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HIV-1 envelope glycoprotein (Env), which consists of trimeric (gp160) 3 cleaved to (gp120 and gp41) 3 , interacts with the primary receptor CD4 and a coreceptor (such as chemokine receptor CCR5) to fuse viral and target-cell membranes. The gp120–coreceptor interaction has previously been proposed as the most crucial trigger for unleashing the fusogenic potential of gp41. Here we report a cryo-electron microscopy structure of a full-length gp120 in complex with soluble CD4 and unmodified human CCR5, at 3.9 Å resolution. The V3 loop of gp120 inserts into the chemokine-binding pocket formed by seven transmembrane helices of CCR5, and the N terminus of CCR5 contacts the CD4-induced bridging sheet of gp120. CCR5 induces no obvious allosteric changes in gp120 that can propagate to gp41; it does bring the Env trimer close to the target membrane. The N terminus of gp120, which is gripped by gp41 in the pre-fusion or CD4-bound Env, flips back in the CCR5-bound conformation and may irreversibly destabilize gp41 to initiate fusion. The coreceptor probably functions by stabilizing and anchoring the CD4-induced conformation of Env near the cell membrane. These results advance our understanding of HIV-1 entry into host cells and may guide the development of vaccines and therapeutic agents. The cryo-electron microscopy structure of the gp120 component of the HIV-1 envelope glycoprotein, in complex with the primary receptor CD4 and coreceptor CCR5, provides insight into the cell-entry mechanism of HIV-1.

The envelope glycoprotein trimer (Env) on the surface of HIV-1 recognizes CD4⁺ T cells and mediates viral entry. During this process, Env undergoes substantial conformational rearrangements, making it difficult to study in its native state. Soluble stabilized trimers have provided valuable insights into the Env structure, but they lack the hydrophobic membrane proximal external region (MPER, an important target of broadly neutralizing antibodies), the transmembrane domain, and the cytoplasmic tail. Here we present (i) a cryogenic electron microscopy (cryo-EM) structure of a clade B virus Env, which lacks only the cytoplasmic tail and is stabilized by the broadly neutralizing antibody PGT151, at a resolution of 4.2 angstroms and (ii) a reconstruction of this form of Env in complex with PGT151 and MPER-targeting antibody 10E8 at a resolution of 8.8 angstroms. These structures provide new insights into the wild-type Env structure.

New high-resolution cryo-electron microscopy structures of the HIV-1 envelope protein provide a detailed description and understanding of how the HIV-1 fusion machinery functions and how it changes its structure over time to convert from the pre-fusion to the fusion-intermediate conformation. Understanding the HIV-1 envelope The envelope glycoprotein on the surface of HIV (Env) binds to its cellular receptor CD4 and co-receptor CXCR4/CCR5. Upon receptor binding it undergoes structural rearrangements that result in fusion between the lipid bilayer of the virus and the host cell membrane. Several previous studies have revealed static pre-fusion, intermediate and post-fusion states of HIV-1 Env. In this study, Andrew Ward and colleagues present a variety of new high resolution cryo-electron microscopy structures of Env, which together provide a detailed description and understanding of how the HIV-1 fusion machinery functions and how it changes its structure over time to convert from the pre- to the post-fusion confirmation. For many enveloped viruses, binding to a receptor(s) on a host cell acts as the first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for replication 1 , 2 . The envelope glycoprotein (Env) trimer on the surface of HIV is responsible for receptor binding and fusion. Although Env can tolerate a high degree of mutation in five variable regions (V1–V5), and also at N-linked glycosylation sites that contribute roughly half the mass of Env, the functional sites for recognition of receptor CD4 and co-receptor CXCR4/CCR5 are conserved and essential for viral fitness. Soluble SOSIP Env trimers are structural and antigenic mimics of the pre-fusion native, surface-presented Env 3 , 4 , and are targets of broadly neutralizing antibodies. Thus, they are attractive immunogens for vaccine development 5 , 6 , 7 , 8 . Here we present high-resolution cryo-electron microscopy structures of subtype B B41 SOSIP Env trimers in complex with CD4 and antibody 17b, or with antibody b12, at resolutions of 3.7 Å and 3.6 Å, respectively. We compare these to cryo-electron microscopy reconstructions of B41 SOSIP Env trimers with no ligand or in complex with either CD4 or the CD4-binding-site antibody PGV04 at 5.6 Å, 5.2 Å and 7.4 Å resolution, respectively. Consequently, we present the most complete description yet, to our knowledge, of the CD4–17b-induced intermediate and provide the molecular basis of the receptor-binding-induced conformational change required for HIV-1 entry into host cells. Both CD4 and b12 induce large, previously uncharacterized conformational rearrangements in the gp41 subunits, and the fusion peptide becomes buried in a newly formed pocket. These structures provide key details on the biological function of the type I viral fusion machine from HIV-1 as well as new templates for inhibitor design.

