Optimized protocol for detection of native, full‐length <scp>HIV</scp>‐1 envelope on the surface of transfected cells

Altman, Jennie; Liu, X.; Itri, Vincenza; Zolla‐Pazner, Susan; Powell, Rebecca

doi:10.1002/hsr2.74

Cited by 4 publications

(5 citation statements)

References 32 publications

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“…Analysis for site-specific glycosylation was performed as in [ 105 ]. Briefly, 293T- and PBMC-derived infectious virus stocks were concentrated by sucrose cushion centrifugation.…”

Section: Methodsmentioning

confidence: 99%

“…The SDS-PAGE gel bands were washed with 100% acetonitrile and water three times. A proteomics-based strategy was used to assess the degree of glycan processing and the degree of site-occupancy of each glycosite [ 105 ]. The proteins in the gel bands were denatured and alkylated by 10 mM dithiothreitol (DTT) and 55 mM iodoacetamide in 25 mM ammonium bicarbonate, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting proteins were digested with the combination of trypsin and chymotrypsin at an enzyme/substrate ratio of 1:15 (w/w) and 1:10 (w/w) respectively in 25 mM ammonium bicarbonate. Sequential treatment with two endoglycosidases was then performed to introduce novel mass signatures for peptides that contain glycans of high-mannose types and complex-type glycans [ 105 ]. First, the Env peptides were digested with Endo H to cleave high-mannose (and hybrid) glycans between the innermost GlcNAc residues, leaving a GlcNAc attached to Asn (N+203).…”

Section: Methodsmentioning

confidence: 99%

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Signal peptide of HIV-1 envelope modulates glycosylation impacting exposure of V1V2 and other epitopes

et al. 2020

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HIV-1 envelope (Env) is a trimer of gp120-gp41 heterodimers, synthesized from a precursor gp160 that contains an ER-targeting signal peptide (SP) at its amino-terminus. Each trimer is swathed by ~90 N-linked glycans, comprising complex-type and oligomannose-type glycans, which play an important role in determining virus sensitivity to neutralizing antibodies. We previously examined the effects of single point SP mutations on Env properties and functions. Here, we aimed to understand the impact of the SP diversity on glycosylation of virus-derived Env and virus neutralization by swapping SPs. Analyses of site-specific glycans revealed that SP swapping altered Env glycan content and occupancy on multiple N-linked glycosites, including conserved N156 and N160 glycans in the V1V2 region at the Env trimer apex and N88 at the trimer base. Virus neutralization was also affected, especially by antibodies against V1V2, V3, and gp41. Likewise, SP swaps affected the recognition of soluble and cell-associated Env by antibodies targeting distinct V1V2 configurations, V3 crown, and gp41 epitopes. These data highlight the contribution of SP sequence diversity in shaping the Env glycan content and its impact on the configuration and accessibility of V1V2 and other Env epitopes.

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“…Analysis for site-specific glycosylation was performed as in [ 105 ]. Briefly, 293T- and PBMC-derived infectious virus stocks were concentrated by sucrose cushion centrifugation.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Signal peptide of HIV-1 envelope modulates glycosylation impacting exposure of V1V2 and other epitopes

et al. 2020

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“…An assay to detect antibody binding to cell surface expressed Env was performed as described ( 56 , 82 , 83 ). Briefly, monolayers of 293T cells (4 × 10 6 ) seeded in 100 mm tissue culture dishes were transfected with 20 µg of gp160 expression plasmid using jetPEI (DNA:jetPEI ratio of 1:3) following manufacturer’s instructions (Polyplus, New York, NY, USA).…”

Section: Methodsmentioning

confidence: 99%

Broadly neutralizing antibody epitopes on HIV-1 particles are exposed after virus interaction with host cells

