Vaccinia Virus Infection Requires Maturation of Macropinosomes

The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live‐cell imaging, targeted RNAi screening and perturbants of endosome maturation, we analyzed the properties and maturation pathway of the macropinocytic vacuoles containing VACV MVs in HeLa cells. The vacuoles first acquired markers of early endosomes [Rab5, early endosome antigen 1 and phosphatidylinositol(3)P]. Prior to release of virus cores into the cytoplasm, they contained markers of late endosomes and lysosomes (Rab7a, lysosome‐associated membrane protein 1 and sorting nexin 3). RNAi screening of endocytic cell factors emphasized the importance of late compartments for VACV infection. Follow‐up perturbation analysis showed that infection required Rab7a and PIKfyve, confirming that VACV is a late‐penetrating virus dependent on macropinosome maturation. VACV EV infection was inhibited by depletion of many of the same factors, indicating that both infectious particle forms share the need for late vacuolar conditions for penetration.      

Cellular senescence contributes to age‐dependent changes in circulating extracellular vesicle cargo and function

Extracellular vesicles (EVs) have emerged as important regulators of inter‐cellular and inter‐organ communication, in part via the transfer of their cargo to recipient cells. Although circulating EVs have been previously studied as biomarkers of aging, how circulating EVs change with age and the underlying mechanisms that contribute to these changes are poorly understood. Here, we demonstrate that aging has a profound effect on the circulating EV pool, as evidenced by changes in concentration, size, and cargo. Aging also alters particle function; treatment of cells with EV fractions isolated from old plasma reduces macrophage responses to lipopolysaccharide, increases phagocytosis, and reduces endothelial cell responses to vascular endothelial growth factor compared to cells treated with young EV fractions. Depletion studies indicate that CD63⁺ particles mediate these effects. Treatment of macrophages with EV‐like particles revealed that old particles increased the expression of EV miRNAs in recipient cells. Transfection of cells with microRNA mimics recapitulated some of the effects seen with old EV‐like particles. Investigation into the underlying mechanisms using bone marrow transplant studies revealed circulating cell age does not substantially affect the expression of aging‐associated circulating EV miRNAs in old mice. Instead, we show that cellular senescence contributes to changes in particle cargo and function. Notably, senolytic treatment of old mice shifted plasma particle cargo and function toward that of a younger phenotype. Collectively, these results demonstrate that senescent cells contribute to changes in plasma EVs with age and suggest a new mechanism by which senescent cells can affect cellular functions throughout the body.     

Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV‐1 infection

Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV‐1‐infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re‐routed to non‐viral EVs in a Nef‐dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.     

RNA Interference and Single Particle Tracking Analysis of Hepatitis C Virus Endocytosis

Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets.     

Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner

Ebolavirus (EBOV) is an enveloped, single-stranded, negative-sense RNA virus that causes severe hemorrhagic fever with mortality rates of up to 90% in humans and nonhuman primates. Previous studies suggest roles for clathrin- or caveolae-mediated endocytosis in EBOV entry; however, ebolavirus virions are long, filamentous particles that are larger than the plasma membrane invaginations that characterize clathrin- or caveolae-mediated endocytosis. The mechanism of EBOV entry remains, therefore, poorly understood. To better understand Ebolavirus entry, we carried out internalization studies with fluorescently labeled, biologically contained Ebolavirus and Ebolavirus-like particles (Ebola VLPs), both of which resemble authentic Ebolavirus in their morphology. We examined the mechanism of Ebolavirus internalization by real-time analysis of these fluorescently labeled Ebolavirus particles and found that their internalization was independent of clathrin- or caveolae-mediated endocytosis, but that they co-localized with sorting nexin (SNX) 5, a marker of macropinocytosis-specific endosomes (macropinosomes). Moreover, the internalization of Ebolavirus virions accelerated the uptake of a macropinocytosis-specific cargo, was associated with plasma membrane ruffling, and was dependent on cellular GTPases and kinases involved in macropinocytosis. A pseudotyped vesicular stomatitis virus possessing the Ebolavirus glycoprotein (GP) also co-localized with SNX5 and its internalization and infectivity were affected by macropinocytosis inhibitors. Taken together, our data suggest that Ebolavirus is internalized into cells by stimulating macropinocytosis in a GP-dependent manner. These findings provide new insights into the lifecycle of Ebolavirus and may aid in the development of therapeutics for Ebolavirus infection.     