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.Cryo-EM resolution of HIV-1 Env trimer bound to CD4 and a tyrosine-sulfated, coreceptor-mimicking antibody reveals two configurations of gp120–gp41 protomers that create asymmetric Env trimer conformations on the path to membrane fusion.

The HIV-1 envelope glycoprotein (Env) trimer, composed of gp120 and gp41 subunits, mediates viral entry into cells. Recombinant Env trimers have been studied structurally, but characterization of Env embedded in intact virus membranes has been limited to low resolution. Here, we deploy cryo-electron tomography and subtomogram averaging to determine the structures of Env trimers on aldrithiol-2 (AT-2)-inactivated virions in ligand-free, antibody-bound and CD4-bound forms at subnanometer resolution. Tomographic reconstructions document molecular features consistent with high-resolution structures of engineered soluble and detergent-solubilized Env trimers. One of three conformational states previously predicted by smFRET was not observed by cryo-ET, potentially owing to AT-2 inactivation. We did observe Env trimers to open in situ in response to CD4 binding, with an outward movement of gp120-variable loops and an extension of a critical gp41 helix. Overall features of Env trimer embedded in AT-2-treated virions appear well-represented by current engineered trimers.Cryo-ET and subtomogram averaging analyses are used to determine structures of HIV-1 Env trimers on the surface of inactivated virions.

During the first steps of HIV infection the Env subunit gp41 is thought to establish contact between the membranes and to be the main driver of fusion. Here we investigated in liquid crystalline membranes the structure and cholesterol recognition of constructs made of a gp41 external region carrying a cholesterol recognition amino acid consensus (CRAC) motif and a hydrophobic membrane anchoring sequence. CD- und ATR-FTIR spectroscopies indicate that the constructs adopt a high degree of helical secondary structure in membrane environments. Furthermore, 15 N and 2 H solid-state NMR spectra of gp41 polypeptides reconstituted into uniaxially oriented bilayers agree with the CRAC domain being an extension of the transmembrane helix. Upon addition of cholesterol the CRAC NMR spectra remain largely unaffected when being associated with the native gp41 transmembrane sequence but its topology changes when anchored in the membrane by a hydrophobic model sequence. The 2 H solid-state NMR spectra of deuterated cholesterol are indicative of a stronger influence of the model sequence on this lipid when compared to the native gp41 sequence. These observations are suggestive of a strong coupling between the transmembrane and the membrane proximal region of gp41 possibly enforced by oligomerization of the transmembrane helical region.

