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

ABSTRACTPrimary 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.IMPORTANCEThe 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.

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Shedding-Resistant HIV-1 Envelope Glycoproteins Adopt Downstream Conformations That Remain Responsive to Conformation-Preferring Ligands

Journal of Virology ◽

10.1128/jvi.00597-20 ◽

2020 ◽

Vol 94 (17) ◽

Author(s):

Maolin Lu ◽

Xiaochu Ma ◽

Nick Reichard ◽

Daniel S. Terry ◽

James Arthos ◽

...

Keyword(s):

Structural Characterization ◽

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

Conformational State ◽

Broadly Neutralizing Antibodies ◽

Membrane Bound ◽

State 1 ◽

Hiv 1 ◽

Conformation State

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer of gp120-gp41 heterodimers mediates virus entry into CD4-positive (CD4+) cells. Single-molecule fluorescence resonance energy transfer (smFRET) has revealed that native Env on the surface of viruses predominantly exists in a pretriggered conformation (state 1) that is preferentially recognized by many broadly neutralizing antibodies (bNAbs). Env is activated by binding receptor CD4, which drives transitions through a default intermediate conformation (state 2) into the three-CD4-bound open conformation (state 3). The application of smFRET to assess the conformational state of existing Env constructs and ligand complexes recently revealed that all current high-resolution structures correspond to downstream states 2 and 3. The structure of state 1, therefore, remains unknown. We sought to identify conditions whereby HIV-1 Env could be stabilized in the pretriggered state 1 for possible structural characterization. Shedding of gp120, known to severely complicate structural studies, can be prevented by using the uncleaved gp160JR-FL precursor with alterations in the protease cleavage site (R508S/R511S) or by introducing a disulfide bridge between gp120 and gp41 designated “SOS” (A501C/T605C). smFRET demonstrated that both shedding-preventing modifications shifted the conformational landscape of Env downstream toward states 2 and 3. However, both membrane-bound Env proteins on the surface of intact viruses remained conformationally dynamic, responsive to state-stabilizing ligands, and able to be stabilized in state 1 by specific ligands such as the Bristol-Myers Squibb (BMS) entry inhibitors. The here-described identification of state 1-stabilizing conditions may enable structural characterization of the state 1 conformation of HIV-1 Env. IMPORTANCE The HIV-1 envelope glycoprotein (Env) opens in response to receptor CD4 binding from a pretriggered (state 1) conformation through a necessary intermediate to the three-CD4-bound conformation. The application of smFRET to test the conformational state of existing Env constructs and ligand complexes used for high-resolution structures recently revealed that they correspond to the downstream conformations. The structure of the pretriggered Env conformation, preferentially recognized by broadly neutralizing antibodies, remains unknown. Here, we identify experimental conditions that stabilize membrane-bound and shedding-resistant virus Env trimers in state 1, potentially facilitating structural characterization of this unknown conformational state.

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Stabilizing the HIV-1 envelope glycoprotein State 2A conformation

Journal of Virology ◽

10.1128/jvi.01620-20 ◽

2020 ◽

Author(s):

Dani Vézina ◽

Shang Yu Gong ◽

William D. Tolbert ◽

Shilei Ding ◽

Dung Nguyen ◽

...

Keyword(s):

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

Coreceptor Binding ◽

Viral Particles ◽

A Cell ◽

Infected Cells ◽

Cluster A ◽

State 1 ◽

Hiv 1 ◽

Conformation State

The HIV-1 envelope glycoprotein (Env) trimer [(gp120/gp41)3] is a metastable complex expressed at the surface of viral particles and infected cells that samples different conformations. Before engaging CD4, Env adopts an antibody-resistant “closed” conformation (State 1). CD4 binding triggers an intermediate conformation (State 2) and then a more “open” conformation (State 3) that can be recognized by non-neutralizing antibodies (nnAbs) such as those that recognize the coreceptor binding site (CoRBS). Binding of antibodies to the CoRBS permits another family of nnAbs, the anti-cluster A family of Abs which target the gp120 inner domain, to bind and stabilize an asymmetric conformation (State 2A). Cells expressing Env in this conformation are susceptible to antibody-dependent cellular cytotoxicity (ADCC). This conformation can be stabilized by small-molecule CD4 mimetics (CD4mc) or soluble CD4 (sCD4) in combination with anti-CoRBS Ab and anti-cluster A antibodies. The precise stoichiometry of each component that permits this sequential opening of Env remains unknown. Here, we used a cell-based ELISA (CBE) assay to evaluate each component individually. In this assay we used a “trimer mixing” approach by combining wild-type (wt) subunits with subunits impaired for CD4 or CoRBS Ab binding. This enabled us to show that State 2A requires all three gp120 subunits to be bound by sCD4/CD4mc and anti-CoRBS Abs. Two of these subunits can then bind anti-cluster A Abs. Altogether, our data suggests how this antibody vulnerable Env conformation is stabilized. Importance Stabilization of HIV-1 Env State 2A has been shown to sensitize infected cells to ADCC. State 2A can be stabilized by a “cocktail” composed of CD4mc, anti-CoRBS and anti-cluster A Abs. We present evidence that optimal State 2A stabilization requires all three gp120 subunits to be bound by both CD4mc and anti-CoRBS Abs. Our study provides valuable information on how to stabilize this ADCC-vulnerable conformation. Strategies aimed at stabilizing State 2A might have therapeutic utility.

