Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint

McCaul, Nicholas; Quandte, Matthias; Bontjer, Ilja; Zadelhoff, Guus van; Land, Aafke; Crooks, Ema T.; Binley, James Μ.; Sanders, Rogier W.; Braakman, Ineke

doi:10.1016/j.celrep.2021.109646

Cited by 7 publications

(6 citation statements)

References 105 publications

(153 reference statements)

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“…This difference is underlined by the distinct limiting factors of membrane trimers (i.e., expression) versus SOSIPs (i.e., "closed" trimers), which is reflected in the fact that different strains make better prototypes e.g., JR-FL and BG505, respectively. The fact that codon optimization, modified signal peptides [ 118 ], different expression plasmids and lentiviral vectors were unhelpful—all suggest that the expression bottleneck is not at the level of transcription, but relates to protein folding. Various domain swaps were also unhelpful, perhaps because, unlike rigidified SOSIP, native trimers are flexible, increasing the possibility that engraftments can perturb quaternary folding.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—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 (PV) 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 eliminate 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-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. 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 glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, 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. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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“…The Env SP is cleaved off by cellular signal peptidases. Beyond functionality in trafficking and its role as an insertion signal, specific timing of cleavage of the SP may also act as a quality control for properly folded Envs [ 87 ]. The addition of cleavable signal peptides has also been shown to enhance packaging efficiency of pseudotyped viral particles [ 88 ].…”

Section: Hallmark Features Of Gamma-type Envsmentioning

confidence: 99%

Unique Structure and Distinctive Properties of the Ancient and Ubiquitous Gamma-Type Envelope Glycoprotein

Hogan

Johnson

2023

Viruses

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After the onset of the AIDS pandemic, HIV-1 (genus Lentivirus) became the predominant model for studying retrovirus Env glycoproteins and their role in entry. However, HIV Env is an inadequate model for understanding entry of viruses in the Alpharetrovirus, Gammaretrovirus and Deltaretrovirus genera. For example, oncogenic model system viruses such as Rous sarcoma virus (RSV, Alpharetrovirus), murine leukemia virus (MLV, Gammaretrovirus) and human T-cell leukemia viruses (HTLV-I and HTLV-II, Deltaretrovirus) encode Envs that are structurally and functionally distinct from HIV Env. We refer to these as Gamma-type Envs. Gamma-type Envs are probably the most widespread retroviral Envs in nature. They are found in exogenous and endogenous retroviruses representing a broad spectrum of vertebrate hosts including amphibians, birds, reptiles, mammals and fish. In endogenous form, gamma-type Envs have been evolutionarily coopted numerous times, most notably as placental syncytins (e.g., human SYNC1 and SYNC2). Remarkably, gamma-type Envs are also found outside of the Retroviridae. Gp2 proteins of filoviruses (e.g., Ebolavirus) and snake arenaviruses in the genus Reptarenavirus are gamma-type Env homologs, products of ancient recombination events involving viruses of different Baltimore classes. Distinctive hallmarks of gamma-type Envs include a labile disulfide bond linking the surface and transmembrane subunits, a multi-stage attachment and fusion mechanism, a highly conserved (but poorly understood) “immunosuppressive domain”, and activation by the viral protease during virion maturation. Here, we synthesize work from diverse retrovirus model systems to illustrate these distinctive properties and to highlight avenues for further exploration of gamma-type Env structure and function.

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Section: Introductionmentioning

confidence: 99%

“…The general paradigm is that an N-terminal signal peptide (SP) directs their co-translational translocation into the endoplasmic reticulum, where they fold and oligomerize into trimers that remain membrane-anchored via a C-terminal transmembrane (TM) segment. The membrane-inserted SP is cleaved by a host signalase [12]. The protomers are further proteolyzed by an additional host protease to generate two subunits, an N-terminal, peripheral subunit (N-SU), which recognizes a cellular receptor or attachment factor, and a C-terminal, viral membrane anchored subunit (C-SU), which is maintained in a metastable, energy loaded state within the primed trimer [11].…”

Section: Introductionmentioning

confidence: 99%

Structures of the Foamy virus fusion protein reveal an unexpected link with the F protein of paramyxo- and pneumoviruses

Fernández,

Bontems,

Brun

et al. 2024

Preprint

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Foamy viruses (FVs) constitute a subfamily of retroviruses. Their envelope glycoprotein (Env) drives the merger of viral and cellular membranes during entry into cells. The only available structures of retroviral Envs are those from human and simian immunodeficiency viruses from the subfamily of orthoretroviruses, which are only distantly related to the FVs. We report here the cryo-EM structures of the FV Env ectodomain in the pre- and post-fusion states, which demonstrate structural similarity with the fusion protein (F) of paramyxo- and pneumoviruses, implying an evolutionary link between the two viral fusogens. Based on the structural information on the FV Env in two states, we propose a mechanistic model for its conformational change, highlighting how the interplay of its structural elements could drive the structural rearrangement. The structural knowledge on the FV Env now provides a framework for functional investigations such as the FV cell tropism and molecular features controlling the Env fusogenicity, which can benefit the design of FV Env variants with improved features for use as gene therapy vectors.

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Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint

Abstract: Highlights d Intramolecular quality control via two post-targeting roles of gp120 signal peptide d Assembly of N and C termini of HIV-1 gp120 triggers signalpeptide cleavage d Redox-active signal-peptide cysteine sustains conformational plasticity

Cited by 7 publications

References 105 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

Unique Structure and Distinctive Properties of the Ancient and Ubiquitous Gamma-Type Envelope Glycoprotein

Structures of the Foamy virus fusion protein reveal an unexpected link with the F protein of paramyxo- and pneumoviruses

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