Extracellular vesicles from Kaposi Sarcoma-associated herpesvirus lymphoma induce long-term endothelial cell reprogramming

Extracellular signaling is a mechanism that higher eukaryotes have evolved to facilitate organismal homeostasis. Recent years have seen an emerging interest in the role of secreted microvesicles, termed extracellular vesicles (EV) or exosomes in this signaling network. EV contents can be modified by the cell in response to stimuli, allowing them to relay information to neighboring cells, influencing their physiology. Here we show that the tumor virus Kaposi’s Sarcoma-associated herpesvirus (KSHV) hijacks this signaling pathway to induce cell proliferation, migration, and transcriptome reprogramming in cells not infected with the virus. KSHV-EV activates the canonical MEK/ERK pathway, while not alerting innate immune regulators, allowing the virus to exert these changes without cellular pathogen recognition. Collectively, we propose that KSHV establishes a niche favorable for viral spread and cell transformation through cell-derived vesicles, all while avoiding detection.


Author summary
The role of extracellular vesicles (EV) has received considerable attention in recent years. The contents of these cell-derived vesicles have been shown to be modulated upon challenge by a virus or neoplastic transformation, and can influence the behavior of recipient cells. Here we demonstrate that purified EV from an AIDS-associated, virus-driven lymphoma induces unique cellular signaling, motility, and gene expression reprogramming in recipient endothelial cells. This was accomplished without activation of innate immune activation, even after prolonged exposure to these EV. Collectively our results point toward a model in which tumor-derived EV condition neighboring cell physiology, while avoiding detection by immune regulators. Introduction Extracellular communication is pivotal to maintain organismal homeostasis and a disease state. One of the mechanisms by which cells communicate to their surroundings is through extracellular vesicles (EV). Cells package a number of biological molecules into EV such as proteins, nucleic acids, lipids, and metabolites. EV are released into the microenvironment as well as into the circulation via the blood and lymphatic vessels. All vessels are lined with endothelial cells (EC), which are permanently exposed to EV, and uptake the EV-loaded cargo. This may induce changes in differentiation, metabolism, migration, and gene expression (reviewed in [1], for further examples see [2]). Ample experimental evidence has connected EV to cancer metastasis, immune signaling and the response to invading pathogens, though the molecular details of EV biology are far from established and tend to differ dramatically among experimental systems [1]. As in many aspects of modern biology it is difficult to come up with experimental approaches that are both tractable and physiologically relevant. Kaposi Sarcoma (KS) and Kaposi-sarcoma-associated herpesvirus (KSHV) lymphomas, specifically primary effusion lymphoma (PEL), represent one such system to study EV biology. KS is one of the most angiogenic cancers in humans, and was one of the first identifiable markers for AIDS (reviewed in [3]). KSHV is the etiological agent of KS and PEL, which induce a unique tumor microenvironment that remodels tumor and lymphatic vasculature [4][5][6]. Of note, the transdifferentiation induced by the virus is as dramatic in uninfected neighboring cells as in virus-infected cells. We had shown that KSHV-infected cells release EV containing all viral micro RNAs (miRNA), but not the virus itself, and that the viral miRNAs are present at high concentrations in KSHV-lymphoma derived EV (KSHV-EV) in culture and in patients [7]. This establishes the KSHV-EV:EC interaction as physiologically relevant, experimentally robust, and as we show here, highly tractable.
EV membranes are enriched for phosphatidylserine (PS), which is recognized by Annexin-V and plays a role in EV adsorption. Tetraspanins, such as CD9, CD81, and CD63, are found on the majority of EV and have been used to affinity-purify EV [8][9][10][11][12]. Alix and Flotillins-1 and 2 are additional molecules that define EV (see http://www.exocarta.org/). Multiple EV purification methods have been devised [13]. These purification schemes yield comparable preparation of EV though the resultant fractions can be quite heterogeneous and need to be carefully characterized in each experiment. Exosomes are a subtype of EV that is defined by their intracellular biogenesis. Exosomes originate from the inward budding of the late endosome into the multivesicular body and traffic from there to the plasma membrane where they are released. When studies use primary patient material and/or cell culture supernatant, the origin of the EV cannot unequivocally be attributed to the multivesicular body; and the term EV rather than exosome is used. To date, no EV-specific receptors have been defined; evidence for tissue-specific uptake is limited to specific scenarios such as neuronal or immunological synapses [14]. Unlike viruses, EV are believed to be able to enter all cell types. Viruses that modulate EV maturation and content include Human and Simian immunodeficiency virus (HIV and SIV), vaccinia virus, hepatitis C virus, and herpesviruses [1,7,12]. This led Gould et al. to propose the trojan horse hypothesis [15], whereby viruses use EV to modulate the cell physiology of neighboring cells in order to further infection.