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Activation and Inactivation of Primary Human Immunodeficiency Virus Envelope Glycoprotein Trimers by CD4-Mimetic Compounds

Journal of Virology ◽

10.1128/jvi.01880-16 ◽

2016 ◽

Vol 91 (3) ◽

Cited By ~ 20

Author(s):

Navid Madani ◽

Amy M. Princiotto ◽

Connie Zhao ◽

Fatemeh Jahanbakhshsefidi ◽

Max Mertens ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Small Molecule ◽

Conformational Changes ◽

Envelope Glycoprotein ◽

Virus Entry ◽

Envelope Glycoproteins ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the viral envelope glycoproteins (Env), a trimer of three gp120 exterior glycoproteins, and three gp41 transmembrane glycoproteins. The metastable Env is triggered to undergo entry-related conformational changes when gp120 binds sequentially to the receptors, CD4 and CCR5, on the target cell. Small-molecule CD4-mimetic compounds (CD4mc) bind gp120 and act as competitive inhibitors of gp120-CD4 engagement. Some CD4mc have been shown to trigger Env prematurely, initially activating Env function, followed by rapid and irreversible inactivation. Here, we study CD4mc with a wide range of anti-HIV-1 potencies and demonstrate that all tested CD4mc are capable of activating as well as inactivating Env function. Biphasic dose-response curves indicated that the occupancy of the protomers in the Env trimer governs viral activation versus inactivation. One CD4mc bound per Env trimer activated HIV-1 infection. Envs with two CD4mc bound were activated for infection of CD4-negative, CCR5-positive cells, but the infection of CD4-positive, CCR5-positive cells was inhibited. Virus was inactivated when all three Env protomers were occupied by the CD4mc, and gp120 shedding from the Env trimer was increased in the presence of some CD4mc. Env reactivity and the on rates of CD4mc binding to the Env trimer were found to be important determinants of the potency of activation and entry inhibition. Cross-sensitization of Env protomers that do not bind the CD4mc to neutralization by an anti-V3 antibody was not evident. These insights into the mechanism of antiviral activity of CD4mc should assist efforts to optimize their potency and utility. IMPORTANCE The trimeric envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) mediate virus entry into host cells. Binding to the host cell receptors, CD4 and CCR5, triggers changes in the conformation of the HIV-1 envelope glycoprotein trimer important for virus entry. Small-molecule CD4-mimetic compounds inhibit HIV-1 infection by multiple mechanisms: (i) direct blockade of the interaction between the gp120 exterior envelope glycoprotein and CD4; (ii) premature triggering of conformational changes in the envelope glycoproteins, leading to irreversible inactivation; and (iii) exposure of cryptic epitopes to antibodies, allowing virus neutralization. The consequences of the binding of the CD4-mimetic compound to the HIV-1 envelope glycoproteins depends upon how many of the three subunits of the trimer are bound and upon the propensity of the envelope glycoproteins to undergo conformational changes. Understanding the mechanistic factors that influence the activity of CD4-mimetic compounds can help to improve their potency and coverage of diverse HIV-1 strains.

