X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer

The structural and biochemical characterization of broadly neutralizing anti-HIV-1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV-1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high-mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high-resolution structures. 3.5 and 3.9 Å resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation were solved, revealing a glycan shield of high-mannose and complex-type N-glycans that were used to define the complete epitopes of two bNAbs. Here, the refinement of the N-glycans in the crystal structures is discussed and comparisons are made with glycan densities in glycosylated Env structures derived by single-particle cryo-electron microscopy

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

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

X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer

The structural and biochemical characterization of broadly neutralizing anti-HIV-1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV-1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high-mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high-resolution structures. 3.5 and 3.9 Å resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation were solved, revealing a glycan shield of high-mannose and complex-type N-glycans that were used to define the complete epitopes of two bNAbs. Here, the refinement of the N-glycans in the crystal structures is discussed and comparisons are made with glycan densities in glycosylated Env structures derived by single-particle cryo-electron microscopy

Cryo-EM structure of a CD4-bound open HIV-1 envelope trimer reveals structural rearrangements of the gp120 V1V2 loop

The HIV-1 envelope (Env) glycoprotein, a trimer of gp120–gp41 heterodimers, relies on conformational flexibility to function in fusing the viral and host membranes. Fusion is achieved after gp120 binds to CD4, the HIV-1 receptor, and a coreceptor, capturing an open conformational state in which the fusion machinery on gp41 gains access to the target cell membrane. In the well-characterized closed Env conformation, the gp120 V1V2 loops interact at the apex of the Env trimer. Less is known about the structure of the open CD4-bound state, in which the V1V2 loops must rearrange and separate to allow access to the coreceptor binding site. We identified two anti–HIV-1 antibodies, the coreceptor mimicking antibody 17b and the gp120–gp41 interface-spanning antibody 8ANC195, that can be added as Fabs to a soluble native-like Env trimer to stabilize it in a CD4-bound conformation. Here, we present an 8.9-Å cryo-electron microscopy structure of a BG505 Env–sCD4–17b–8ANC195 complex, which reveals large structural rearrangements in gp120, but small changes in gp41, compared with closed Env structures. The gp120 protomers are rotated and separated in the CD4-bound structure, and the three V1V2 loops are displaced by ∼40 Å from their positions at the trimer apex in closed Env to the sides of the trimer in positions adjacent to, and interacting with, the three bound CD4s. These results are relevant to understanding CD4-induced conformational changes leading to coreceptor binding and fusion, and HIV-1 Env conformational dynamics, and describe a target structure relevant to drug design and vaccine efforts

Coexistence of potent HIV-1 broadly neutralizing antibodies and antibody-sensitive viruses in a viremic controller

Some HIV-1–infected patients develop broad and potent HIV-1 neutralizing antibodies (bNAbs) that when passively transferred to mice or macaques can treat or prevent infection. However, bNAbs typically fail to neutralize coexisting autologous viruses due to antibody-mediated selection against sensitive viral strains. We describe an HIV-1 controller expressing HLA-B57*01 and HLA-B27*05 who maintained low viral loads for 30 years after infection and developed broad and potent serologic activity against HIV-1. Neutralization was attributed to three different bNAbs targeting nonoverlapping sites on the HIV-1 envelope trimer (Env). One of the three, BG18, an antibody directed against the glycan-V3 portion of Env, is the most potent member of this class reported to date and, as revealed by crystallography and electron microscopy, recognizes HIV-1 Env in a manner that is distinct from other bNAbs in this class. Single-genome sequencing of HIV-1 from serum samples obtained over a period of 9 years showed a diverse group of circulating viruses, 88.5% (31 of 35) of which remained sensitive to at least one of the temporally coincident autologous bNAbs and the individual’s serum. Thus, bNAb-sensitive strains of HIV-1 coexist with potent neutralizing antibodies that target the virus and may contribute to control in this individual. When administered as a mix, the three bNAbs controlled viremia in HIV-1_(YU2)–infected humanized mice. Our finding suggests that combinations of bNAbs may contribute to control of HIV-1 infection

Mechanism of assembly of a substrate-transfer complex during tail-anchored protein targeting

Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C terminus, are post-translationally targeted to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. In yeast, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 complex (Get4/5), which tethers the co-chaperone Sgt2 to the targeting factor, the Get3 ATPase. Binding of Get4/5 to Get3 is critical for efficient TA targeting; however, questions remain about the formation of the Get3·Get4/5 complex. Here we report crystal structures of a Get3·Get4/5 complex from Saccharomyces cerevisiae at 2.8 and 6.0 Å that reveal a novel interface between Get3 and Get4 dominated by electrostatic interactions. Kinetic and mutational analyses strongly suggest that these structures represent an on-pathway intermediate that rapidly assembles and then rearranges to the final Get3·Get4/5 complex. Furthermore, we provide evidence that the Get3·Get4/5 complex is dominated by a single Get4/5 heterotetramer bound to one monomer of a Get3 dimer, uncovering an intriguing asymmetry in the Get4/5 heterotetramer upon Get3 binding. Ultrafast diffusion-limited electrostatically driven Get3·Get4/5 association enables Get4/5 to rapidly sample and capture Get3 at different stages of the GET pathway

Nanoparticles presenting clusters of CD4 expose a universal vulnerability of HIV-1 by mimicking target cells

CD4-based decoy approaches against HIV-1 are attractive options for long-term viral control, but initial designs, including soluble CD4 (sCD4) and CD4-Ig, were ineffective. To evaluate a therapeutic that more accurately mimics HIV-1 target cells compared with monomeric sCD4 and dimeric CD4-Ig, we generated virus-like nanoparticles that present clusters of membrane-associated CD4 (CD4-VLPs) to permit high-avidity binding of trimeric HIV-1 envelope spikes. In neutralization assays, CD4-VLPs were >12,000-fold more potent than sCD4 and CD4-Ig and >100-fold more potent than the broadly neutralizing antibody (bNAb) 3BNC117, with >12,000-fold improvements against strains poorly neutralized by 3BNC117. CD4-VLPs also neutralized patient-derived viral isolates that were resistant to 3BNC117 and other bNAbs. Intraperitoneal injections of CD4-CCR5-VLP produced only subneutralizing plasma concentrations in HIV-1–infected humanized mice but elicited CD4-binding site mutations that reduced viral fitness. All mutant viruses showed reduced sensitivity to sCD4 and CD4-Ig but remained sensitive to neutralization by CD4-VLPs in vitro. In vitro evolution studies demonstrated that CD4-VLPs effectively controlled HIV-1 replication at neutralizing concentrations, and viral escape was not observed. Moreover, CD4-VLPs potently neutralized viral swarms that were completely resistant to CD4-Ig, suggesting that escape pathways that confer resistance against conventional CD4-based inhibitors are ineffective against CD4-VLPs. These findings suggest that therapeutics that mimic HIV-1 target cells could prevent viral escape by exposing a universal vulnerability of HIV-1: the requirement to bind CD4 on a target cell. We propose that therapeutic and delivery strategies that ensure durable bioavailability need to be developed to translate this concept into a clinically feasible functional cure therapy

Nanoparticles presenting clusters of CD4 expose a universal vulnerability of HIV-1 by mimicking target cells

CD4-based decoy approaches against HIV-1 are attractive options for long-term viral control, but initial designs, including soluble CD4 (sCD4) and CD4-Ig, were ineffective. To evaluate a therapeutic that more accurately mimics HIV-1 target cells compared with monomeric sCD4 and dimeric CD4-Ig, we generated virus-like nanoparticles that present clusters of membrane-associated CD4 (CD4-VLPs) to permit high-avidity binding of trimeric HIV-1 envelope spikes. In neutralization assays, CD4-VLPs were >12,000-fold more potent than sCD4 and CD4-Ig and >100-fold more potent than the broadly neutralizing antibody (bNAb) 3BNC117, with >12,000-fold improvements against strains poorly neutralized by 3BNC117. CD4-VLPs also neutralized patient-derived viral isolates that were resistant to 3BNC117 and other bNAbs. Intraperitoneal injections of CD4-CCR5-VLP produced only subneutralizing plasma concentrations in HIV-1–infected humanized mice but elicited CD4-binding site mutations that reduced viral fitness. All mutant viruses showed reduced sensitivity to sCD4 and CD4-Ig but remained sensitive to neutralization by CD4-VLPs in vitro. In vitro evolution studies demonstrated that CD4-VLPs effectively controlled HIV-1 replication at neutralizing concentrations, and viral escape was not observed. Moreover, CD4-VLPs potently neutralized viral swarms that were completely resistant to CD4-Ig, suggesting that escape pathways that confer resistance against conventional CD4-based inhibitors are ineffective against CD4-VLPs. These findings suggest that therapeutics that mimic HIV-1 target cells could prevent viral escape by exposing a universal vulnerability of HIV-1: the requirement to bind CD4 on a target cell. We propose that therapeutic and delivery strategies that ensure durable bioavailability need to be developed to translate this concept into a clinically feasible functional cure therapy