Epstein-Barr virus LMP1 manipulates the content and functions of extracellular vesicles to enhance metastatic potential of recipient cells.

Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal vesicle content contribute to function and disease initiation or progression. The ability to package a variety of cargo and transmit molecular information between cells renders EVs important mediators of cell-to-cell crosstalk. Latent membrane protein 1 (LMP1) is a chief viral oncoprotein expressed in most Epstein-Barr virus (EBV)-associated cancers and is released from cells at high levels in EVs. LMP1 containing EVs have been demonstrated to promote cell growth, migration, differentiation, and regulate immune cell function. Despite these significant changes in recipient cells induced by LMP1 modified EVs, the mechanism how this viral oncogene modulates the recipient cells towards these phenotypes is not well understood. We hypothesize that LMP1 alters EV content and following uptake of the LMP1-modified EVs by the recipient cells results in the activation of cell signaling pathways and increased gene expression which modulates the biological properties of recipient cell towards a new phenotype. Our results show that LMP1 expression alters the EV protein and microRNA content packaged into EVs. The LMP1-modified EVs also enhance recipient cell adhesion, proliferation, migration, invasion concomitant with the activation of ERK, AKT, and NF-κB signaling pathways. The LMP1 containing EVs induced transcriptome reprogramming in the recipient cells by altering gene expression of different targets including cadherins, matrix metalloproteinases 9 (MMP9), MMP2 and integrin-α5 which contribute to extracellular matrix (ECM) remodeling. Altogether, our data demonstrate the mechanism in which LMP1-modified EVs reshape the tumor microenvironment by increasing gene expression of ECM interaction proteins.

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.

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.

CD63 Regulates Epstein-Barr Virus LMP1 Exosomal Packaging, Enhancement of Vesicle Production, and Noncanonical NF-κB Signaling.

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomal packaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-κB and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-κB activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling.IMPORTANCE EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma, Burkitt's lymphoma, and Hodgkin's lymphoma. In the context of cancer, EBV hijacks the exosomal pathway to modulate cell-to-cell signaling by secreting viral components such as an oncoprotein, LMP1, into host cell membrane-bound EVs. Trafficking of LMP1 into exosomes is associated with increased oncogenicity of these secreted vesicles. However, we have only a limited understanding of the mechanisms surrounding exosomal cargo packaging, including viral proteins. Here, we describe a role of LMP1 in EV production that requires CD63 and provide an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Finally, we present further evidence of the role of CD63 in limiting LMP1-induced noncanonical NF-κB and ERK activation. Our findings have implications for future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in viral-associated tumorigenesis.

Epstein-Barr Virus Orchestrates Spatial Reorganization and Immunomodulation within the Classic Hodgkin Lymphoma Tumor Microenvironment.

Classic Hodgkin Lymphoma (cHL) is a tumor composed of rare malignant Hodgkin and Reed-Sternberg (HRS) cells nested within a T-cell rich inflammatory immune infiltrate. cHL is associated with Epstein-Barr Virus (EBV) in 25% of cases. The specific contributions of EBV to the pathogenesis of cHL remain largely unknown, in part due to technical barriers in dissecting the tumor microenvironment (TME) in high detail. Herein, we applied multiplexed ion beam imaging (MIBI) spatial pro-teomics on 6 EBV-positive and 14 EBV-negative cHL samples. We identify key TME features that distinguish between EBV-positive and EBV-negative cHL, including the relative predominance of memory CD8 T cells and increased T-cell dysfunction as a function of spatial proximity to HRS cells. Building upon a larger multi-institutional cohort of 22 EBV-positive and 24 EBV-negative cHL samples, we orthogonally validated our findings through a spatial multi-omics approach, coupling whole transcriptome capture with antibody-defined cell types for tu-mor and T-cell populations within the cHL TME. We delineate contrasting transcriptomic immunological signatures between EBV-positive and EBV-negative cases that differently impact HRS cell proliferation, tumor-immune interactions, and mecha-nisms of T-cell dysregulation and dysfunction. Our multi-modal framework enabled a comprehensive dissection of EBV-linked reorganization and immune evasion within the cHL TME, and highlighted the need to elucidate the cellular and molecular fac-tors of virus-associated tumors, with potential for targeted therapeutic strategies.

PARK2 Microdeletion or Duplications Have Been Implicated in Different Neurological Disorders Including Early Onset Parkinson Disease.