Extracellular vesicle formation in Lactococcus lactis is stimulated by prophage-encoded holin–lysin system

Gram-positive bacterial extracellular membrane vesicles (EVs) have been drawing more attention in recent years. However, mechanistic insights are still lacking on how EVs are released through the cell walls in Gram-positive bacteria. In this study, we characterized underlying mechanisms of EV production and provide evidence for a role of prophage activation in EV release using the Gram-positive bacterium Lactococcus lactis as a model. By applying a standard EV isolation procedure, we observed the presence of EVs in the culture supernatant of a lysogenic L. lactis strain FM-YL11, for which the prophage-inducing condition led to an over 10-fold increase in EV production in comparison with the non-inducing condition. In contrast, the prophage-encoded holin–lysin knockout mutant YL11ΔHLH and the prophage-cured mutant FM-YL12 produced constantly low levels of EVs. Under the prophage-inducing condition, FM-YL11 did not show massive cell lysis. Defective phage particles were found to be released in and associated with holin–lysin-induced EVs from FM-YL11, as demonstrated by transmission electron microscopic images, flow cytometry and proteomics analysis. Findings from this study further generalized the EV-producing phenotype to Gram-positive L. lactis, and provide additional insights into the EV production mechanism involving prophage-encoded holin–lysin system. The knowledge on bacterial EV production can be applied to all Gram-positive bacteria and other lactic acid bacteria with important roles in fermentations and probiotic formulations, to enable desired release and delivery of cellular components with nutritional values or probiotic effects.     

Generation of the heterogeneity of extracellular vesicles by membrane organization and sorting machineries

BackgroundCells secrete heterogeneous populations of extracellular vesicles (EVs) via unknown mechanisms. EV biogenesis has been postulated to involve lipid-protein clusters, also known as membrane microdomains.MethodsMembrane properties and heterogeneity of melanoma-derived EVs were analyzed by a detergent solubilization assay, sucrose density gradient ultracentrifugation and immunoprecipitation. EV secretion was modulated by RNA interference and pharmacological treatments.ResultsWe identified two EV membranes (low-density exosomal detergent-insoluble membranes [EV-DIMs]; EV detergent-soluble membranes [EV-DSMs]) and discovered an abundant, novel type of high-density EV-DIMs. The high-density EV-DIMs accumulated the microdomain-resident protein flotillin-1, as well as a disintegrin and metalloproteinase domain containing protein 10 (Adam10), the hepatocyte growth factor receptor Met and its proteolytic fragments. Low-density EV-DIMs also contained flotillin-1. EV-DSMs were enriched with tetraspanin CD81, melanogenic enzymes and proteolytic fragments of Adam10. Intact and fragmented forms of Adam10, which resided in distinct membrane types, were secreted by different EVs. The fragmented form of Met was associated with DIMs much more efficiently than the intact form and they were secreted by distinct EVs. We identified that the endosomal sorting complexes required for transport machinery was indispensable for EV secretion of both mature and fragmented forms of Adam10 and Met.ConclusionThe findings of this study reveal the role of the interplay between membrane organization and sorting machineries in generating the heterogeneity of EVs.General significanceThis study provides novel insights into important aspects of EV biogenesis.     

Functional Comparison of SCARB2 and PSGL1 as Receptors for Enterovirus 71

Human scavenger receptor class B, member 2 (SCARB2), and P-selectin glycoprotein ligand-1 (PSGL1) have been identified to be the cellular receptors for enterovirus 71 (EV71). We compared the EV71 infection efficiencies of mouse L cells that expressed SCARB2 (L-SCARB2) and PSGL1 (L-PSGL1) and the abilities of SCARB2 and PSGL1 to bind to the virus. L-SCARB2 cells bound a reduced amount of EV71 compared to L-PSGL1 cells. However, EV71 could infect L-SCARB2 cells more efficiently than L-PSGL1 cells. The results suggested that the difference in the binding capacities of the two receptors was not the sole determinant of the infection efficiency and that SCARB2 plays an essential role after attaching to virions. Therefore, we examined the viral entry into L-SCARB2 cells and L-PSGL1 cells by immunofluorescence microscopy. In both cells, we detected internalized EV71 virions that colocalized with an early endosome marker. We then performed a sucrose density gradient centrifugation analysis to evaluate viral uncoating. After incubating the EV71 virion with L-SCARB2 cells or soluble SCARB2 under acidic conditions below pH 6.0, we observed that part of the native virion was converted into an empty capsid that lacked both genomic RNA and VP4 capsid proteins. The results suggested that the uncoating of EV71 requires both SCARB2 and an acidic environment and occurs after the internalization of the virus-receptor complex into endosomes. However, the empty capsid formation was not observed after incubation with L-PSGL1 cells or soluble PSGL1 under any of the tested pH conditions. These results indicated that SCARB2 is capable of viral binding, viral internalization, and viral uncoating and that the low infection efficiency of L-PSGL1 cells is due to the inability of PSGL1 to induce viral uncoating. The characterization of SCARB2 as an uncoating receptor greatly contributes to the understanding of the early steps of EV71 infection.     

