Tetherin antagonism by SARS-CoV-2 ORF3a and spike protein enhances virus release.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrate that SARS-CoV-2 infection causes tetherin downregulation and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigate the potential viral proteins involved in abrogating tetherin function and find that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles where Coronaviruses bud, and increases tetherin localisation to late endocytic organelles via reduced retrograde recycling. We also find that expression of Spike protein causes a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

In Vitro Interaction of Melanoma-Derived Extracellular Vesicles with Collagen.

Extracellular vesicles are now considered as active contributors to melanoma progression through their capacity to modify the tumor microenvironment and to favor the formation of a pre-metastatic niche. These prometastatic roles of tumor-derived EVs would pass through their interaction with the extracellular matrix (ECM) and its remodeling, in turn providing a substrate favoring persistent tumor cell migration. Nevertheless, the capacity of EVs to directly interact with ECM components is still questionable. In this study, we use electron microscopy and a pull-down assay to test the capacity of sEVs, derived from different melanoma cell lines, to physically interact with collagen I. We were able to generate collagen fibrils coated with sEVs and to show that melanoma cells release subpopulations of sEVs that can differentially interact with collagen.

Bioengineered small extracellular vesicles deliver multiple SARS-CoV-2 antigenic fragments and drive a broad immunological response.

The COVID-19 pandemic highlighted the clear risk that zoonotic viruses pose to global health and economies. The scientific community responded by developing several efficacious vaccines which were expedited by the global need for vaccines. The emergence of SARS-CoV-2 breakthrough infections highlights the need for additional vaccine modalities to provide stronger, long-lived protective immunity. Here we report the design and preclinical testing of small extracellular vesicles (sEVs) as a multi-subunit vaccine. Cell lines were engineered to produce sEVs containing either the SARS-CoV-2 Spike receptor-binding domain, or an antigenic region from SARS-CoV-2 Nucleocapsid, or both in combination, and we tested their ability to evoke immune responses in vitro and in vivo. B cells incubated with bioengineered sEVs were potent activators of antigen-specific T cell clones. Mice immunised with sEVs containing both sRBD and Nucleocapsid antigens generated sRBD-specific IgGs, nucleocapsid-specific IgGs, which neutralised SARS-CoV-2 infection. sEV-based vaccines allow multiple antigens to be delivered simultaneously resulting in potent, broad immunity, and provide a quick, cheap, and reliable method to test vaccine candidates.

CD63 sorts cholesterol into endosomes for storage and distribution via exosomes.

Extracellular vesicles such as exosomes are now recognized as key players in intercellular communication. Their role is influenced by the specific repertoires of proteins and lipids, which are enriched when they are generated as intraluminal vesicles (ILVs) in multivesicular endosomes. Here we report that a key component of small extracellular vesicles, the tetraspanin CD63, sorts cholesterol to ILVs, generating a pool that can be mobilized by the NPC1/2 complex, and exported via exosomes to recipient cells. In the absence of CD63, cholesterol is retrieved from the endosomes by actin-dependent vesicular transport, placing CD63 and cholesterol at the centre of a balance between inward and outward budding of endomembranes. These results establish CD63 as a lipid-sorting mechanism within endosomes, and show that ILVs and exosomes are alternative providers of cholesterol.

Lack of involvement of CD63 and CD9 tetraspanins in the extracellular vesicle content delivery process.

Extracellular vesicles (EVs) are thought to mediate intercellular communication by transferring cargoes from donor to acceptor cells. The EV content-delivery process within acceptor cells is still poorly characterized and debated. CD63 and CD9, members of the tetraspanin family, are highly enriched within EV membranes and are respectively enriched within multivesicular bodies/endosomes and at the plasma membrane of the cells. CD63 and CD9 have been suspected to regulate the EV uptake and delivery process. Here we used two independent assays and different cell models (HeLa, MDA-MB-231 and HEK293T cells) to assess the putative role of CD63 and CD9 in the EV delivery process that includes uptake and cargo delivery. Our results suggest that neither CD63, nor CD9 are required for this function.

ER membrane contact sites support endosomal small GTPase conversion for exosome secretion.

