Xiaochu Ma

Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions

HIVs shape-shifting envelope protein HIVs envelope protein (Env) coats virus particles and allows HIV to enter host cells. HIV entry is highly dynamic. Env proteins work in groups of three (called trimers), which bind to the viral receptor and co-receptor, both expressed by host cells. Viral receptor binding causes a structural rearrangement in the trimer that allows for co-receptor binding and finally, viral entry. To visualize dynamic changes in Env conformation during viral entry, Munro et al. added differently colored fluorescent tags to two different regions of individual HIV trimers. Single-molecule fluorescence resonance entry transfer revealed three distinct Env conformations before cell entry. Occupation of particular conformations depended on host receptor binding. Science, this issue p. 759 Single-molecule fluorescence studies reveal how HIV’s viral spike protein varies its conformation as it binds to receptors. The HIV-1 envelope (Env) mediates viral entry into host cells. To enable the direct imaging of conformational dynamics within Env, we introduced fluorophores into variable regions of the glycoprotein gp120 subunit and measured single-molecule fluorescence resonance energy transfer within the context of native trimers on the surface of HIV-1 virions. Our observations revealed unliganded HIV-1 Env to be intrinsically dynamic, transitioning between three distinct prefusion conformations, whose relative occupancies were remodeled by receptor CD4 and antibody binding. The distinct properties of neutralization-sensitive and neutralization-resistant HIV-1 isolates support a dynamics-based mechanism of immune evasion and ligand recognition.

Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env

As the sole viral antigen on the HIV-1–virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1–Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.

Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody

An antibody to block viral fusion A small fraction of HIV-1–infected individuals develop broad and potent antibodies that bind the HIV-1 envelope protein (Env). These antibodies recognize a limited set of conserved epitopes on Env, such as Envs host receptor-binding site. Kong et al. now report a neutralizing antibody isolated from an HIV-1–infected individual that binds to the fusion peptide of Env. This is unexpected because viruses often try to mask such key components of their cell entry machinery from antibody attack. Crystal structures of the antibody bound to the fusion peptide and to Env itself define the epitope, provide insight into the specific mechanism of antibody binding, and may inform HIV-1 vaccine design. Science, this issue p. 828 A neutralizing antibody against HIV-1 unexpectedly targets a key component of the virus’ cell entry machinery. The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. Here, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showed that the N-terminal portion of the fusion peptide can be solvent-exposed. These results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.

Release of gp120 Restraints Leads to an Entry-Competent Intermediate State of the HIV-1 Envelope Glycoproteins

ABSTRACT Primary human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimers [(gp120/gp41)3] typically exist in a metastable closed conformation (state 1). Binding the CD4 receptor triggers Env to undergo extensive conformational changes to mediate virus entry. We identified specific gp120 residues that restrain Env in state 1. Alteration of these restraining residues destabilized state 1, allowing Env to populate a functional conformation (state 2) intermediate between state 1 and the full CD4-bound state (state 3). Increased state 2 occupancy was associated with lower energy barriers between the states. State 2 was an obligate intermediate for all transitions between state 1 and state 3. State 2-enriched Envs required lower CD4 concentrations to trigger virus entry and more efficiently infected cells expressing low levels of CD4. These Envs were resistant to several broadly neutralizing antibodies and small-molecule inhibitors. Thus, state 2 is an Env conformation on the virus entry pathway; sampling state 2 increases the adaptability of HIV-1 to different host cell receptor levels and immune environments. Our results provide new insights into the conformational regulation of HIV-1 entry. IMPORTANCE The envelope glycoproteins (Env) of HIV-1 mediate virus entry and are the sole targets of neutralizing antibodies. Understanding the way that Env promotes HIV-1 entry can expedite drug and vaccine development. By destabilizing Env, we found that it assumes an intermediate state that is functional and obligate for transitions to entry-competent conformations. Increased sampling of this state enhances the ability of HIV-1 to infect cells that express low levels of the CD4 receptor and allows the virus to evade neutralizing antibodies and small-molecule inhibitors. These findings provide new mechanistic insights into the function and inhibition of HIV-1 Env and will contribute to ongoing therapeutic and prevention efforts to combat HIV-1. The envelope glycoproteins (Env) of HIV-1 mediate virus entry and are the sole targets of neutralizing antibodies. Understanding the way that Env promotes HIV-1 entry can expedite drug and vaccine development. By destabilizing Env, we found that it assumes an intermediate state that is functional and obligate for transitions to entry-competent conformations. Increased sampling of this state enhances the ability of HIV-1 to infect cells that express low levels of the CD4 receptor and allows the virus to evade neutralizing antibodies and small-molecule inhibitors. These findings provide new mechanistic insights into the function and inhibition of HIV-1 Env and will contribute to ongoing therapeutic and prevention efforts to combat HIV-1.

HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations

HIV-1 entry into cells requires binding of the viral envelope glycoprotein (Env) to receptor CD4 and coreceptor. Imaging of individual Env molecules on native virions shows Env trimers to be dynamic, spontaneously transitioning between three distinct well-populated conformational states: a pre-triggered Env (State 1), a default intermediate (State 2) and a three-CD4-bound conformation (State 3), which can be stabilized by binding of CD4 and coreceptor-surrogate antibody 17b. Here, using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we show the default intermediate configuration to be asymmetric, with individual protomers adopting distinct conformations. During entry, this asymmetric intermediate forms when a single CD4 molecule engages the trimer. The trimer can then transition to State 3 by binding additional CD4 molecules and coreceptor.

The β20–β21 of gp120 is a regulatory switch for HIV-1 Env conformational transitions

The entry of HIV-1 into target cells is mediated by the viral envelope glycoproteins (Env). Binding to the CD4 receptor triggers a cascade of conformational changes in distant domains that move Env from a functionally “closed” State 1 to more “open” conformations, but the molecular mechanisms underlying allosteric regulation of these transitions are still elusive. Here, we develop chemical probes that block CD4-induced conformational changes in Env and use them to identify a potential control switch for Env structural rearrangements. We identify the gp120 β20–β21 element as a major regulator of Env transitions. Several amino acid changes in the β20–β21 base lead to open Env conformations, recapitulating the structural changes induced by CD4 binding. These HIV-1 mutants require less CD4 to infect cells and are relatively resistant to State 1-preferring broadly neutralizing antibodies. These data provide insights into the molecular mechanism and vulnerability of HIV-1 entry.Binding of viral envelope glycoproteins (Env) to the host cell CD4 receptor mediates HIV-1 entry. Here, the authors develop compounds that inhibit the CD4-induced conformational changes in Env and show that the gp120 β20-β21 element is a key regulator for Env transitions.

Publisher Correction: The β20–β21 of gp120 is a regulatory switch for HIV-1 Env conformational transitions

The original version of this Article contained an error in the spelling of the author Amos B. Smith, III, which was incorrectly given as Amos B. SmithIII. This has now been corrected in both the PDF and HTML versions of the Article.

Conformational Changes in HIV-1 Env Trimer Induced by a Single CD4 as Revealed by Cryo-EM

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA 4. Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA.

Conformational Dynamics of Single HIV-1 Envelope Proteins on the Surface of Native Virions

The HIV-1 envelope (Env) mediates viral entry into host cells. While static images of Env in unliganded and ligand-bound forms define distinct conformations, direct observations of Env dynamics have yet to be realized. Here we apply single-molecule fluorescence resonance energy transfer (smFRET) imaging to elucidate the dynamics of native Env trimers on the surface of HIV-1 virions. Our observations indicated that unliganded HIV-1 Env transitions between three distinct pre-fusion conformations, which are affected by the viral receptor and co-receptors. Differences in conformational dynamics and ligand responsiveness of neutralization-sensitive and neutralization-resistant HIV-1 isolates delineated a dynamics-based mechanism of immune evasion. Broadly neutralizing antibodies stabilized one distinct pre-fusion conformation of Env, indicating the importance of the observed dynamics to HIV-1 Env function.