Visualization of Retroviral Envelope Spikes in Complex with the V3 Loop Antibody 447-52D on Intact Viruses by Cryo-Electron Tomography

Dutta, Moumita; Li, Jun; Roux, Kenneth H.; Taylor, Kenneth A.

doi:10.1128/jvi.01596-14

Cited by 7 publications

(6 citation statements)

References 91 publications

(126 reference statements)

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“…We found that V3 peptide binding was again associated with tier 1A NAb titers ( P = .0035 and R = 0.72, Spearman rank correlation test) (Figure 5 C), whereas binding to CC loop and cytoplasmic tail peptides were not. These data are consistent with the reported exquisite sensitivity of SF162.LS and MW965.26 to the monoclonal antibody 447-52D (<0.02 ID 50 titer [µg/L]) [ 8 ], a well described V3-loop antibody [ 12 , 13 ], as well as the sensitivity of TH023.6 to V3-specific neutralizing antibodies isolated from subjects in the RV144 vaccine trial [ 14 ].…”

Section: Resultssupporting

confidence: 87%

Antibody Responses After Analytic Treatment Interruption in Human Immunodeficiency Virus-1-Infected Individuals on Early Initiated Antiretroviral Therapy

Stephenson

Neubauer²,

Bricault³

et al. 2016

Open Forum Infectious Diseases

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The examination of antibody responses in human immunodeficiency virus (HIV)-1-infected individuals in the setting of antiretroviral treatment (ART) interruption can provide insight into the evolution of antibody responses during viral rebound. In this study, we assessed antibody responses in 20 subjects in AIDS Clinical Trials Group A5187, wherein subjects were treated with antiretroviral therapy during acute/early HIV-1 infection, underwent analytic treatment interruption, and subsequently demonstrated viral rebound. Our data suggest that early initiation of ART arrests the maturation of HIV-1-specific antibody responses, preventing epitope diversification of antibody binding and the development of functional neutralizing capacity. Antibody responses do not appear permanently blunted, however, because viral rebound triggered the resumption of antibody maturation in our study. We also found that antibody responses measured by these assays did not predict imminent viral rebound. These data have important implications for the HIV-1 vaccine and eradication fields.

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Section: Resultssupporting

confidence: 87%

Antibody Responses After Analytic Treatment Interruption in Human Immunodeficiency Virus-1-Infected Individuals on Early Initiated Antiretroviral Therapy

Stephenson

Neubauer²,

Bricault³

et al. 2016

Open Forum Infectious Diseases

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“…In the present case and one described previously (66), spike analysis by more conventional methods produced what can be described as "watered-down" versions of the class averages reported here, which indicated that incomplete ligand binding and/or conformational mobility was operating. Just how incomplete the ligand binding was can be seen in Fig.…”

supporting

confidence: 74%

“…An important mechanism for neutralization resistance derives from the strain-specific shielding of V3 by the V1/V2 loop (55)(56)(57)(58)(59)(60)(61)(62)(63)(64). The interactions of several antibodies with the HIV-1 V3 loop have been studied at the structural level (7,13,17,20,40,65,66), but there are currently no structural studies of antibody interactions with the V3 loop of SIV gp120.…”

mentioning

confidence: 99%

Structure of Simian Immunodeficiency Virus Envelope Spikes Bound with CD4 and Monoclonal Antibody 36D5

Roux

et al. 2017

J Virol

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The human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) envelope spike (Env) mediates viral entry into host cells. The V3 loop of the gp120 component of the Env trimer contributes to the coreceptor binding site and is a target for neutralizing antibodies. We used cryo-electron tomography to visualize the binding of CD4 and the V3 loop monoclonal antibody (MAb) 36D5 to gp120 of the SIV Env trimer. Our results show that 36D5 binds gp120 at the base of the V3 loop and suggest that the antibody exerts its neutralization effect by blocking the coreceptor binding site. The antibody does this without altering the dynamics of the spike motion between closed and open states when CD4 is bound. The interaction between 36D5 and SIV gp120 is similar to the interaction between some broadly neutralizing anti-V3 loop antibodies and HIV-1 gp120. Two conformations of gp120 bound with CD4 are revealed, suggesting an intrinsic dynamic nature of the liganded Env trimer. CD4 binding substantially increases the binding of 36D5 to gp120 in the intact Env trimer, consistent with CD4-induced changes in the conformation of gp120 and the antibody binding site. Binding by MAb 36D5 does not substantially alter the proportions of the two CD4-bound conformations. The position of MAb 36D5 at the V3 base changes little between conformations, indicating that the V3 base serves as a pivot point during the transition between these two states. Glycoprotein spikes on the surfaces of SIV and HIV are the sole targets available to the immune system for antibody neutralization. Spikes evade the immune system by a combination of a thick layer of polysaccharide on the surface (the glycan shield) and movement between spike domains that masks the epitope conformation. Using SIV virions whose spikes were "decorated" with the primary cellular receptor (CD4) and an antibody (36D5) at part of the coreceptor binding site, we visualized multiple conformations trapped by the rapid freezing step, which were separated using statistical analysis. Our results show that the CD4-induced conformational dynamics of the spike enhances binding of the antibody.

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“…Already, the feasibility of antibody labeling for purified macromolecules, viruses, and isolated organelles has been demonstrated (Beck et al 2007;Roos et al 1996). These studies have enabled investigators to determine the 3D structures of low molecular weight proteins by single particle cryo-EM (Wu et al 2012), where proteins localize on specific regions of a virus (Roos et al 1996), and the structural rearrangements that occur when a neutralizing antibody binds with the target antigen Dutta et al 2014;Harris et al 2013;Lin et al 2013;Tran et al 2012). However, similar procedures have not been widely applied to studies of whole, intact mammalian cells because of the concerns associated with retaining cell viability during immunolabeling; the thicknesses of cells and the impact this has on cryo-preservation and cryo-EM imaging; and early reports of antibodyinduced membrane protein 'capping' on live cells (Ash et al 1977;Ferrante and Thong 1979).…”

Section: Introductionmentioning

confidence: 99%

Native Immunogold Labeling of Cell Surface Proteins and Viral Glycoproteins for Cryo-Electron Microscopy and Cryo-Electron Tomography Applications

Strauss

et al. 2015

J Histochem Cytochem.

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SummaryNumerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells. (J Histochem Cytochem 63:780-792, 2015) Keywords cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), immuno-electron microscopy (immuno-EM), transmission electron microscopy (TEM)

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Visualization of Retroviral Envelope Spikes in Complex with the V3 Loop Antibody 447-52D on Intact Viruses by Cryo-Electron Tomography

Cited by 7 publications

References 91 publications

Antibody Responses After Analytic Treatment Interruption in Human Immunodeficiency Virus-1-Infected Individuals on Early Initiated Antiretroviral Therapy

Antibody Responses After Analytic Treatment Interruption in Human Immunodeficiency Virus-1-Infected Individuals on Early Initiated Antiretroviral Therapy

Structure of Simian Immunodeficiency Virus Envelope Spikes Bound with CD4 and Monoclonal Antibody 36D5

Native Immunogold Labeling of Cell Surface Proteins and Viral Glycoproteins for Cryo-Electron Microscopy and Cryo-Electron Tomography Applications

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