Electron Tomography of the Contact between T Cells and SIV/HIV-1: Implications for Viral Entry

Sougrat, Rachid; Bartesaghi, Alberto; Lifson, Jeffrey D.; Bennett, Adam E.; Bess, Julian W.; Zabransky, Daniel J.; Subramaniam, Sriram

doi:10.1371/journal.ppat.0030063

Cited by 173 publications

(197 citation statements)

References 33 publications

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“…We imaged virions of SIV strain mac239, selected because of the high levels of envelope glycoprotein incorporation in the membranes of these virions (22)(23)(24). Consistent with these previous electron microscopic analyses, the typical SIV virion is 120 nm in diameter and can be studded with as many as 100 envelope spikes of ϳ13 nm in height, and ϳ12 nm wide at the distal end (Fig.…”

Section: Resultsmentioning

confidence: 54%

Cryoelectron Tomographic Analysis of an HIV-neutralizing Protein and Its Complex with Native Viral gp120

Bennett

Ryk

et al. 2007

Journal of Biological Chemistry

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Identifying structural determinants of human immunodeficiency virus (HIV) neutralization is an important component of rational drug and vaccine design. We used cryoelectron tomography and atomic force microscopy to characterize the structure of an extremely potent HIV-neutralizing protein, D1D2-Ig␣tp (abbreviated as D1D2-IgP), a polyvalent antibody construct that presents dodecameric CD4 in place of the Fab regions. We show that D1D2-IgP has a novel structure, displaying greater flexibility of its antibody arms than the closely related IgM. Using simian immunodeficiency virus in complex with D1D2-IgP, we present unequivocal evidence that D1D2-IgP can cross-link surface spikes on the same virus and on neighboring viruses. The observed binding to the viral envelope spikes is the result of specific CD4-gp120 interaction, because binding was not observed with MICA-IgP, a construct that is identical to D1D2-IgP except that major histocompatibility complex Class I-related Chain A (MICA) replaces the CD4 moiety. CD4-mediated binding was also associated with a significantly elevated proportion of ruptured viruses. The ratio of inactivated to CD4-liganded gp120-gp41 spikes can be much greater than 1:1, because all gp120-gp41 spikes on the closely apposed surfaces of cross-linked viruses should be incapable of accessing the target cell surface and mediating entry, as a result of inter-virus spike cross-linking. These results implicate flexibility rather than steric bulk or polyvalence per se as a structural explanation for the extreme potency of D1D2-IgP and thus suggest polyvalence presented on a flexible scaffold as a key design criterion for small molecule HIV entry inhibitors. Human immunodeficiency virus type 1 (HIV-1)3 infection of target cells is initiated by binding of the viral envelope glycoprotein gp120 to the cell surface receptor CD4 (1-3). The first step in the infection process, gp120-CD4 binding is an attractive drug target because the CD4 binding site of gp120 is well conserved and because de novo infection of target cells would be blocked, preventing all subsequent stages of the viral life cycle. No therapeutic agents that target gp120-CD4 binding are currently available, although certain small molecules are in clinical trials (reviewed in Ref. 4).Soluble monomeric CD4 (sCD4) neutralizes primary isolates poorly at pharmacologically realizable concentrations (5) and is therefore not useful as a therapeutic agent against HIV-1. Minimally passaged clinical primary isolates, which best model the in vivo virus, are unaffected by physiologically sustainable concentrations of sCD4 in infectivity assays (6). Recent reports suggest that the sCD4-induced conformational fixation in gp120 (7-9) makes sCD4 binding energetically unfavorable (6).In contrast to the soluble monomeric form, CD4 on the cell surface is thought to be clustered (10, 11). The clustered CD4 may mediate an avidity effect, so that soluble CD4 competes poorly with target cell CD4 for viral gp120 (6). To mimic the clustering of cell surface CD4, Ar...

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Section: Resultsmentioning

confidence: 54%

Cryoelectron Tomographic Analysis of an HIV-neutralizing Protein and Its Complex with Native Viral gp120

Bennett

Ryk

et al. 2007

Journal of Biological Chemistry

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“…This model is also consistent with the evidence that HIV-1 alters endolysosomal traffic in dendritic cells (33), although virions themselves appear not to be present in endolysosomes. Contact of virions localized within surface-accessible membrane invaginations in the dendritic cell with microvillar/filopodial extensions from the T cell that are likely enriched for CD4 and CCR5/CXCR4 (30) initiates transfer of viruses onto the T-cell surface, leading to formation of entry claw structures (34) and membrane fusion. In the absence of CD4 availability on the T cell or when gp120 on the viral surface is prevented from binding CD4, the viruses remain associated with the dendritic cell, perhaps via interactions with dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) (35,36) (other receptors are possible) until the formation of productive synapses with T cells that are capable of supporting gp120-CD4 interaction.…”

Section: Discussionmentioning

confidence: 99%

3D visualization of HIV transfer at the virological synapse between dendritic cells and T cells

Felts

Narayan

Estes

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4 + T cells and virus-bearing antigen-presenting cells such as macrophages or dendritic cells via specialized structures known as virological synapses. Using ion abrasion SEM, electron tomography, and superresolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T cells. We demonstrate the striking envelopment of T cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4 + T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T cells, but virions are not released passively at the synapse; instead, virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T cells from the extracellular milieu, the burial of the site of HIV transfer, and the receptor-dependent initiation of virion transfer by T cells highlight unique aspects of cell-cell HIV transmission.