In this study, we sought to characterize how EV taken from the HIV-associated PEL, either cell culture or primary patient fluid, influence EC behavior. We discovered that affinity purified EV mediated cell migration, proliferation, and secretion of human interleukin-6 (IL-6) through the extracellular signal related kinases (ERK1/2) pathway. This was accomplished without tripping of innate sensors such as interferon regulatory factor 3 (IRF3), stimulator of interferon genes (STING) [16][17][18][19][20][21], or nuclear factor kappa B (NF-κB). It allows KSHV to  monitored for migration into the bottom chamber, which contained the cytokine in serumfree media. KSHV-EV significantly enhanced cell migration in response VEGF and IL-6, but not PDGF-β or SDF1-α (S12 Fig). Third, a scratch assay was performed. The hTERT-HU-VECs were grown in the presence of EV, the confluent monolayer disrupted, and closure monitored over time. KSHV-EV and primary PEL EV enhanced migration, EV from HD did not (Fig 5C). The positive control, VEGF, alone or added to EV from HD yielded the same degree of scratch closures as induced by KSHV-EV (Fig 5D and 5E). Fourth, to test the hypothesis that EV induced cytokines, which could amplify or mediate the migration phenotype, supernatants were analyzed for IL-6, IL-10, which are implicated in KS biology, as well as the immune response cytokines IL-18, IL-1β, and interferon alpha (IFN-α). Only IL-6 was significantly induced in response to KSHV-EV and primary PEL EV (Fig 5F and S13 Fig). These experiments establish that KSHV-EV, but not EV circulating in healthy patients contributes to the pathophysiology of KS by modulating EC function and inducing human IL-6. https://doi.org/10.1371/journal.ppat.1007536.g002 Secreted vesicles from an AIDS-lymphoma reprogram cell physiology

KSHV-EV do not activate innate immune signaling pathways
To test whether KSHV-EV elicited an innate immune response in EC, a number of known innate immune signaling pathways were examined. On the one hand, such a phenotype would be expected as PEL and KS represent an inflammatory microenvironment (reviewed in [3]); on the other hand, pro-as well as anti-inflammatory phenotypes have been reported for infected-cell derived EV from different viruses (reviewed [1]). The innate immune response involves membrane-bound receptors, such as toll-like receptors (TLRs), as well as cytoplasmic RIG-like receptors, such as RIG-I and MDR-5. Both pathways ultimately converge onto interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB). IRF3 and NF-κB are  normally sequestered in the cytoplasm. Upon stimulation, they translocate to the nucleus. KSHV-EV, or primary PEL EV did not induce nuclear translocation of IRF3 (Fig 6A and 6B) and did not induce IRF3 phosphorylation (Fig 6C). This was in contrast to infection with West Nile Virus or stimulation with Polyinosinic:polycytidylic acid (PolyI:C). KSHV-EV did not inhibit the phosphorylation of IRF3 in response to PolyI:C (Fig 6D), suggesting the KSHV EV did not actively inhibit signaling. KSHV-EV, or primary PEL EV did not induce nuclear translocation of NF-κB either (S14 Fig). Likewise, targeted transcriptional profiling of ninety NF-κB-regulated genes showed no response to KSHV-EV (S15 Fig). The IRF3 and NF-κB transcription factors represent the endpoint of a multitude of RNA sensing pathways. As neither was activated, it is unlikely that any of the upstream receptors (TLR, RLR) were activated to the degree that authentic viral infection would. KSHV is a DNA virus, and its reactivation from latency is curbed by cGAS/STING [25]. To test for the induction of cGAS-STING signaling by KSHV-EV, we monitored the induction of interferon-beta (IFN-β). KSHV-EV did not change IFN-β transcript levels, and KSHV-EV did not modulate the cGAS/STING response to Interferon Stimulatory DNA (ISD) or Poly I:C (Fig 7A). As a second, independent measure of cGAS/STING activation we measured TANK binding kinase (TBK). TANK is phosphorylated upon recognition of cytosolic nucleic acids in a MAVS dependent manner, and physically interacts with STING. KSHV-EV did not affect phospho-TBK levels alone or in conjunction with the positive inducers (Fig 7B and 7C). In sum, neither TLR, RIG-I, nor cGAS/STING pathways were activated by KSHV-EV; at least under these conditions KSHV-EV did not inhibit the activation of these pathways by physiological triggers either.

