Correction for Lu et al., “Shedding-Resistant HIV-1 Envelope Glycoproteins Adopt Downstream Conformations That Remain Responsive to Conformation-Preferring Ligands”

Lu, Maolin; Ma, Xiaochu; Reichard, Nick; Terry, Daniel S.; Arthos, James; Smith, Amos B.; Sodroski, Joseph; Blanchard, Scott C.; Mothes, Walther

doi:10.1128/jvi.01661-20

Cited by 5 publications

(1 citation statement)

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“…Notably, PGT145 sensitivity of gp160CT SOS trimers was somewhat reduced in all cases, as neutralization depends on a tightly folded apex that is slightly perturbed by both the SOS and CT mutations. Overall, our findings are consistent with a slightly activated SOS trimer state (116) that is V2-sensitive and not overtly V3-sensitive.…”

Section: Gp160ct/sossupporting

confidence: 83%

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

Crooks

Almanza

D’Addabbo

et al. 2021

Preprint

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HIV-1 vaccine immunofocusing strategies have the potential to induce broadly reactive nAbs. Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets of neutralizing antibodies (NAbs), the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-neutralizing antibodies, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the V2 loop's C-strand. Notably, a 167N mutation improved V2-sensitivity. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing other glycans in some cases had local and global "ripple" effects on glycan maturation and sequon occupation in the gp120 outer domain and gp41. V2 mAb CH01 selectively bound trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a N49 glycan perturbs gp41 glycans via a distal glycan network effect, increasing FP NAb sensitivity - and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30-40 spikes. Overall, we identified 7 diverse trimers with a range of sensitivities to two targets that should enable rigorous testing of immunofocusing vaccine concepts.

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Section: Gp160ct/sossupporting

confidence: 83%

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

Crooks

Almanza

D’Addabbo

et al. 2021

Preprint

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An intact amber-free HIV-1 system for in-virus protein bioorthogonal click labeling that delineates envelope conformational dynamics

Grover

Han

et al. 2023

Preprint

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The HIV-1 envelope (Env) glycoprotein is conformationally dynamic and mediates membrane fusion required for cell entry. Single-molecule fluorescence resonance energy transfer (smFRET) of Env using peptide tags has provided mechanistic insights into the dynamics of Env conformations. Nevertheless, using peptide tags risks potential effects on structural integrity. Here, we aim to establish minimally invasive smFRET systems of Env on the virus by combining genetic code expansion and bioorthogonal click chemistry. Amber stop-codon suppression allows site-specifically incorporating noncanonical/unnatural amino acids (ncAAs) at introduced amber sites into proteins. However, ncAA incorporation into Env (or other HIV-1 proteins) in the virus context has been challenging due to low copies of Env on virions and incomplete amber suppression in mammalian cells. Here, we developed an intact amber-free virus system that overcomes impediments from preexisting ambers in HIV-1. Using this system, we successfully incorporated dual ncAAs at amber-introduced sites into Env on intact virions. Dual-ncAA incorporated Env retained similar neutralization sensitivities to neutralizing antibodies as wildtype. smFRET of click-labeled Env on intact amber-free virions recapitulated conformational profiles of Env. The amber-free HIV-1 infectious system also permits in-virus protein bioorthogonal labeling, compatible with various advanced microscopic studies of virus entry, trafficking, and egress in living cells.Amber-free HIV-1 infectious systems actualized minimal invasive Env tagging for smFRET, versatile for in-virus bioorthogonal click labeling in advanced microscopic studies of virus-host interactions.

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Asymmetric Structures and Conformational Plasticity of the Uncleaved Full-Length Human Immunodeficiency Virus Envelope Glycoprotein Trimer

Zhang

Wang

et al. 2021

J Virol

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The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41) 3 ] is produced by cleavage of a conformationally flexible gp160 precursor. Gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pre-triggered, “closed” (State-1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more “open” Env conformations (States 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and crosslinking decreased the exposure of pNAb epitopes on cell-surface gp160; however, after detergent solubilization, crosslinked and BMS-806-treated gp160 sampled non-State-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1 N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume State-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a State-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs. IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage to a persistent virus like HIV-1.

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Correction for Lu et al., “Shedding-Resistant HIV-1 Envelope Glycoproteins Adopt Downstream Conformations That Remain Responsive to Conformation-Preferring Ligands”

Cited by 5 publications

References 0 publications

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

An intact amber-free HIV-1 system for in-virus protein bioorthogonal click labeling that delineates envelope conformational dynamics

Asymmetric Structures and Conformational Plasticity of the Uncleaved Full-Length Human Immunodeficiency Virus Envelope Glycoprotein Trimer

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