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

Platt, Emily J.; Gomes, Michelle M.; Kabat, David

doi:10.1073/pnas.1109728109

Cited by 37 publications

(39 citation statements)

References 41 publications

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“…To test this, we adsorbed virions onto HeLa-CD4/CCR5(⌬18) cells at 4°C and then either treated the cultures immediately with pronase or incubated them at 37°C for 1 to 4 h in the presence or absence of dynasore before pronase treatment. We then visualized the virions by immunofluorescence in a deconvolution microscope using a monoclonal antibody to p24 Gag (12,34,38).…”

Section: Resultsmentioning

confidence: 99%

“…We conclude that this reversible peptide-dependent system is a powerful means to investigate pathways of HIV-1 entry and mechanisms of entry inhibitor function. We recently used a variant of this system to show that the neutralizing monoclonal antibody 2G12 binds and neutralizes HIV-1 reversibly by slowing the CCR5-dependent entry steps (38).…”

Section: Discussionmentioning

confidence: 99%

“…HIV-1 JRCSF (Ad) uptake and the effect of the endocytosis inhibitor dynasore were investigated using HIV-1 p24 Gag immunostaining and deconvolution microscopy (12,38). At 24 h before the assay, glass coverslips (diameter, 13 mm; VWR International, Radnor, PA) were placed in the wells of 24-well plates and HeLa-CD4/CCR5(⌬18) cells were seeded at 3.0 ϫ 10 4 cells per well.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…Control cultures were placed on ice after spinoculation, rinsed, immediately subjected to pronase digestion without incubation at 37°C, and then fixed. Fixed cells were permeabilized with 0.5% NP-40 -10% sucrose in PBS and stained for HIV-1 Gag proteins using the 183-H12-5C p24 monoclonal antibody and goat antimouse Alexa 594 (Invitrogen Life Technologies Corp., Grand Island, NY), as described previously (38). The fixed cells on coverslips were mounted on glass slides and viewed with a Zeiss Axiovert 200M deconvolution microscope, and z-stacks were acquired using the ϫ40 objective and AxioVision software (release 4.6.3; Carl Zeiss).…”

Section: Cells and Virusesmentioning

confidence: 99%

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Reversible and Efficient Activation of HIV-1 Cell Entry by a Tyrosine-Sulfated Peptide Dissects Endocytic Entry and Inhibitor Mechanisms

Platt

Gomes

Kabat

2014

J Virol

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HIV-1 membranes contain gp120-gp41 trimers. Binding of gp120 to CD4 and a coreceptor (CCR5 or CXCR4) reduces the constraint on metastable gp41, enabling a series of conformational changes that cause membrane fusion. An analytic difficulty occurs because these steps occur slowly and asynchronously within cohorts of adsorbed virions. We previously isolated HIV-1 JRCSF variants that efficiently use CCR5 mutants severely damaged in the tyrosine-sulfated amino terminus or extracellular loop 2. Surprisingly, both independent adaptations included gp120 mutations S298N, F313L, and N403S, supporting other evidence that they function by weakening gp120's grip on gp41 rather than by altering gp120 binding to specific CCR5 sites. Although several natural HIV-1 isolates reportedly use CCR5(⌬18) (CCR5 with a deletion of 18 N-terminal amino acids, including the tyrosine-sulfated region) when the soluble tyrosine-sulfated peptide is present, we show that HIV-1 JRCSF with the adaptive mutations [HIV-1 JRCSF (Ad)] functions approximately 100 times more efficiently and that coreceptor activation is reversible, enabling synchronous efficient entry control under physiological conditions. This system revealed that three-stranded gp41 folding intermediates susceptible to the inhibitor enfuvirtide form slowly and asynchronously on cell surface virions but resolve rapidly, with virions generally forming only one target. Adsorbed virions asynchronously and transiently become competent for entry at 37°C but are inactivated if the CCR5 peptide is absent during their window of opportunity. This competency is conferred by endocytosis, which results in inactivation if the peptide is absent. For both wild-type and adapted HIV-1 isolates, early gp41 refolding steps obligatorily occur on cell surfaces, whereas the final step(s) is endosomal. This system powerfully dissects HIV-1 entry and inhibitor mechanisms. IMPORTANCEWe present a powerful means to reversibly and efficiently activate or terminate HIV-1 entry by adding or removing a tyrosinesulfated CCR5 peptide from the culture medium. This system uses stable cell clones and a variant of HIV-1 JRCSF with three adaptive mutations. It enabled us to show that CCR5 coreceptor activation is rapidly reversible and to dissect aspects of entry that had previously been relatively intractable. Our analyses elucidate enfuvirtide (T-20) function and suggest that HIV-1 virions form only one nonredundant membrane fusion complex on cell surfaces. Additionally, we obtained novel and conclusive evidence that HIV-1 entry occurs in an assembly line manner, with some steps obligatorily occurring on cell surfaces and with final membrane fusion occurring in endosomes. Our results were confirmed for wild-type HIV-1. Thus, our paper provides major methodological and mechanistic insights about HIV-1 infection. H uman immunodeficiency virus type 1 (HIV-1) envelope glycoproteins are trimeric heterodimers containing a surface subunit gp120 that binds receptors and a transmembrane subunit gp41 that mediates membra...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Cells and Virusesmentioning

confidence: 99%

Section: Cells and Virusesmentioning

confidence: 99%

See 2 more Smart Citations

Reversible and Efficient Activation of HIV-1 Cell Entry by a Tyrosine-Sulfated Peptide Dissects Endocytic Entry and Inhibitor Mechanisms