KSHV-EC activate ERK1/2
To identify molecular pathways that could explain KSHV-EV induced endothelial cell migration, we explored ERK1/2 signaling. ERK1/2 has been implicated in IL-6 signaling as well as EC migration [26,27]. Treatment of hTERT-HUVEC with KSHV-EV and primary PEL EV, but not HD EV induced ERK1/2 phosphorylation (p-ERK1/2) (Fig 8A). The EV themselves did not contain p-ERK1/2 (Fig 8B). To exclude the possibility that IL-6 induced ERK1/2 phosphorylation as part of a secondary feedback loop, the experiment was repeated in the presence of antagonistic anti-IL-6 receptor antibodies. Despite blocking IL-6 signaling, ERK1/2 became phosphorylated upon KSHV-EV exposure (Fig 8C). Pre-incubation with Annexin-V, which blocks EV adsorption, significantly reduced p-ERK1/2 levels (Fig 8C). This result is consistent with the notion that ERK activation was a direct result of EV exposure. ERK1/2 is phosphorylated by MEK, which can be targeted pharmacologically. To test the hypothesis that MEK kinase activity was required for p-ERK1/2 in response to KSHV-EV and primary PEL EV, we used AZD6244. Pre-treatment of cells with AZD6244 blocked ERK1/2 activation by primary PEL EV relative to DMSO control (Fig 8D). AZD6244 also reduced primary PEL EV-and KSHV-EV-dependent cell migration (Fig 8E and 8F). To ensure that AZD6244 did not exert off-target effects, we repeated the cell migration assay with a different MEK inhibitor, PD184352. Treatment with PD184352 antagonized the enhanced cell migration phenotype of hTERT-HUVECs treated with KSHV-EV (S16 Fig). These experiments (C) KSHV-EV or primary PEL EV accelerates wound healing. hTERT-HUVECs were grown to confluency and an artificial wound was created using a scratch. 2 hours prior to the scratch, cells were incubated with HD EV, KSHV-EV, or primary PEL EV. Images were taken immediately after the scratch (0 hour), 6 hour, and 12 hours in the absence of VEGF. (D) Same as (C), except with the addition of VEGF as a control (E) Quantification of wound closure at 6 and 12 hours with the various treatments. Wound boundaries from (C) and (D) were imaged using ImageJ for quantification (F) KSHV-EV or primary PEL EV induce IL-6 secretion. hTERT-HUVECs were incubated with EV from HD, KSHV, or primary PEL for 24 hours and cell supernatants were harvested and levels of IL-6 were determined by ELISA (See also S13 Fig). Secreted vesicles from an AIDS-lymphoma reprogram cell physiology demonstrate that KSHV-EV activate the Ras/Raf/MEK/ERK pathway, which leads to EC activation, proliferation, and migration.

KSHV-EV stably reprograms endothelial cells
In PEL and KS patients, KSHV-EV are continually released into the microenvironment and systemically into the blood and lymphatic circulation. Next to hemangioma, KS is the most angiogenic cancer in humans (reviewed in [3]). PEL grow as effusions, bathing the cavity walls in KSHV-EV. Thus, a physiological relevant experimental design would expose EC repeatedly to KSHV-EV. Such a design measures long-term cellular reprogramming (Fig 9A). Here, hTERT-HUVECs were exposed to KSHV-EV or BJAB-derived EV over a period of 12 days. Every 24 hours the media was replenished with fresh KSHV-EV or control-tumor EV, and cellular programming was analyzed by RNAseq. The time course was divided into an acute phase (day 2 and day 4), an intermediate phase (day 6 and day 8), and a chronic phase (day 10 and 12). KSHV-EV induced synchronized, progressive, and directional transcription profile changes compared to control (Fig 9B, Table). Heatmap representation of these altered genes show distinct contrasts between the treatment groups (Fig 9D-left), which were maintained over the 12 days of KSHV-EV exposure.