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Exposing HIV-1 Env: Implications for therapeutic strategies

Clinical & Investigative Medicine ◽

10.25011/cim.v42i4.33109 ◽

2019 ◽

Vol 42 (4) ◽

pp. E2-E6 ◽

Cited By ~ 1

Author(s):

Andrés Finzi

Keyword(s):

Neutralizing Antibodies ◽

Conformational States ◽

Open Conformation ◽

Coreceptor Binding ◽

Immunodeficiency Virus ◽

Infected Cells ◽

State 1 ◽

Hiv 1 ◽

Coreceptor Binding Site ◽

Conformation State

The human immunodeficiency virus (HIV-1) envelope glycoprotein trimer (Env) is exposed on the surfaces of both virions and infected cells. Thus, Env is the principal target for neutralizing antibodies and antibodies able to mediate antibodydependent cellular cytotoxicity (ADCC). The HIV-1 Env is a flexible molecule known to exist in at least three different conformational states: states 1, 2 and 3. Before interacting with the primary receptor, CD4, Env preferentially adopts a compact, “closed” conformation (state 1) that is largely antibody-resistant. The CD4 binding “opens” Env increasing the vulnerability of infected cells to ADCC mediated by non-neutralizing antibodies, as these easily-elicited antibodies preferentially recognize epitopes exposed in the open conformational states (states 2/3). These antibodies include the anti-coreceptor binding site and the anti-cluster A families of antibodies that, in combination with small CD4-mimetic compounds, stabilize a new asymmetric Env conformation (state 2A) that is vulnerable to ADCC. Approaches aimed at stabilizing this “open” conformation represent new interventional approaches to fight HIV-1 infection.

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HIV-1 Envelope Glycoproteins Proteolytic Cleavage Protects Infected Cells from ADCC Mediated by Plasma from Infected Individuals

Viruses ◽

10.3390/v13112236 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2236

Author(s):

Jérémie Prévost ◽

Halima Medjahed ◽

Dani Vézina ◽

Hung-Ching Chen ◽

Beatrice H. Hahn ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Proteolytic Cleavage ◽

Antibody Dependent Cellular Cytotoxicity ◽

Furin Cleavage ◽

Closed Conformation ◽

Furin Cleavage Site ◽

Infected Cells ◽

State 1 ◽

Hiv 1 ◽

Conformation State

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible but controls its transition from the unbound “closed” conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature “opening” of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the “closed” conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.

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HIV-1 envelope glycoproteins proteolytic cleavage protects infected cells from ADCC mediated by plasma from infected individuals

10.1101/2021.10.26.465908 ◽

2021 ◽

Author(s):

Jeremie Prevost ◽

Halima Medjahed ◽

Dani Vezina ◽

Hung-Ching Chen ◽

Beatrice H Hahn ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Proteolytic Cleavage ◽

Antibody Dependent Cellular Cytotoxicity ◽

Furin Cleavage ◽

Closed Conformation ◽

Furin Cleavage Site ◽

Infected Cells ◽

State 1 ◽

Hiv 1 ◽

Conformation State

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible, but controls its transition from the unbound closed conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature opening of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the closed conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.

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Small-molecule inhibitors of HIV-1 entry block receptor-induced conformational changes in the viral envelope glycoproteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0307953101 ◽

2004 ◽

Vol 101 (14) ◽

pp. 5036-5041 ◽

Cited By ~ 183

Author(s):

Z. Si ◽

N. Madani ◽

J. M. Cox ◽

J. J. Chruma ◽

J. C. Klein ◽

...

Keyword(s):

Small Molecule ◽

Conformational Changes ◽

Small Molecule Inhibitors ◽

Viral Envelope ◽

Envelope Glycoproteins ◽

Hiv 1 ◽

Entry Block

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Small Molecule Inhibitors of Influenza Virus Entry

Pharmaceuticals ◽

10.3390/ph14060587 ◽

2021 ◽

Vol 14 (6) ◽

pp. 587

Author(s):

Zhaoyu Chen ◽

Qinghua Cui ◽

Michael Caffrey ◽

Lijun Rong ◽

Ruikun Du

Keyword(s):

Influenza Virus ◽

Membrane Fusion ◽

Small Molecule ◽

Drug Target ◽

Small Molecule Inhibitors ◽

Virus Entry ◽

Critical Role ◽

Structural Basis ◽

Future Directions ◽

The Past

Hemagglutinin (HA) plays a critical role during influenza virus receptor binding and subsequent membrane fusion process, thus HA has become a promising drug target. For the past several decades, we and other researchers have discovered a series of HA inhibitors mainly targeting its fusion machinery. In this review, we summarize the advances in HA-targeted development of small molecule inhibitors. Moreover, we discuss the structural basis and mode of action of these inhibitors, and speculate upon future directions toward more potent inhibitors of membrane fusion and potential anti-influenza drugs.

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