Zika Virus Hijacks Extracellular Vesicle Tetraspanin Pathways for Cell-to-Cell Transmission.

Extracellular vesicles (EVs) are membrane-encapsulated structures released by cells which carry signaling factors, proteins, and microRNAs that mediate intercellular communication. Accumulating evidence supports an important role of EVs in the progression of neurological conditions and both the spread and pathogenesis of infectious diseases. It has recently been demonstrated that EVs from hepatitis C virus (HCV)-infected individuals and cells contained replicative-competent viral RNA that was capable of infecting hepatocytes. Being a member of the same viral family, it is likely the Zika virus also hijacks EV pathways to package viral components and secrete vesicles that are infectious and potentially less immunogenic. As EVs have been shown to cross blood-brain and placental barriers, it is possible that Zika virus could usurp normal EV biology to gain access to the brain or developing fetus. Here, we demonstrate that Zika virus-infected cells secrete distinct EV subpopulations with specific viral protein profiles and infectious genomes. Zika virus infection resulted in the enhanced production of EVs with various sizes and densities compared to those released from noninfected cells. We also show that the EV-enriched tetraspanin CD63 regulates the release of EVs and Zika viral genomes and capsids following infection. Overall, these findings provide evidence for an alternative means of Zika virus transmission and demonstrate the role of EV biogenesis and trafficking proteins in the modulation of Zika virus infection and virion morphogenesis. IMPORTANCE Zika virus is a reemerging infectious disease that spread rapidly across the Caribbean and South America. Infection of pregnant women during the first trimester has been linked to microcephaly, a neurological condition where babies are born with smaller heads due to abnormal brain development. Babies born with microcephaly can develop convulsions and suffer disabilities as they age. Despite the significance of Zika virus, little is known about how the virus infects the fetus or causes disease. Extracellular vesicles (EVs) are membrane-encapsulated structures released by cells that are present in all biological fluids. EVs carry signaling factors, proteins, and microRNAs that mediate intercellular communication. EVs have been shown to be a means by which some viruses can alter cellular environments and cross previously unpassable cellular barriers. Thus, gaining a greater understanding of how Zika virus affects EV cargo may aid in the development of better diagnostics, targeted therapeutics, and/or prophylactic treatments.

Epstein-Barr Virus LMP1 Modulates the CD63 Interactome.

Tetraspanin CD63 is a cluster of cell surface proteins with four transmembrane domains; it is associated with tetraspanin-enriched microdomains and typically localizes to late endosomes and lysosomes. CD63 plays an important role in the cellular trafficking of different proteins, EV cargo sorting, and vesicle formation. We have previously shown that CD63 is important in LMP1 trafficking to EVs, and this also affects LMP1-mediated intracellular signaling including MAPK/ERK, NF-κB, and mTOR activation. Using the BioID method combined with mass spectrometry, we sought to define the broad CD63 interactome and how LMP1 modulates this network of interacting proteins. We identified a total of 1600 total proteins as a network of proximal interacting proteins to CD63. Biological process enrichment analysis revealed significant involvement in signal transduction, cell communication, protein metabolism, and transportation. The CD63-only interactome was enriched in Rab GTPases, SNARE proteins, and sorting nexins, while adding LMP1 into the interactome increased the presence of signaling and ribosomal proteins. Our results showed that LMP1 alters the CD63 interactome, shifting the network of protein enrichment from protein localization and vesicle-mediated transportation to metabolic processes and translation. We also show that LMP1 interacts with mTOR, Nedd4 L, and PP2A, indicating the formation of a multiprotein complex with CD63, thereby potentially regulating LMP1-dependent mTOR signaling. Collectively, the comprehensive analysis of CD63 proximal interacting proteins provides insights into the network of partners required for endocytic trafficking and extracellular vesicle cargo sorting, formation, and secretion.

BioID Combined with Mass Spectrometry to Study Herpesvirus Protein-Protein Interaction Networks.

Herpes viruses are important human pathogens that cause a wide range of diseases from skin lesions to malignancies. Protein interactions drive many cellular events and mediate a number of biochemical pathways leading to different physiological outcomes. Protein interactions between viral proteins and host proteins play significant roles in viral entry, replication and suppression of host-immune responses. Therefore, the study of virus-host interactions promises significant advancement in designing therapeutics to control infection and disease. Various approaches are employed in the field to study and identify protein interactions that combine affinity purification along with different detection methods. Advancements in protein purification and high-throughput detection methods have resulted in an unprecedented level of discovery. Here we detail the use of proximity dependent biotinylation (BioID) as a means of affinity purification coupled with the use of LC-MS/MS for the detection and identification of protein-protein interaction networks.

Differential Effects of Extracellular Vesicles of Lineage-Specific Human Pluripotent Stem Cells on the Cellular Behaviors of Isogenic Cortical Spheroids.

Extracellular vesicles (EVs) contribute to a variety of signaling processes and the overall physiological and pathological states of stem cells and tissues. Human induced pluripotent stem cells (hiPSCs) have unique characteristics that can mimic embryonic tissue development. There is growing interest in the use of EVs derived from hiPSCs as therapeutics, biomarkers, and drug delivery vehicles. However, little is known about the characteristics of EVs secreted by hiPSCs and paracrine signaling during tissue morphogenesis and lineage specification. Methods: In this study, the physical and biological properties of EVs isolated from hiPSC-derived neural progenitors (ectoderm), hiPSC-derived cardiac cells (mesoderm), and the undifferentiated hiPSCs (healthy iPSK3 and Alzheimer's-associated SY-UBH lines) were analyzed. Results: Nanoparticle tracking analysis and electron microscopy results indicate that hiPSC-derived EVs have an average size of 100-250 nm. Immunoblot analyses confirmed the enrichment of exosomal markers Alix, CD63, TSG101, and Hsc70 in the purified EV preparations. MicroRNAs including miR-133, miR-155, miR-221, and miR-34a were differently expressed in the EVs isolated from distinct hiPSC lineages. Treatment of cortical spheroids with hiPSC-EVs in vitro resulted in enhanced cell proliferation (indicated by BrdU+ cells) and axonal growth (indicated by ß-tubulin III staining). Furthermore, hiPSC-derived EVs exhibited neural protective abilities in Aß42 oligomer-treated cultures, enhancing cell viability and reducing oxidative stress. Our results demonstrate that the paracrine signaling provided by tissue context-dependent EVs derived from hiPSCs elicit distinct responses to impact the physiological state of cortical spheroids. Overall, this study advances our understanding of cell‒cell communication in the stem cell microenvironment and provides possible therapeutic options for treating neural degeneration.