To test the hypothesis that KSHV-EV mediate any or all of the transcriptionally changes hitherto ascribed to KSHV miRNA expression within the infected cell, we analyzed transcriptional changes in genes that were previously identified in KSHV-infected EC [6,28,29]. Changes in these particular sets of mRNAs signal BEC to LEC transcription upon direct infection of KSHV. These mRNAs remained largely unchanged (S18 Fig). The notable exception was MAF1 mRNA, which was previously shown to be a direct target of multiple KSHV miR-NAs with eleven predicted target sites in the 3'UTR [6], which was robustly down-regulated. As a control we analyzed a predefined set of interferon stimulatory genes (ISGs) [30] (Fig 9D,  right). These showed induction during the acute phase for a few genes; however, these did not differ between control and KSHV-EV. This induction was not maintained over time, consistent with the idea that KSHV-EV reprogram EC towards a proliferative, activated phenotype, which is different from phenotypic changes induced by inflammation.
Time course analysis allowed for the identification of patterns of transcriptional changes for individual genes. The minimal set of EV-exposure biomarkers was comprised of genes with the highest and most consistent mRNA changes over time (Fig 10A and 10B). Among them were CD9 and JUNB, which we chose to validate at the level of protein expression ( Fig  10C). CD9 protein levels were greatly reduced in KSHV-EV treated cells, particularly in the intermediate and chronic time points. As an internal control, we monitored the protein levels of a separate EV protein Tsg101, which remained constant in both treatment groups over time.

Fig 7. PEL EV do not Activate nor Antagonize IFN-β Expression or STING activation in response to stimulation. (A)
hTERT-HUVECs were treated with BJAB (control) EV or KSHV-EV (or mock treated), and subsequently with mock, interferon stimulatory DNA fragment (ISD), or Poly I:C. Relative IFN-β mRNA levels were quantified and standardized to β-Actin [25]. (B) PEL EV do not Inhibit Phosphorylation of TBK1 in Response to ISD Stimulation. hTERT-HUVECs were treated with BJAB (control) EV or KSHV-EV (or mock treated), and subsequently with mock or ISD. Cells were lysed at various time points post stimulation and contents were run out for western blot analysis for total TBK1, phosphorylated TBK1 (p-TBK1), and β-Actin as a loading control. (C) PEL EV does not Inhibit Phosphorylation of TBK1 in Response to Poly I:C Stimulation. hTERT-HUVECs were treated with BJAB (control) EV or KSHV-EV (or mock treated), and subsequently with mock or Poly I:C. Cells were lysed at various time points post stimulation and contents were run out for western blot analysis for total TBK1, phosphorylated TBK1 (p-TBK1), and β-Actin as a loading control. https://doi.org/10.1371/journal.ppat.1007536.g007 Secreted vesicles from an AIDS-lymphoma reprogram cell physiology The advantage of genome-wide transcriptional profiling lies in the identification of signaling networks, rather than individual genes. Hence, the significantly altered genes were clustered into gene ontology (GO) pathways [31]. The preeminent pathways identified herein related to extracellular matrix modulation, cell adhesion, growth and migration (Fig 11). Next, we explored phenotypic changes that would be consistent with the transcriptional pathways that dominated GO analysis. These are summarized in S3 Table. Experiments showing increased cell growth and migration (and thus decreased adhesion) in response to KSHV-EV, but not control-EV were already noted above. CD9 is a tetraspanin, which is involved in cell adhesion, motility and junctional integrity. It is also involved in EV biogenesis. To test whether the KSHV-EV-induced downregulation of CD9 at the mRNA and protein level led to a reduction in EV secretion in the recipient cells, we measured EV in the supernatant of hTERT-HU-VEC cells at 24 hours after treatment with KSHV-EV. This experiment was possible because earlier studies had shown that exogenously added EV, analogous to liposomal transfection, are taken up within a few hours after addition to media [12,32]. There were no changes in the biophysical characteristics of the hTERT-HUVEC-derived EV, but the total amount was reduced by comparison to control (Fig 12 and S18 Fig). To test the hypothesis that KSHV-EV induce some, but not all phenotypes as KSHV infection, morphological differences in the recipient hTERT-HUVEC were evaluated. HTERT-HUVEC were exposed to KSHV-EV, mock, or BJAB-derived, control EV for four consecutive days. Chronically infected HUVECs served as a control. KSHV-EV treatment did not alter Tubulin (Fig 13A) or Actin (Fig 13B) organization. KSHV LANA was present in infected, but not EV-treated cells (Fig 13C). By contrast, the proliferation marker Ki-67 was dramatically induced by KSHV-EV, but not control EV. Ki-67 positivity was similar to KSHV infected cells (Fig 13D and 13E). H&E stain revealed a greater cell density in KSHV-EV compared to mock or BJAB-EV treated cells (S20 Fig). KSHVinfected hTERT cells exhibited greatly increased cell size, as previously described and consistent with mTOR/S6K activation [33]. Overall these results mirror the dramatic dysregulation of infected as well as uninfected EC in KS lesions, where the normal vasculature and extracellular environment is essentially destroyed and slit-like empty spaces develop. This analysis demonstrated that KSHV-EV inducing a long-lasting reprogramming of EC, which results in transcription signatures and pathway alterations consistent with the phenotypic changes observed in KS lesions.