Vaccinia virus A26 and A27 proteins form a stable complex tethered to mature virions by association with the A17 transmembrane protein

During vaccinia virus replication, mature virions (MVs) are wrapped with cellular membranes, transported to the periphery, and exported as extracellular virions (EVs) that mediate spread. The A26 protein is unusual in that it is present in MVs but not EVs. This distribution led to a proposal that A26 negatively regulates wrapping. A26 also has roles in the attachment of MVs to the cell surface and incorporation of MVs into proteinaceous A-type inclusions in some orthopoxvirus species. However, A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV, regulates incorporation of the MV into inclusions, and possibly prevents EV formation. Here, we provide evidence that A26 forms a disulfide-bonded complex with A27 that is anchored to the MV through a noncovalent interaction with the A17 transmembrane protein. In the absence of A27, A26 was unstable, and only small amounts were detected. The interaction of A26 with A27 depended on a C-terminal segment of A26 with 45% amino acid identity to A27. Deletion of A26 failed to enhance EV formation by vaccinia virus, as had been predicted. Nevertheless, the interaction of A26 and A27 may have functional significance, since each is thought to mediate binding to cells through interaction with laminin and heparan sulfate, respectively. We also found that A26 formed a noncovalent complex with A25, a truncated form of the cowpox virus A-type inclusion matrix protein. The latter association suggests a mechanism for incorporation of virions into A-type inclusions in other orthopoxvirus strains.     

Micromorphological Aspects of the Development of Rubella Virus in BHK-21 Cells

Sequential effects of rubella virus infection in BHK-21 cells were studied by electron microscopy of thin sections of control and infected cells, 2 to 7 days after infection. Vacuolization of cytoplasm in Golgi areas apparently preceded budding of virions from vacuole membranes and involvement of the endoplasmic reticulum. Newly formed endoplasmic reticulum cisternae encircled and segregated virionforming vacuoles together with other cellular elements. Large vacuolar complexes with numerous virus particles developed, and virus release from these areas occurred with disruption at the cell periphery. The viral particles, with a mean diameter of about 56 nm, consisted of an electron-dense core surrounded by a less dense capsid, enveloped by a typical unit membrane derived from the vacuole membrane.     

Mechanisms of extracellular vesicle uptake in stressed retinal pigment epithelial cell monolayers

PurposeExtracellular vesicles (EVs) can mediate long-distance communication in polarized RPE monolayers. Specifically, EVs from oxidatively stressed donor cells (stress EVs) rapidly reduced barrier function (transepithelial resistance, TER) in naïve recipient monolayers, when compared to control EVs. This effect on TER was dependent on dynamin-mediated EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Here, we further determined molecular mechanisms involved in uptake of EVs by naïve RPE cells.MethodsRPE cells were grown as monolayers in media supplemented with 1% FBS followed by transfer to FBS-free media. Cultures were used to collect control or stress EVs upon treatment with H₂O₂, others served as naïve recipient cells. In recipient monolayers, TER was used to monitor EV-uptake-based activity, live-cell imaging confirmed uptake. EV surface proteins were quantified by protein chemistry.ResultsClathrin-independent, lipid raft-mediated internalization was excluded as an uptake mechanism. Known ligand-receptor interactions involved in clathrin-dependent endocytosis include integrins and proteoglycans. Desialylated glycans and integrin-receptors on recipient cells were necessary for EV uptake and subsequent reduction of TER in recipient cells. Protein quantifications confirmed elevated levels of ligands and neuraminidase on stress EVs. However, control EVs could confer activity in the TER assay if exogenous neuraminidase or additional ligand was provided.ConclusionsIn summary, while EVs from both stressed cells and control contain cargo to communicate stress messages to naive RPE cells, stress EVs contain surface ligands that confer rapid uptake by recipient cells. We propose that EVs potentially contribute to RPE dysfunction in aging and disease.     