Platt

Gomes

Kabat

2014

J Virol

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“…Thus, isolation of antibodies that bind the high mannose moieties of the gp120 was one of the biggest breakthroughs in the glycobiology field [50,51]. The 2G12 clone is the best described broadly neutralizing antibody, which slows the entry of the virus into cells and inhibits replication [52]. The PG9 clone binds to the glycans from both gp120 and gp140 envelope proteins.…”

Section: Human Immunodeficiency Virusmentioning

confidence: 99%

Exploitation of Glycobiology in Anti-Adhesion Approaches against Biothreat Agents

Utratna¹,

Deegan²,

Joshi³

2016

J Bioterror Biodef

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Pathogen adherence to a host cell is one of the first essential steps for establishing invasion, colonization and release of virulence factors such as toxins. Understanding the mechanisms used by pathogens and toxins to adhere and invade human cells could lead to the development of new strategies for preventing and controlling the spread of infectious diseases. This review focuses on carbohydrate-lectin interactions utilized by selected biothreat agents to bind and invade host cells. The principle of using anti-adhesion molecules, based on glycobiology research, has already been shown to be effective in the treatment of influenza. Therefore, translating the same principle to other biothreat agents that mediate invasion of a host cell through carbohydrate-lectin mechanisms is a very promising strategy. We investigate recent literature to highlight the latest developments in the field of glycobiology focused on inhibiting the initial steps of pathogen invasion, with examples for bacteria, toxin and virus interactions. The successful glycomimetics and glycoconjugates represent strategies for interruption of adhesion by single molecules and in multivalent systems against uropathogenic E. coli, several toxins (Shiga-like, cholera, botulinum) and wellknown or emerging viruses (influenza, HIV, Ebola, and Zika). This review provides promising directions and prophylactic as well as therapeutic potential of anti-adhesive strategies against selected biothreat targets.

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Surface DEAE groups facilitate protein transport on polymer chains in DEAE‐modified‐and‐DEAE‐dextran‐grafted resins

Gong

Bai

et al. 2016

AIChE Journal

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in Wiley Online Library (wileyonlinelibrary.com)Sepharose FF was modified with diethylaminoethyl-dextran (DEAE-dextran, DexD) and/or DEAE (D) to fabricate three types of ion exchangers FF-DexD (grafting-ligand resin), FF-D (surface-ligand resin), and FF-D-DexD (mixed-ligand resin), for protein adsorption equilibria and kinetics study. It was found that both adsorption capacity and uptake rate (effective diffusivity, D e ) were significantly enhanced by grafting DEAE-dextran. Notably, the D e values on FF-DexD and FF-D-DexD (D e /D 0 > 1.4) were six times greater than those on FF-D (D e /D 0 < 0.3). More importantly, the increase of surface-ligand density greatly enhanced uptake kinetics on FF-D-DexD. The results indicate that the surface ligands assisted the transport of bound proteins on polymer chains in the mixed-ligand resins. That is, surface ligands worked as "transfer stations" between two neighboring chains, resulting in enhanced transport of bound proteins on chains. The research thus disclosed the unique role of surface ligands in facilitating protein uptake kinetics onto polymergrafted ion-exchangers.The static adsorption equilibrium experiments of BSA on the resins were carried out in 20 mmol/L Tris-HCl (pH 8.0) as described earlier. 24 The adsorption density of protein (q) was calculated by mass balance with the free protein concentration in equilibrium (c), and the Langmuir model was used to describe the adsorption isotherms,The black lines represent agarose matrix, the blue curves represent dextran chains, the green lines represent the spacers, the red circles represent ion-exchange groups, and the pink and purple circles represent bound proteins. The dashed blue curves and faded circles represent the grafted polymer chains with adsorbed proteins after swing, and the dashed black boxes highlight the sites for the "relay"/delivery of proteins. The quantities of the dextran chains, spacers and the ion-exchange groups in this figure do not represent their real densities in the resins. In (a) it shows that the upper protein (pink) on the chain is delivered while the lower one (purple) is not. In (b) it shows that chain delivery occurs for the lower protein (purple), which was mediated by the surface DEAE ligands. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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

Cited by 37 publications

References 41 publications

Reversible and Efficient Activation of HIV-1 Cell Entry by a Tyrosine-Sulfated Peptide Dissects Endocytic Entry and Inhibitor Mechanisms

Reversible and Efficient Activation of HIV-1 Cell Entry by a Tyrosine-Sulfated Peptide Dissects Endocytic Entry and Inhibitor Mechanisms

Exploitation of Glycobiology in Anti-Adhesion Approaches against Biothreat Agents

Surface DEAE groups facilitate protein transport on polymer chains in DEAE‐modified‐and‐DEAE‐dextran‐grafted resins

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