Discussion
KS is an incredibly angiogenic cancer, second only to hemangioma [3]. It is driven by KSHVinfected EC and defined by a unique molecular mechanism that manifests itself in aberrant EC behavior. Many studies have focused on the cell autonomous roles of KSHV [6,28,29,[33][34][35][36][37][38]. In addition, studies by Mesri and others (reviewed in [39]) have established that the KS phenotype depends to a large degree on paracrine signaling mechanisms to reprogram neighboring uninfected EC. This report establishes that KSHV-EV mediate some of the paracrine phenotypes of KS (summarized in S3 Table).
signaling pathway inhibits EV-dependent cell migration. hTERT-HUVECs seeded in a specialized xCelligence CIM plate in the presence of DMSO or AZD6244. 6 hours later, bottom well media was replaced with media containing HD EV, KSHV-EV, or primary PEL EV. (F) Quantitation of cell migration in (E) at 12, 24, and 36 hours post exposure to HD EV, KSHV-EV, or primary PEL EV in the presence of DMSO (black) or AZD6244 (red) (see also S16 Fig). https://doi.org/10.1371/journal.ppat.1007536.g008 Secreted vesicles from an AIDS-lymphoma reprogram cell physiology EV mediate a large variety of phenotypes in the immediate microenvironment as well as at distant sites. EV have established roles in cell differentiation, angiogenesis, cell migration as well as metastasis [40][41][42][43][44]. We had shown earlier that KSHV miRNAs are present in systemically circulating EV in KS patients, PEL fluid as well as in transgenic mice, which carry the KSHV miRNAs, but are not competent to make virions [7]. PEL are of post-GC lineage lymphoma, approaching almost plasmablastic stage. They grow i.p. (in body cavities), unlike Burkitt lymphoma, which are also post-GC lymphoma, but pre-plasmablastic and grow as a solid mass in lymph nodes, not as an effusion. This may explain the prominence that EV have in the biology of PEL and KSHV vis-a-vis other tumors.
Crucial to the study of EV is a well-validated purification pipeline [22,45]. In the context of virus infections, it is important to exclude viral particles, which tend to co-purify with EV in ultracentrifugation, crowding-agent, and size exclusion chromatography approaches. Hence, we added affinity purification using antibody-coated beads directed against CD63 or other tetraspanins as the final purification step. This step depletes virions to below the limit of detection [7,12], as it positively retains EV on a column rather than collecting a precipitate. It also reduces the complexity of the EV populations [10,46] to only those EV that are of narrow size, tetraspanin-positive, and inhibited by Annexin-V. Adding an RNAse and DNAse step as well as size exclusion chromatography eliminated contaminating free RNA and DNA and selected against vesicles that are released non-specifically by dying cells. This has not always been done and may explain reports of EV preparations that induce heavy DNA and RNA-dependent immune stimulation in recipient cells. We believe the final product of our purification pipeline represents biologically-relevant KSHV-EV at or slightly below physiological concentration.
It has been a matter of debate as to whether EV induce or suppress the innate immune response. This phenotype depends largely on the specific virus and target cell. Professional immune cells, such as dendritic cells and macrophages are known to receive and transmit proinflammatory signals through EV [47][48][49]. RNA viruses induce a dramatic innate response and large amounts of secondary messengers, such as IFN-β or cGAMP [48]. DNA viruses, such as herpesviruses may also transmit pro-inflammatory signals through EV, which can be sensed by professional antigen presenting cells [47,50]. While we cannot exclude that lytically replicating cells or professional antigen presenting cells infected with KSHV behave differently, these experiments demonstrate that EC, the primary target of KSHV infection, do not become activated by EV from KSHV-infected lymphoma cells or by EV from primary PEL fluid. They may become activated by cytokines or small soluble molecules, though a nonactivated phenotype of uninfected cells would also be consistent with the biology of KSHV as most primary KSHV infections are clinically asymptomatic and not associated with mononucleosis-like symptoms or autoimmunity as seen with EBV or human cytomegalovirus infection.