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“…HIV virions exist as roughly spherical nanoparticles ϳ100 nm in diameter and are coated by the viral envelope membrane (1). The viral membrane is a lipid bilayer ϳ4 nm thick, interspersed with membrane-embedded glycoproteins.…”

Section: The Virus and Its Targetmentioning

confidence: 99%

“…Specific lipids in the viral and target cell membranes are not indicated. B, docking of the virus via gp120 to the cell surface receptor gives rise to Env conformational changes (72,73) and aggregation of Env proteins (1,43). C, further conformational changes lead to lipid mixing (47) and redistribution of small aqueous dyes (53,100).…”

Section: Hiv Env Structure On the Atomic Scale And Nanoscalementioning

confidence: 99%

HIV Entry and Envelope Glycoprotein-mediated Fusion

Blumenthal

Durell

Viard

2012

Journal of Biological Chemistry

186

203

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HIV entry involves binding of the trimeric viral envelope glycoprotein (Env) gp120/gp41 to cell surface receptors, which triggers conformational changes in Env that drive the membrane fusion reaction. The conformational landscape that the lipids and Env navigate en route to fusion has been examined by biophysical measurements on the microscale, whereas electron tomography, x-rays, and NMR have provided insights into the process on the nanoscale and atomic scale. However, the coupling between the lipid and protein pathways that give rise to fusion has not been resolved. Here, we discuss the known and unknown about the overall HIV Env-mediated fusion process. The Virus and Its TargetHIV virions exist as roughly spherical nanoparticles ϳ100 nm in diameter and are coated by the viral envelope membrane (1). The viral membrane is a lipid bilayer ϳ4 nm thick, interspersed with membrane-embedded glycoproteins. The HIV matrix protein Gag Pr55 drives viral assembly by recruiting all the building blocks, which include both viral and cellular components required for the formation of a fully infectious virion (2). A typical HIV viral membrane contains ϳ300,000 lipids, with a rather unique distribution of lipids compared with the lipid composition of cells from which it is derived (3,4). In addition to the virally encoded envelope glycoprotein gp120/ gp41, the HIV membrane also incorporates a plethora of cell membrane proteins in the process of assembly and budding (5). The HIV envelope proteins are expressed in the endoplasmic reticulum as a precursor protein, gp160, which transits the Golgi apparatus, where glycosylation is completed (6, 7). The precursor is cleaved in the trans-Golgi by the cellular protease furin into two proteins, gp120 and gp41 (8). These proteins are present on the cell surface as the envelope complex, a mushroomshaped trimer of heterodimers of gp120 and gp41, incorporated into the viral envelope through the transmembrane region of gp41 as virus particles bud from the cell surface (9).The number of gp120/gp41 trimers per virion ranges between 10 and 100 depending on the isolate (10). HIV gp120 is ϳ50% carbohydrate and is one of the most glycosylated proteins known (11). The number of accessory HIV membrane proteins has, in general, not been determined, with the exception of HLA class II in HIV-1 MN derived from H9 cells, which has ϳ50 native HLA class II complexes (12). The lipids and proteins are glycoconjugated, providing an additional shield for HIV against immune and environmental challenges. The viral genome is thus comfortably ensconced in a wrapper of lipid and protein covered by carbohydrate. However, mannose-type carbohydrates on HIV are recognized by DC-SIGN (dendritic cellspecific intercellular adhesion molecule-3-grabbing non-integrin), which is a C-type lectin receptor present on dendritic cells (13). The dendritic cells internalize HIV-DC-SIGN complexes and migrate to the lymph nodes, where they interact with T cells. The HIV-DC-SIGN complexes are then recycled to the cell periphery, and ...

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Electron Tomography of the Contact between T Cells and SIV/HIV-1: Implications for Viral Entry

Cited by 173 publications

References 33 publications

Cryoelectron Tomographic Analysis of an HIV-neutralizing Protein and Its Complex with Native Viral gp120

Cryoelectron Tomographic Analysis of an HIV-neutralizing Protein and Its Complex with Native Viral gp120

3D visualization of HIV transfer at the virological synapse between dendritic cells and T cells

HIV Entry and Envelope Glycoprotein-mediated Fusion

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