Protein Composition Reflects Extracellular Vesicle Heterogeneity

Extracellular vesicles (EVs) are membrane‐enclosed particles that are released by virtually all cells from all living organisms. EVs shuttle biologically active cargo including protein, RNA, and DNA between cells. When shed by cancer cells, they function as potent intercellular messangers with important functional consequences. Cells produce a diverse spectrum of EVs, spanning from small vesicles of 40–150 nm in diameter, to large vesicles up to 10 μm in diameter. While this diversity was initially considered to be purely based on size, it is becoming evident that different classes of EVs, and different populations within one EV class may harbor distinct molecular cargo and play specific functions. Furthermore, there are considerable cell type‐dependent differences in the cargo and function of shed EVs. This review focuses on the most recent proteomic studies that have attempted to capture the EV heterogeneity by directly comparing the protein composition of different EV classes and EV populations derived from the same cell source. Recent studies comparing protein composition of the same EV class(es) derived from different cell types are also summarized. Emerging approaches to study EV heterogeneity and their important implications for future studies are also discussed.     

Anti‐human CD9 antibody Fab fragment impairs the internalization of extracellular vesicles and the nuclear transfer of their cargo proteins

The intercellular communication mediated by extracellular vesicles (EVs) has gained international interest during the last decade. Interfering with the mechanisms regulating this cellular process might find application particularly in oncology where cancer cell‐derived EVs play a role in tumour microenvironment transformation. Although several mechanisms were ascribed to explain the internalization of EVs, little is our knowledge about the fate of their cargos, which are crucial to mediate their function. We recently demonstrated a new intracellular pathway in which a fraction of endocytosed EV‐associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into the nucleoplasmic reticulum. Silencing tetraspanin CD9 both in EVs and recipient cells strongly decreased the endocytosis of EVs and abolished the nuclear transfer of their cargos. Here, we investigated whether monovalent Fab fragments derived from 5H9 anti‐CD9 monoclonal antibody (referred hereafter as CD9 Fab) interfered with these cellular processes. To monitor the intracellular transport of proteins, we used fluorescent EVs containing CD9‐green fluorescent protein fusion protein and various melanoma cell lines and bone marrow‐derived mesenchymal stromal cells as recipient cells. Interestingly, CD9 Fab considerably reduced EV uptake and the nuclear transfer of their proteins in all examined cells. In contrast, the divalent CD9 antibody stimulated both events. By impeding intercellular communication in the tumour microenvironment, CD9 Fab‐mediated inhibition of EV uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti‐melanoma therapeutic strategies.     

Cerebrospinal Fluid Extracellular Vesicles Undergo Age Dependent Declines and Contain Known and Novel Non-coding RNAs

Brain development requires precise orchestration of cellular events through the coordinate exchange of information between distally located cells. One mechanism by which intercellular communication is achieved is through the transfer of extracellular vesicles (EVs). Exosomes are EVs that carry lipids, nucleic acids, and proteins and are detectable in most biological fluids including cerebrospinal fluid (CSF). Here we report that CSF EV concentrations undergo age dependent fluctuations. We characterized EV RNA content by next generation small RNA sequencing and miRNA microarray analysis and identified a temporal shift in CSF EV content. CSF EVs encapsulated miRNAs that contain a conserved hnRNPA2/B1 recognition sequence. We found that hnRNPA2/B1-containing EVs were produced by choroid plexus epithelial cells and that hnRNPA2/B1 containing EVs decreased with age. These results provide insight into EV exchange of miRNAs within the central nervous system and a framework to understand how changes in EVs may have an important impact on brain development.     

Tyrosine phosphorylation events during coxsackievirus B3 replication.

In order to study cellular and viral determinants of pathogenicity, interactions between coxsackievirus B3 (CVB3) replication and cellular protein tyrosine phosphorylation were investigated. During CVB3 infection of HeLa cells, distinct proteins become phosphorylated on tyrosine residues, as detected by the use of antiphosphotyrosine Western blotting. Two proteins of 48 and 200 kDa showed enhanced tyrosine phosphorylation 4 to 5 h postinfection (p.i.), although virus-induced inhibition of cellular protein synthesis had already occurred 3 to 4 h p.i. Subcellular fractionation experiments revealed distinct localization of tyrosine-phosphorylated proteins of 48 and 200 kDa in the cytosol and membrane fractions of infected cells, respectively. In addition, in Vero cells infected with CVB3, echovirus (EV)11, or EV12, increased tyrosine phosphorylation of a 200-kDa protein was detected 6 h p.i. Herbimycin A, a specific inhibitor of Src-like protein tyrosine kinases, was shown to inhibit virus-induced tyrosine phosphorylations and to reduce the production of progeny virions. In contrast, in cells treated with the inhibitors staurosporine and calphostin C, the synthesis of progeny virions was not affected. Immunoprecipitation experiments suggested that the tyrosine-phosphorylated 200-kDa protein in CVB3-infected cells is of cellular origin. In summary, these investigations have begun to unravel the effect of CVB3 as well as EV11 and EV12 replication on cellular tyrosine phosphorylation and support the importance of tyrosine phosphorylation events for effective virus replication. Such cellular phosphorylation events triggered in the course of enterovirus infection may enhance virus replication.     