The PARK2 gene is located on 6q26, encodes ubiquitin-E3- ligase, and is a transcriptional repressor of p53. It contains 12 exons. PARK2 copy number variants has been reported in various types of neurodevelopmental disorders, namely schizophrenia, Parkinson's disease (PD), autism spectrum disorder (ASD), and attention-deficit/hyperactivity disorder (ADHD). In this retrospective study, nine cases (five with microdeletion and four with microduplication) are reported with 6q26 deletion disrupting the PARK2 gene. Microdeletion sizes ranged between 215 Kb and 356 Kb, and duplication between 279 Kb and 726 Kb. These were present within the exons 7-10. Family follow up with FISH probes revealed paternal inheritance in two cases, maternal in two cases, and de novo origin in one case. Our results support previous studies showing that patients with PARK2 CNVs involving exons 5-12 might be more deleterious and cause a unique syndrome. Comprehensive analysis of additional case studies is needed to have a full characterization of this neurological disorder syndrome.

Efficient Lung Cancer Molecular Diagnostics by Combining Next Generation Sequencing with Reflex Idylla Genefusion Assay Testing.

Molecular diagnostics for lung cancer is a well-established standard of care, but how to use the available diagnostic tools for optimal and cost-effective patient care remains unresolved. Here, we show that DNA-only, small gene next-generation sequencing (sNGS) panels (<50 genes) combined with ultra-rapid reflex testing for common fusion transcripts using the Idylla Genefusion assay provide a cost-effective and sufficiently comprehensive testing modality for the majority of lung cancer cases. We also demonstrate the need for additional reflex testing capability on larger DNA and fusion panels for a small subset of lung cancers bearing rare single-nucleotide variants, indels and fusion transcripts and secondary, post-treatment resistance mutations. A similar testing workflow could be adopted for other solid tumor types for which extensive gene/fusion variant profiles are available both in the treatment-naïve and post-therapy settings.

Common clonal origin of three distinct hematopoietic neoplasms in a single patient: B-cell lymphoma, T-cell lymphoma, and polycythemia vera.

The potential for more than one distinct hematolymphoid neoplasm to arise from a common mutated stem or precursor cell has been proposed based on findings in primary human malignancies. Particularly, angioimmunoblastic T-cell lymphoma (AITL), which shares a somatic mutation profile in common with other hematopoietic malignancies, has been reported to occur alongside myeloid neoplasms or clonal B-cell proliferations, with identical mutations occurring in more than one cell lineage. Here we report such a case of an elderly woman who was diagnosed over a period of 8 years with diffuse large B-cell lymphoma, polycythemia vera, and AITL, each harboring identical somatic mutations in multiple genes. Overall, at least five identical nucleotide mutations were shared across multiple specimens, with two identical mutations co-occurring at variable variant allele frequencies in all three specimen types. These findings lend credence to the theory that a common mutated stem cell could give rise to multiple neoplasms through parallel hematopoietic differentiation pathways.

Incidental discovery of acute myeloid leukemia during liquid biopsy of a lung cancer patient.

Liquid biopsy is considered an alternative to standard next-generation sequencing (NGS) of solid tumor samples when biopsy tissue is inadequate for testing or when testing of a peripheral blood sample is preferred. A common assumption of liquid biopsies is that the NGS data obtained on circulating cell-free DNA is a high-fidelity reflection of what would be found by solid tumor testing. Here, we describe a case that challenges this widely held assumption. A patient diagnosed with lung carcinoma showed pathogenic IDH1 and TP53 mutations by liquid biopsy NGS at an outside laboratory. Subsequent in-house NGS of a metastatic lymph node fine-needle aspiration (FNA) sample revealed two pathogenic EGFR mutations. Morphologic and immunophenotypic assessment of the patient's blood sample identified acute myeloid leukemia, with in-house NGS confirming and identifying pathogenic IDH1, TP53, and BCOR mutations, respectively. This case, together with a few similar reports, demonstrates that caution is needed when interpreting liquid biopsy NGS results, especially if they are inconsistent with the presumptive diagnosis. Our case suggests that routine parallel sequencing of peripheral white blood cells would substantially increase the fidelity of the obtained liquid biopsy results.

Utility of Select Gene Mutation Detection in Tumors by the Idylla Rapid Multiplex PCR Platform in Comparison to Next-Generation Sequencing.