Exosomes are endosome-derived extracellular vesicles involved in intercellular communication. They are generated as intraluminal vesicles within endosomal compartments that fuse with the plasma membrane (PM). The molecular events that generate secretory endosomes and lead to the release of exosomes are not well understood. We identified a subclass of non-proteolytic endosomes at prelysosomal stage as the compartment of origin of CD63 positive exosomes. These compartments undergo a Rab7a/Arl8b/Rab27a GTPase cascade to fuse with the PM. Dynamic endoplasmic reticulum (ER)-late endosome (LE) membrane contact sites (MCS) through ORP1L have the distinct capacity to modulate this process by affecting LE motility, maturation state, and small GTPase association. Thus, exosome secretion is a multi-step process regulated by GTPase switching and MCS, highlighting the ER as a new player in exosome-mediated intercellular communication.

Tetherin antagonism by SARS-CoV-2 enhances virus release: multiple mechanisms including ORF3a-mediated defective retrograde traffic.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrated that SARS-CoV-2 infection causes tetherin downregulation, and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigated the potential viral proteins involved in abrogating tetherin function and found that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles via reduced retrograde recycling and increases tetherin localisation to late endocytic organelles. By removing tetherin from the Coronavirus budding compartments, ORF3a enhances virus release. We also found expression of Spike protein caused a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9.

Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.

Rapid Isolation of Rare Isotype-Switched Hybridoma Variants: Application to the Generation of IgG2a and IgG2b MAb to CD63, a Late Endosome and Exosome Marker.

CD63, a member of the tetraspanin superfamily, is used as a marker of late endosomes and lysosome-related organelles, as well as a marker of exosomes. Here, we selected rare isotype variants of TS63 by sorting hybridoma cells on the basis of their high expression of surface immunoglobulins of the IgG2a and IgG2b subclass. Pure populations of cells secreting IgG2a and IgG2b variants of TS63 (referred to as TS63a and TS63b) were obtained using two rounds of cell sorting and one limited dilution cloning step. We validate that these new TS63 variants are suitable for co-labeling with mAb of the IgG1 subclass directed to other molecules, using anti mouse subclass antibodies, and for the labeling of exosomes through direct binding to protein A-coated gold particles. These mAbs will be useful to study the intracellular localization of various proteins and facilitate electron microscopy analysis of CD63 localization.

To be or not to be... secreted as exosomes, a balance finely tuned by the mechanisms of biogenesis.

The release of extracellular vesicles such as exosomes provides an attractive intercellular communication pathway. Exosomes are 30- to 150-nm membrane vesicles that are generated in endosomal compartment and act as intercellular mediators in both physiological and pathological context. Despite the growing interest in exosome functions, the mechanisms responsible for their biogenesis and secretion are still not completely understood. Knowledge about these mechanisms is important because they control the composition, and hence the function and secretion, of exosomes. Exosomes are produced as intraluminal vesicles in extremely dynamic endosomal organelles, which undergo various maturation processes in order to form multivesicular endosomes. Notably, the function of multivesicular endosomes is balanced between exosome secretion and lysosomal degradation. In the present review, we present and discuss each intracellular trafficking pathway that has been reported or proposed as regulating exosome biogenesis, with a particular focus on the importance of endosomal dynamics in sorting out cargo proteins to exosomes and to the secretion of multivesicular endosomes. An overall picture reveals several key mechanisms, which mainly act at the crossroads of endosomal pathways as regulatory checkpoints of exosome biogenesis.

Liver Metastasis Is Facilitated by the Adherence of Circulating Tumor Cells to Vascular Fibronectin Deposits.

The interaction between circulating tumor cells (CTC) and endothelial cells during extravasation is a critical process during metastatic colonization, but its mechanisms remain poorly characterized. Here we report that the luminal side of liver blood vessels contains fibronectin deposits that are enriched in mice bearing primary tumors and are also present in vessels from human livers affected with metastases. Cancer cells attached to endothelial fibronectin deposits via talin1, a major component of focal adhesions. Talin1 depletion impaired cancer cell adhesion to the endothelium and transendothelial migration, resulting in reduced liver metastasis formation in vivo Talin1 expression levels in patient CTC's correlated with prognosis and therapy response. Together, our findings uncover a new mechanism for liver metastasis formation involving an active contribution of hepatic vascular fibronectin and talin1 in cancer cells. Cancer Res; 77(13); 3431-41. ©2017 AACR.

Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells.

Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.