Identification of a human SCARB2 region that is important for enterovirus 71 binding and infection

We previously identified human scavenger receptor class B, member 2 (SCARB2), as a cellular receptor for enterovirus 71 (EV71). Expression of human SCARB2 (hSCARB2) permitted mouse L929 cells to efficiently bind to virions and to produce both viral proteins and progeny viruses upon EV71 infection. Mouse Scarb2 (mScarb2) exhibited 85.8% amino acid identity and 99.9% similarity to hSCARB2. The expression of mScarb2 in L929 cells conferred partial susceptibility. Very few virions bound to mScarb2-expressing cells. The viral titer in L929 cells expressing mScarb2 was approximately 40- to 100-fold lower than that in L929 cells expressing hSCARB2. Using hSCARB2-mScarb2 chimeric mutants, we attempted to map the region that was important for efficient EV71 infection. L929 cells expressing chimeras that carried amino acids 142 to 204 from the human sequence were susceptible to EV71, while chimeras that carried the mouse sequence in this region were not. Moreover, this region was also critical for binding to virions. The determination of this region in hSCARB2 that is important for EV71 binding and infection greatly contributes to the understanding of virus-receptor interactions. Further studies will clarify the early steps of EV71 infection.     

Comparative Analysis of Technologies for Quantifying Extracellular Vesicles (EVs) in Clinical Cerebrospinal Fluids (CSF)

Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs <150 nm in diameter, NTA detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2–3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations.     

Extracellular vesicles concentration is a promising and important parameter for industrial bioprocess monitoring

Extracellular vesicles (EVs) are membrane vesicles that are produced by cells to be released into their microenvironment. In this study, we present the EV concentration as a new factor for optimization of industrial bioprocess control. The release of EVs depends on many cell properties, including cell activation and stress status, and cell death. Therefore, the EV concentration might provide a readout for identification of the cell state and the conditions during a bioprocess. Our data show that the EV concentration increased during the bioprocess, which indicated deteriorating conditions in the bioreactor. This increase in EV concentration in the fermentation broth was the consequence of two different processes: cell activation, and cell death. However, the release of EVs from activated living cells had a much weaker impact on EV concentration in the bioreactor than those released during cell death. EVs and cells in the bioprocess environment were quantified by flow cytometry. The most accurate data were obtained directly from unprocessed samples, making the monitoring of the EV concentration a rapid, easy, and cheap method. These EV concentrations reflect the conditions in the bioreactor and provide new information regarding the state of the bioprocess. Therefore, we suggest EV concentration as a new and important parameter for the monitoring of industrial bioprocesses.     

Human SCARB2-mediated entry and endocytosis of EV71

Enterovirus (EV) 71 infection is known to cause hand-foot-and-mouth disease (HFMD) and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2) has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2) to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus.     

Radioiodination of extravesicular surface constituents to study the biocorona,cell trafficking and storage stability of extracellular vesicles

BackgroundExtracellular vesicles (EVs) are produced by all cell types and serve as biological packets delivering a wide variety of molecules for cell-to-cell communication. However, the biology of the EV extravesicular surface domain that we have termed EV ‘biocorona’ remains underexplored. Upon cell secretion, EVs possess an innate biocorona containing membrane integral and peripheral constituents that is modified by acquired constituents post secretion. This distinguishes EVs from synthetic nanoparticulate biomaterials that are limited to an adsorption-based, acquired biocorona.MethodsThe EV biocorona molecular constituents were radiolabeled with ¹²⁵I to study biocorona constituents and its surface dynamics. As example toolset applications, ¹²⁵I-EVs were utilized to study EV cell trafficking and the stability of the EV biocorona during storage.ResultsThe biocorona of EVs consisted of proteins, lipids, DNA and RNA. The cellular uptake of ¹²⁵I-EVs was temperature dependent and internalized ¹²⁵I-EVs were rapidly recycled by cells. When ¹²⁵I-EVs were stored in a purified state, they exhibited time and temperature dependent biocorona shedding and proteolytic degradation that was partially inhibited in the presence of serum.ConclusionThe EV biocorona is complex and dynamic. Radiolabeling of the EV biocorona enables a unique platform methodology to study the biocorona and will facilitate unlocking EV's full clinical translation potential.General significanceThe EV biocorona affects EV mediated biological processes in health and disease. Acquiring knowledge of the EV biocorona composition, dynamics, stability and structure not only informs the diagnostic and therapeutic translation of EVs but also aids in designing biomimetic nanomaterials for drug delivery.