A V3 Loop-Dependent gp120 Element Disrupted by CD4 Binding Stabilizes the Human Immunodeficiency Virus Envelope Glycoprotein Trimer

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119

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, a membrane-fusing machine, mediates virus entry into host cells and is the sole virus-specific target for neutralizing antibodies. Binding the receptors, CD4 and CCR5/CXCR4, triggers Env conformational changes from the metastable unliganded state to the fusion-active state. We used cryo-electron microscopy to obtain a 6-Å structure of the membrane-bound, heavily glycosylated HIV-1 Env trimer in its uncleaved and unliganded state. The spatial organization of secondary structure elements reveals that the unliganded conformations of both glycoprotein (gp)120 and gp41 subunits differ from those induced by receptor binding. The gp120 trimer association domains, which contribute to interprotomer contacts in the unliganded Env trimer, undergo rearrangement upon CD4 binding. In the unliganded Env, intersubunit interactions maintain the gp41 ectodomain helical bundles in a "spring-loaded" conformation distinct from the extended helical coils of the fusion-active state. Quaternary structure regulates the virus-neutralizing potency of antibodies targeting the conserved CD4-binding site on gp120. The Env trimer architecture provides mechanistic insights into the metastability of the unliganded state, receptor-induced conformational changes, and quaternary structure-based strategies for immune evasion.H uman immunodeficiency virus type 1 (HIV-1) is an enveloped retrovirus that causes AIDS (1, 2). To enter host cells, HIV-1 uses a metastable, trimeric envelope glycoprotein (Env) spike to engage cellular receptors and to fuse the viral and target cell membranes (3-5). During synthesis and folding in the endoplasmic reticulum of the virus-producing cell, the Env precursor [glycoprotein (gp)160] is heavily modified by N-linked glycosylation and assembles into trimers (6). In most HIV-1 strains, more than 27 potential N-linked glycosylation sites are present in each gp160 protomer. After further glycan processing in the Golgi apparatus, the gp160 Env trimer precursors are proteolytically cleaved and transported to the cell surface for incorporation into virions (7). Each protomer composing the trimeric Env spike thus consists of a gp120 exterior subunit and a gp41 transmembrane subunit. The sequential binding of gp120 to the target cell receptors, CD4 and chemokine receptor (either CCR5 or CXCR4), allows the metastable Env complex to transit into fusion-active conformations (3,(8)(9)(10)(11). These conformational transitions expose the hydrophobic gp41 N-terminal fusion peptide, promoting its insertion into the target cell membrane, and further permit the formation of a highly stable gp41 six-helix bundle that mediates viral-cell membrane fusion (12)(13)(14). The Env spike is the only virus-specific component potentially accessible to neutralizing antibodies and thus has evolved a protective "glycan shield" and a high degree of interstrain variability.High-resolution structures are available for monomeric HIV-1 gp120 core fragments in the CD4-boun...

Section: Resultsmentioning

confidence: 84%

Molecular architecture of the uncleaved HIV-1 envelope glycoprotein trimer

Mao

Wang

et al. 2013

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119

“…Despite their definition as variable loops, the V2 and V3 regions of gp120 contain highly conserved domains (33). Because several lines of evidence suggest that V3 establishes direct interaction with V2 (12)(13)(14)(15)(16)(17)(18)(19)(20)(21), and the conserved base of V3 is the binding site for the N-terminal region of the CCR5 coreceptor (34), we looked for potential structural homology between V2 and CCR5. We recognized that the conserved central region of V2 contains two tyrosine residues (Tyr173 and Tyr177) with spacing identical to that of two tyrosines (Tyr10 and Tyr14) present in the N-terminal region of CCR5 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, despite these advances, many critical aspects related to the structural mechanisms of HIV-1 immune vulnerability and evasion remain unresolved. In particular, the fine molecular details of the interaction between the second and third variable loops (V2 and V3, respectively) of gp120, which are believed to play a critical role in stabilizing the prefusion envelope structure (19)(20)(21), are elucidated only partially. Functionally, V2 and V3 cooperate in the formation of quaternary epitopes targeted Significance Despite intensive efforts, the structure of the native HIV-1 envelope trimer-the sole relevant target for vaccine designhas remained elusive.…”