Rao,

Lambert,

Upadhyay

2023

J Virol

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The envelope (Env) glycoproteins on HIV-1 virions are the sole target of broadly neutralizing antibodies (bNAbs) and the focus of vaccines. However, many cross-reactive conserved epitopes are often occluded on virus particles, contributing to the evasion of humoral immunity. This study aimed to identify the Env epitopes that are exposed/occluded on HIV-1 particles and to investigate the mechanisms contributing to their masking. Using a flow cytometry-based assay, three HIV-1 isolates, and a panel of antibodies, we show that only select epitopes, including V2i, the gp120-g41 interface, and gp41-MPER, are accessible on HIV-1 particles, while V3, V2q, and select CD4bs epitopes are masked. These epitopes become accessible after allosteric conformational changes are induced by the pre-binding of select Abs, prompting us to test if similar conformational changes are required for these Abs to exhibit their neutralization capability. We tested HIV-1 neutralization where the virus-mAb mix was pre-incubated/not pre-incubated for 1 hour prior to adding the target cells. Similar levels of neutralization were observed under both assay conditions, suggesting that the interaction between virus and target cells sensitizes the virions for neutralization via bNAbs. We further show that lectin-glycan interactions can also expose these epitopes. However, this effect is dependent on the lectin specificity. Given that, bNAbs are ideal for providing sterilizing immunity and are the goal of current HIV-1 vaccine efforts, these data offer insight on how HIV-1 may occlude these vulnerable epitopes from the host immune response. In addition, the findings can guide the formulation of effective antibody combinations for therapeutic use. IMPORTANCE The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein mediates viral entry and is the sole target of neutralizing antibodies. Our data suggest that antibody epitopes including V2q (e.g., PG9, PGT145), CD4bs (e.g., VRC01, 3BNC117), and V3 (2219, 2557) are masked on HIV-1 particles. The PG9 and 2219 epitopes became accessible for binding after conformational unmasking was induced by the pre-binding of select mAbs. Attempts to understand the masking mechanism led to the revelation that interaction between virus and host cells is needed to sensitize the virions for neutralization by broadly neutralizing antibodies (bNAbs). These data provide insight on how bNAbs may gain access to these occluded epitopes to exert their neutralization effects and block HIV-1 infection. These findings have important implications for the way we evaluate the neutralizing efficacy of antibodies and can potentially guide vaccine design.

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“…Cell-associated Env binding assay. An assay to detect antibody binding to cell surface expressed Env was performed as described (66,86). Briefly, monolayers of 293T cells (4 × 10 6 ) seeded in 100-mm tissue culture dishes were transfected with 20 μg of gp160 expression plasmid using jetPEI (DNA:jetPEI ratio of 1:3) following manufacturer's instructions (Polyplus, New York, NY).…”

Section: Virus-associated Env Binding Assay (Vaeba)mentioning

confidence: 99%

Broadly Neutralizing Antibody Epitopes on HIV-1 Particles are exposed after Virus Interaction with Host Cells

Rao

Lambert

Upadhyay

2023

Preprint

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HIV-1 envelope glycoproteins (Env) are critical for infection and are key targets for vaccine development. Env proteins displayed on virions are conformationally diverse, comprising both functional and non-functional forms. These heterogeneous Env populations have important implications for neutralizing and non-neutralizing antibody elicitation and functions. This study aimed to interrogate the antigenic composition of Env on virions. Using a flow cytometry-based assay we show that only some epitopes including V2i, gp120-g41 interface, and gp41-MPER are accessible on HIV-1 particles, while V3, V2q, and select CD4bs epitopes are obscured for monoclonal antibody (mAb) binding. To investigate the mechanisms contributing to the masking of these epitopes we first asked whether time-dependent dynamics of Env can affect their exposure. Extending the time of virus-mAb interaction increased the binding of mAbs, epitopes of which were already accessible on virions but not those that are occluded. However, the occluded epitopes became accessible after conformational unmasking was induced by pre-binding of select mAbs, prompting us to test if similar conformational changes are required for these mAbs to exhibit their neutralization capability. We tested HIV-1 neutralization where virus-mAb mix was incubated or not incubated for one-hour prior to adding the target cells. Similar levels of neutralization were observed under both assay conditions, suggesting that interaction between virus and cells sensitizes the virions for neutralization via broadly-neutralizing antibodies (bNAbs). These findings provide insight into how bNAbs may gain access to these occluded epitopes to exert their neutralization effects. Further studies with larger virus and mAb panels are warranted.

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Optimized protocol for detection of native, full‐length HIV‐1 envelope on the surface of transfected cells

Cited by 4 publications

References 32 publications

Signal peptide of HIV-1 envelope modulates glycosylation impacting exposure of V1V2 and other epitopes

Signal peptide of HIV-1 envelope modulates glycosylation impacting exposure of V1V2 and other epitopes

Broadly neutralizing antibody epitopes on HIV-1 particles are exposed after virus interaction with host cells

Broadly Neutralizing Antibody Epitopes on HIV-1 Particles are exposed after Virus Interaction with Host Cells

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