Significance Enveloped viruses infect cells via fusion of viral and host cell membranes mediated by highly conserved fusion proteins. HIV-1 glycoprotein 41 (gp41) represents a family of fusion proteins with similar structures and fusion mechanisms. They couple their energetic folding to draw two membranes close for fusion, forming trimers of helical hairpins. Yet, the energy release, force generation, and kinetics associated with folding of these proteins are poorly quantified. We found that gp41 hairpins fold sequentially but in a kinetically coupled manner and that an anti-HIV drug blocked gp41 folding by a new mechanism. As major proteins on viral surfaces, fusion proteins are primary targets for vaccine development and fusion inhibitors to intervene in major infectious diseases such as AIDS, Ebola, and influenza. HIV-1 glycoprotein 41 (gp41) mediates viral entry into host cells by coupling its folding energy to membrane fusion. Gp41 folding is blocked by fusion inhibitors, including the commercial drug T20, to treat HIV/AIDS. However, gp41 folding intermediates, energy, and kinetics are poorly understood. Here, we identified the folding intermediates of a single gp41 trimer-of-hairpins and measured their associated energy and kinetics using high-resolution optical tweezers. We found that folding of gp41 hairpins was energetically independent but kinetically coupled: Each hairpin contributed a folding energy of ∼−23 k BT, but folding of one hairpin successively accelerated the folding rate of the next one by ∼20-fold. Membrane-mimicking micelles slowed down gp41 folding and reduced the stability of the six-helix bundle. However, the stability was restored by cooperative folding of the membrane-proximal external region. Surprisingly, T20 strongly inhibited gp41 folding by actively displacing the C-terminal hairpin strand in a force-dependent manner. The inhibition was abolished by a T20-resistant gp41 mutation. The energetics and kinetics of gp41 folding established by us provides a basis to understand viral membrane fusion, infection, and therapeutic intervention.

Human and monkey immunodeficiency viruses are studded with \"spikes\" that enable them to infect cells. Structural studies reveal that these spikes are tripod-like assemblies that cluster on the virus surface.

Molecular and structural characterization is reported for a new broad and potent monoclonal antibody against HIV that binds to an epitope bridging the gp41 and gp120 subunits — the antibody affects a step in virus entry after binding to CD4 and before engagement of CCR5. Novel vaccine target on HIV-1 This paper describes a broadly neutralizing HIV-specific monoclonal antibody that binds with high potency to a novel HIV-1 envelope glycoprotein epitope. Molecular and structural characterization of the new antibody, named 35O22, show that it is specific for a new site of vulnerability made up of amino acids and glycans bridging the gp41 and gp120 subunits. The antibody affects a step in virus entry after binding to CD4 and before engagement of CCR5. Serologic analysis indicates that antibodies to this newly recognized site of vulnerability are commonly elicited by natural infection, raising the prospect that in may be a promising potential vaccine target. The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-1 (reviewed in ref. 1 ). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with a half-maximum inhibitory concentration (IC 50 ) <50 μg ml −1 . The median IC 50 of neutralized viruses was 0.033 μg ml −1 , among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120 and gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops 1 – 4 and in fully saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops 3 – 9 . To investigate changes resulting from substoichiometric CD4 binding, we solved single-particle cryo-electron microscopy (cryo-EM) structures of soluble, native-like heterotrimeric Envs bound to one or two CD4 molecules. Most of the Env trimers bound to one CD4 adopted the closed, prefusion Env state, with a minority exhibiting a heterogeneous partially open Env conformation. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state 10 that showed gp120 outward rotation but maintained the prefusion three-stranded gp120 bridging sheet with only partial V1V2 displacement and V3 exposure. We conclude that most of the engagements with one CD4 molecule were insufficient to stimulate CD4-induced conformational changes, whereas binding two CD4 molecules led to Env opening in CD4-bound protomers only. The substoichiometric CD4-bound soluble Env heterotrimer structures resembled counterparts derived from a cryo-electron tomography study of complexes between virion-bound Envs and membrane-anchored CD4 (ref.  11 ), validating their physiological relevance. Together, these results illuminate intermediate conformations of HIV-1 Env and illustrate its structural plasticity. Single-particle cryo-electron microscopy structures of HIV-1 envelope (Env) trimers bound to one or two CD4 receptors identify intermediate Env conformations that precede host–virus fusion and inform the design of therapeutics to prevent HIV-1 infection.