Transmembrane Domains Mediate Intra- and Extracellular Trafficking of Epstein-Barr Virus Latent Membrane Protein 1.

EBV latent membrane protein 1 (LMP1) is released from latently infected tumor cells in small membrane-enclosed extracellular vesicles (EVs). Accumulating evidence suggests that LMP1 is a major driver of EV content and functions. LMP1-modified EVs have been shown to influence recipient cell growth, migration, differentiation, and regulation of immune cell function. Despite the significance of LMP1-modified exosomes, very little is known about how this viral protein enters or manipulates the host EV pathway. In this study, LMP1 deletion mutants were generated to assess protein regions required for EV trafficking. Following transfection of LMP1 or mutant plasmids, EVs were collected by differential centrifugation, and the levels of specific cargo were evaluated by immunoblot analysis. The results demonstrate that, together, the N terminus and transmembrane region 1 of LMP1 are sufficient for efficient sorting into EVs. Consistent with these findings, a mutant lacking the N terminus and transmembrane domains 1 through 4 (TM5-6) failed to be packaged into EVs, and exhibited higher colocalization with endoplasmic reticulum and early endosome markers than the wild-type protein. Surprisingly, TM5-6 maintained the ability to colocalize and form a complex with CD63, an abundant exosome protein that is important for the incorporation of LMP1 into EVs. Other mutations within LMP1 resulted in enhanced levels of secretion, pointing to potential positive and negative regulatory mechanisms for extracellular vesicle sorting of LMP1. These data suggest new functions of the N terminus and transmembrane domains in LMP1 intra- and extracellular trafficking that are likely downstream of an interaction with CD63.IMPORTANCE EBV infection contributes to the development of cancers, such as nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin's disease, and posttransplant lymphomas, in immunocompromised or genetically susceptible individuals. LMP1 is an important viral protein expressed by EBV in these cancers. LMP1 is secreted in extracellular vesicles (EVs), and the transfer of LMP1-modified EVs to uninfected cells can alter their physiology. Understanding the cellular machinery responsible for sorting LMP1 into EVs is limited, despite the importance of LMP1-modified EVs. Here, we illustrate the roles of different regions of LMP1 in EV packaging. Our results show that the N terminus and TM1 are sufficient to drive LMP1 EV trafficking. We further show the existence of potential positive and negative regulatory mechanisms for LMP1 vesicle sorting. These findings provide a better basis for future investigations to identify the mechanisms of LMP1 targeting to EVs, which could have broad implications in understanding EV cargo sorting.

The interactome of EBV LMP1 evaluated by proximity-based BioID approach.

Epstein-Barr virus LMP1 is an oncoprotein required for immortalizing B lymphocytes and also plays important roles in transforming non-lymphoid tissue. The discovery of LMP1 protein interactions will likely generate targets to treat EBV-associated cancers. Here, we define the broader LMP1 interactome using the recently developed BioID method. Combined with mass spectrometry, we identified over 1000 proteins across seven independent experiments with direct or indirect relationships to LMP1. Pathway analysis suggests that a significant number of the proteins identified are involved in signal transduction and protein or vesicle trafficking. Interestingly, a large number of proteins thought to be important in the formation of exosomes and protein targeting were recognized as probable LMP1 interacting partners, including CD63, syntenin-1, ALIX, TSG101, HRS, CHMPs, and sorting nexins. Therefore, it is likely that LMP1 modifies protein trafficking and exosome biogenesis pathways. In support of this, knock-down of syntenin-1 and ALIX resulted in reduced exosomal LMP1.

The Epstein-Barr virus LMP1 interactome: biological implications and therapeutic targets.

The oncogenic potential of Epstein-Barr virus (EBV) is mostly attributed to latent membrane protein 1 (LMP1), which is essential and sufficient for transformation of fibroblast and primary lymphocytes. LMP1 expression results in the activation of multiple signaling cascades like NF-ΚB and MAP kinases that trigger cell survival and proliferative pathways. LMP1 specific signaling events are mediated through the recruitment of a number of interacting proteins to various signaling domains. Based on these properties, LMP1 is an attractive target to develop effective therapeutics to treat EBV-related malignancies. In this review, we focus on LMP1 interacting proteins, associated signaling events, and potential targets that could be exploited for therapeutic strategies.

Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells.IMPORTANCE The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major oncoprotein, LMP1, into vesicles for secretion. We have recently described a role of the host cell protein CD63 in regulating intracellular signaling of the viral oncoprotein by shuttling LMP1 into exosomes. Here, we provide strong evidence of the utility of CD63-dependent EVs in regulating global intracellular signaling, including mTOR activation by LMP1. We also demonstrate a key role of CD63 in coordinating endosomal and autophagic processes to regulate LMP1 levels within the cell. Overall, this study offers new insights into the complex intersection of cellular secretory and degradative mechanisms and the implications of these processes in viral replication.