while at the same time controlling the buffer composition, lipid composition and membrane tension. Moreover, this method does not require a step of protein detachment, greatly simplifying the process of protein encapsulation over existing methods.