Testing of tumors by next generation sequencing (NGS) is impacted by relatively long turnaround times and a need for highly trained personnel. Recently, Idylla oncology assays were introduced to test for BRAF, EGFR, KRAS, and NRAS common hotspot mutations that do not require specialized trained personnel. Moreover, the interpretation of results is fully automated, with rapid turnaround time. Though Idylla testing and NGS have been shown to have high concordance in identifying EGFR, BRAF, KRAS, and NRAS hotspot mutations, there is limited experience on optimal ways the Idylla system can be used in routine practice. We retrospectively evaluated all cases with EGFR, BRAF, KRAS, or NRAS mutations identified in clinical specimens sequenced on two different NGS panels at the University of Rochester Medical Center (URMC) molecular diagnostics laboratory between July 2020 and July 2021 and assessed if these mutations would be detected by the Idylla cartridges if used. We found that the Idylla system could accurately identify Tier 1 or 2 actionable genomic alterations in select associated disease pathologies if used. Yet, in a minority of cases, we would have been unable to detect NGS-identified pathogenic mutations due to their absence on the Idylla panels. We derived algorithmic practice guidelines for the use of the Idylla cartridges. Overall, Idylla molecular testing could be implemented either as a first-line standalone diagnostic tool in select indications or for orthogonal confirmation of uncertain results.

Engineering extracellular vesicles by three-dimensional dynamic culture of human mesenchymal stem cells.

Human mesenchymal stem cell (hMSC) derived extracellular vesicles (EVs) have shown therapeutic potential in recent studies. However, the corresponding therapeutic components are largely unknown, and scale-up production of hMSC EVs is a major challenge for translational applications. In the current study, hMSCs were grown as 3D aggregates under wave motion to promote EV secretion. Results demonstrate that 3D hMSC aggregates promote activation of the endosomal sorting complexes required for transport (ESCRT)-dependent and -independent pathways. mRNA sequencing revealed global transcriptome alterations for 3D hMSC aggregates. Compared to 2D-hMSC-EVs, the quantity of 3D-hMSC-EVs was enhanced significantly (by 2-fold), with smaller sizes, higher miR-21 and miR-22 expression, and an altered protein cargo (e.g., upregulation of cytokines and anti-inflammatory factors) uncovered by proteomics analysis, possibly due to altered EV biogenesis. Functionally, 3D-hMSC-EVs rejuvenated senescent stem cells and exhibited enhanced immunomodulatory potentials. In summary, this study provides a promising strategy for scalable production of high-quality EVs from hMSCs with enhanced therapeutic potential.

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.

Epstein-Barr Virus LMP1 Promotes Syntenin-1- and Hrs-Induced Extracellular Vesicle Formation for Its Own Secretion To Increase Cell Proliferation and Migration.

Extracellular vesicles (EVs) are important mediators of cell-to-cell communication that are involved in both normal processes and pathological conditions. Latent membrane protein 1 (LMP1) is a major viral oncogene that is expressed in most Epstein-Barr virus (EBV)-associated cancers and secreted in EVs. LMP1-modified EVs have the ability to influence recipient cell growth, migration, and differentiation and regulate immune cell function. Despite the significance of LMP1-modified EVs in EBV malignancies, very little is understood about how this protein hijacks the host EV pathway for secretion. Using the biotin identification (BioID) method, we identified LMP1-proximal interacting proteins that are known to play roles in EV formation and protein trafficking. Analysis of the identified LMP1-interacting proteins revealed an enrichment in the ESCRT pathway and associated proteins, including CD63, Syntenin-1, Alix, TSG101, Hrs, and charged multivesicular body proteins (CHMPs). LMP1 transcriptionally upregulated and increased the protein expression of EV biogenesis and secretion genes. Nanoparticle tracking and immunoblot analysis revealed reduced levels of LMP1 EV packaging and of vesicle production following the knockdown of Syntenin-1, Alix, Hrs, and TSG101, with altered endolysosomal trafficking observed when Syntenin-1 and Hrs expression was reduced. Knockdown of specific ESCRT-III subunits (CHMP4B, -5, and -6) impaired LMP1 packaging and secretion into EVs. Finally, we demonstrate that the efficient secretion of LMP1-modified EVs promotes cell attachment, proliferation, and migration and tumor growth. Together, these results begin to shed light on how LMP1 exploits host ESCRT machinery to direct the incorporation of the viral oncoprotein into the EV pathway for secretion to alter the tumor microenvironment.IMPORTANCE LMP1 is a notable viral protein that contributes to the modification of EV content and tumor microenvironment remodeling. LMP1-modified EVs enhance tumor proliferation, migration, and invasion potential and promote radioresistance. Currently, the mechanisms surrounding LMP1 incorporation into the host EV pathways are not well understood. This study revealed that LMP1 utilizes Hrs, Syntenin-1, and specific components of the ESCRT-III complex for release from the cell, enhancement of EV production, and metastatic properties of cancer cells. These findings begin to unravel the mechanism of LMP1 EV trafficking and may provide new targets to control EBV-associated cancers.

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.