Molecular Characterization of the Coproduced Extracellular Vesicles in HEK293 during Virus-Like Particle Production

Lavado‐García, Jesús; Cervera, Laura; Jorge, Inmaculada; Vázquez, Jesús; Gòdia, Francesc

doi:10.1021/acs.jproteome.0c00581

Cited by 21 publications

(16 citation statements)

References 76 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For example, RDIMIS identified 80 vesicle-specific binders for a population of human cell line–derived EVs and showed that the interactome of recombinant and endogenously generated EVs were remarkably similar. This is consistent with previous works showing strong similarities between the proteomic and lipid compositions of recombinant and endogenous EVs ( 26 , 46 ). Therefore, these binders may shed light on the receptor specificity of endogenous EVs and their potential signaling activity.…”

Section: Discussionsupporting

confidence: 93%

A membrane protein display platform for receptor interactome discovery

Cao¹,

Peterson²,

Müller³

et al. 2021

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

View full text Add to dashboard Cite

Cell surface receptors are critical for cell signaling and constitute a quarter of all human genes. Despite their importance and abundance, receptor interaction networks remain understudied because of difficulties associated with maintaining membrane proteins in their native conformation and their typically weak interactions. To overcome these challenges, we developed an extracellular vesicle-based method for membrane protein display that enables purification-free and high-throughput detection of receptor–ligand interactions in membranes. We demonstrate that this platform is broadly applicable to a variety of membrane proteins, enabling enhanced detection of extracellular interactions over a wide range of binding affinities. We were able to recapitulate and expand the interactome for prominent members of the B7 family of immunoregulatory proteins such as PD-L1/CD274 and B7-H3/CD276. Moreover, when applied to the orphan cancer-associated fibroblast protein, LRRC15, we identified a membrane-dependent interaction with the tumor stroma marker TEM1/CD248. Furthermore, this platform enabled profiling of cellular receptors for target-expressing as well as endogenous extracellular vesicles. Overall, this study presents a sensitive and easy to use screening platform that bypasses membrane protein purification and enables characterization of interactomes for any cell surface–expressed target of interest in its native state.

show abstract

Section: Discussionsupporting

confidence: 93%

A membrane protein display platform for receptor interactome discovery

Cao¹,

Peterson²,

Müller³

et al. 2021

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

View full text Add to dashboard Cite

show abstract

“…Further experiments would be needed to determine whether the effect is strictly due to Env expression or associated with the strain of Env (and Gag or Pol) used in these studies. Characterization of extracellular vesicles coexpressed in HEK cells once transfected with HIV-1 VLP expression vectors has shown the act of transfection is sufficient to induce morphological changes in these particles [ 110 ]. Nonetheless, these observations highlight the need for more efficient and scalable methods for the removal of contaminating particles from HIV-1 VLP immunogens in the production of potential VLP vaccines.…”

Section: Discussionmentioning

confidence: 99%

Immunogenicity of HIV-1-Based Virus-Like Particles with Increased Incorporation and Stability of Membrane-Bound Env

et al. 2021

View full text Add to dashboard Cite

An optimal prophylactic vaccine to prevent human immunodeficiency virus (HIV-1) transmission should elicit protective antibody responses against the HIV-1 envelope glycoprotein (Env). Replication-incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present virion-associated Env with a native-like structure during vaccination that closely resembles that encountered on infectious virus. Here, we optimized the incorporation of Env into previously designed mature-form VLPs (mVLPs) and assessed their immunogenicity in mice. The incorporation of Env into mVLPs was increased by replacing the Env transmembrane and cytoplasmic tail domains with those of influenza haemagglutinin (HA-TMCT). Furthermore, Env was stabilized on the VLP surface by introducing an interchain disulfide and proline substitution (SOSIP) mutations typically employed to stabilize soluble Env trimers. The resulting mVLPs efficiently presented neutralizing antibody epitopes while minimizing exposure of non-neutralizing antibody sites. Vaccination of mice with mVLPs elicited a broader range of Env-specific antibody isotypes than Env presented on immature VLPs or extracellular vesicles. The mVLPs bearing HA-TMCT-modified Env consistently induced anti-Env antibody responses that mediated modest neutralization activity. These mVLPs are potentially useful immunogens for eliciting neutralizing antibody responses that target native Env epitopes on infectious HIV-1 virions.

show abstract

“…Primary antibodies used for protein validation were mouse anti‐HIV‐1 p24 (A2‐851‐100, Icosagen, Estonia, 1:1000), rabbit anti‐CNPase antibody (ab183500, Abcam, UK, 1:1000), rabbit anti‐CIT antibody (ab86782, Abcam, UK, 1:2000), rabbit anti‐NEDD4‐2 antibody (ab46521, Abcam, UK, 1:1000) and mouse anti‐ß‐actin antibody (MA5‐15739, Thermo Fisher Scientific, 1:5000) as a loading control. Western blots were performed following the protocol previously reported (Lavado‐García, Jorge, et al, 2020; Lavado‐García, González‐Domínguez, et al, 2020).…”

Section: Methodsmentioning

confidence: 99%

Metabolic engineering of HEK293 cells to improve transient transfection and cell budding of HIV‐1 virus‐like particles

Lavado‐García

Díaz-Maneh

Canal-Paulí

et al. 2021

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

HIV‐1 Gag virus‐like particles (VLPs) are promising candidates for the development of future vaccines. Recent viral outbreaks have manifested the need of robust vaccine production platforms able to adapt to new challenges while achieving mass production capacity. For the rapid production of VLPs, the method of transient gene expression (TGE) have proved highly efficient. Based on a previous characterization of the HEK293 cell line upon transient transfection using multiplexed quantitative proteomics, molecular production bottlenecks and metabolic pathways likely to be optimized were identified. In this study, these molecular components and metabolic pathways have been explored and modulated via transient metabolic engineering using approaches like design of experiments to fully exploit and optimize VLP production, transfection and budding efficiency. Upon overexpression of endosomal sorting complex required for transport accessory proteins like NEDD4L and CIT, VLP production increased 3.3 and 2.9‐fold, respectively. Overexpression of glycosphingolipid precursor enzyme UGCG improved transfection efficiency by 17% and knocking‐down the Gag‐binding protein CNP improved 2.5‐fold VLP specific productivity. Combining CNP inhibition and UGCG overexpression further improved budding efficiency by 37.3%. Modulating VLP production and accessory pathways like intracellular budding, demonstrated the potential of metabolic engineering to optimize and intensify the development of robust production platforms for future vaccines.

show abstract

Molecular Characterization of the Coproduced Extracellular Vesicles in HEK293 during Virus-Like Particle Production

Cited by 21 publications

References 76 publications

A membrane protein display platform for receptor interactome discovery

A membrane protein display platform for receptor interactome discovery

Immunogenicity of HIV-1-Based Virus-Like Particles with Increased Incorporation and Stability of Membrane-Bound Env

Metabolic engineering of HEK293 cells to improve transient transfection and cell budding of HIV‐1 virus‐like particles

Contact Info

Product

Resources

About