mentioning

confidence: 99%

Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

Cimbro¹,

Gallant²,

Dolan

et al. 2014

Elicitation of broadly neutralizing antibodies is essential for the development of a protective vaccine against HIV-1. However, the native HIV-1 envelope adopts a protected conformation that conceals highly conserved sites of vulnerability from antibody recognition. Although high-definition structures of the monomeric core of the envelope glycoprotein subunit gp120 and, more recently, of a stabilized soluble gp140 trimer have been solved, fundamental aspects related to the conformation and function of the native envelope remain unresolved. Here, we show that the conserved central region of the second variable loop (V2) of gp120 contains sulfated tyrosines (Tys173 and Tys177) that in the CD4-unbound prefusion state mediate intramolecular interaction between V2 and the conserved base of the third variable loop (V3), functionally mimicking sulfated tyrosines in CCR5 and anti-coreceptor-binding-site antibodies such as 412d. Recombinant gp120 expressed in continuous cell lines displays low constitutive levels of V2 tyrosine sulfation, which can be enhanced markedly by overexpression of the tyrosyl sulfotransferase TPST2. In contrast, virion-associated gp120 produced by primary CD4 + T cells is inherently highly sulfated. Consistent with a functional role of the V2 sulfotyrosines, enhancement of tyrosine sulfation decreased binding and neutralization of HIV-1 BaL by monomeric soluble CD4, 412d, and anti-V3 antibodies and increased recognition by the trimer-preferring antibodies PG9, PG16, CH01, and PGT145. Conversely, inhibition of tyrosine sulfation increased sensitivity to soluble CD4, 412d, and anti-V3 antibodies and diminished recognition by trimer-preferring antibodies. These results identify the sulfotyrosine-mediated V2-V3 interaction as a critical constraint that stabilizes the native HIV-1 envelope trimer and modulates its sensitivity to neutralization.T he development of a protective vaccine remains a high priority for the global control of the HIV/AIDS epidemic (1). However, the unique biological features of HIV-1 make this task extremely challenging. The main obstacles include the ability of the virus to integrate into the host chromosomes, a remarkable degree of genetic variability, and the cryptic, antibody-shielded conformation adopted by the viral envelope in the native spikes that protrude from the virion surface (2). These spikes are composed of homotrimers of heterodimers of the envelope glycoprotein subunits gp120 and gp41 maintained in an energetically unfavorable, metastable conformation (3, 4). Upon binding to CD4 and a coreceptor such as CCR5 or CXCR4, gp120 undergoes dramatic conformational changes that lead to a low-energy state, creating permissive conditions for activation of the gp41 fusogenic mechanism (3). In the prefusion conformation, gp120 effectively conceals its highly conserved receptor-and coreceptor-binding sites from antibody recognition, imposing a high-entropy penalty for interaction with CD4 or antibodies to the coreceptor-binding site such as 17b; in contrast, in the open, l...

“…4F, derives from the fact that N295 and N332 occur directly adjacent to the disulfide bond on opposite borders of the V3 loop. This is a critical fulcrum for gp120 function because CD4 weakens V3 constraints on gp41 (19) and induces V3 movement toward the apex of the gp120 trimer (16,17,19) where HIV-1 JRCSF -Ad that had been pretreated for 1 h at 37°C ± 4 μg/mL 2G12. Titers were normalized relative to maximum titers at 100 μM Nt (n = 3, error bars are ± SEM).…”

Section: G12 Inhibits Cd4 and Ccr5 Binding And Blocks A Coreceptordementioning

confidence: 99%

“…After CD4 binding, V3 loop regions of gp120 reduce their constraining hold on gp41 and move toward the trimer apex where they interact with coreceptors, thereby playing a pivotal role in controlling HIV-1 entry rates (16)(17)(18)(19). These and additional conformational changes in gp120 enable gp41 to refold by a multistep process that fuses the viral and cellular membranes.…”

mentioning

confidence: 99%

Kinetic mechanism for HIV-1 neutralization by antibody 2G12 entails reversible glycan binding that slows cell entry

Platt

Gomes

Kabat

2012

Despite structural knowledge of broadly neutralizing monoclonal antibodies (NMAbs) complexed to HIV-1 gp120 and gp41 envelope glycoproteins, virus inactivation mechanisms have been difficult to prove, in part because neutralization assays are complex and were previously not understood. Concordant with recent evidence that HIV-1 titers are determined by a race between entry of cell-attached virions and competing inactivation processes, we show that NMAb 2G12, which binds to gp120 N-glycans with α (1, 2)-linked mannose termini and inhibits replication after passive transfer into patients, neutralizes by slowing entry of adsorbed virions. Accordingly, apparent neutralization is attenuated when a kinetically competing virus inactivation pathway is blocked. Moreover, removing 2G12 from media causes its dissociation from virions coupled to accelerated entry and restored infectivity, demonstrating the reversibility of neutralization. A difference between 2G12 dissociation and infectivity recovery rates implies that the inhibited complexes at virus-cell junctions contain several 2G12's that must dissociate before entry commences. Quantitative microscopy of 2G12 binding and dissociation from single virions and studies using a split CCR5 coreceptor suggest that 2G12 competitively inhibits interactions between gp120's V3 loop and the tyrosine sulfate-containing CCR5 amino terminus, thereby reducing assembly of complexes that catalyze entry. These results reveal a unique reversible kinetic mechanism for neutralization by an antibody that binds near a critical V3 region in the glycan shield of gp120.