These results support a model whereby cellular proteins, cellular and viral miRNAs that are carried in KSHV-EV modulate long-term reprogramming of EC in the immediate Secreted vesicles from an AIDS-lymphoma reprogram cell physiology    Whereas prior studies focused on the immediate effects of a single bolus of EV, these experiments were designed to mimic continuous KSHV-EV exposure as seen in KS and PEL patients or patients with a high latent virus burden. The concentration of EV in normal blood is~10 10 -10 11 /mL [23], which is 6 orders of magnitude higher than the median concentration of KSHV in the blood of symptomatic, untreated AIDS-KS patients [51]. We added 10 10 /mL to 10 6 cells (MOI = 10,000). Based on our prior work and Fig 3, we assume that all EV are endocytic-competent and all KSHV-EV carry the KSHV miRNAs. EV adsorption plateaus within hours of exposure [32]. Using a MOI bolus of 4000 vs. 1000 is unlikely to result in a qualitatively different response after 4 days. The novelty of this experimental design is mimicking chronic exposure, as is the case in the KS microenvironment or for any endothelial cell lining the blood vessels. Whereas PEL and KS, in the context of uncontrolled progression to AIDS, are rapidly fatal, de novo KSHV infection per se is not. HIV-negative endemic, pediatric and classic KS have rapid as well as smoldering clinical progression [52]. KSHV-associated multicentric Castleman's disease has a waxing and waning presentation, closely associated with IL-6 levels [53]. IL-6, IL-10, VEGF, PDGF and other inflammatory cytokines are elevated in PEL, KS and MCD, and agents, such as pomalidomide, rapamycin and tocilizumab which modulate their levels, modulate disease [54][55][56]. KSHV-EV consistently induced human IL-6 in uninfected cells. These experiments show that in addition to cytokines EV also transmit pro-growth signals and can reprogram EC.
KSHV-EV induced a much more long-lasting phenotype than acute phase cytokines, which mimics differentiation and trans-differentiation. Whereas it was difficult to identify a single master regulator of this trans-differentiation phenotype, network analysis showed significant changes of transcriptional modules that regulate extracellular matrix remodeling, translation and exosome biogenesis. By comparison, IFN-β and NF-κB transcriptional networks were unaffected. KSHV-EV signaled through MAPK/ERK, which is consistent with MAPK/ERK's role in modulating EC motility and vascular behavior (reviewed in [57]). Our observations are consistent with a recent study by Yogev et al. [58], who showed that KSHV-EV (derived from infected EC) induce metabolic remodeling of nearby uninfected cells. This represents perhaps the initiating step of trans-differentiation. Afterwards, continued KSHV-EV exposure resulted in continued reprogramming as has been described for KSHV-infected and KSHV-miRNA transfected EC [4][5][6]34] and these experiments verified that most KSHV-miRNAs are present in KSHV-EV. Reprogramming here is used to defined an altered state of gene transcription and cell lineage, such as published by Hansen et al. [6], upon transfection of the KSHV miR-NAs into EC, or upon infection of EC with KSHV [4,28,29,[59][60][61]. At this point we do not know if this reprogramming will persist after EV exposure has subsided and if not, how quickly the cells return to their normal state. Clinically, KS lesions and KS-associated edema regress as KSHV is cleared by immune restoration upon cART or lowering of immunosuppressive drugs in the context of transplant KS. Hence, we speculate that that the KSHV-EV induced phenotype likewise is transient. This would be in contrast to permanent lineage reprogramming, which is most commonly associated with epigenetic changes to the cellular DNA. Evidence for KSHV-infection induced chromatin remodeling has been published [62][63][64][65]. If in addition to transcriptional reprogramming, the viral miRNAs (or other molecules) that are (+) served as a control. (D) hTERT-HUVECs were treated with Mock, Control (BJAB)-EV, or KSHV-EV for 4 days and stained for the cell proliferation marker Ki-67 to monitor any changes in intracellular cytoskeleton. Mock treated hTERT-HUVEC KSHV (+) served as a control. (E) Percent positive Ki-67 cells were plotted and statistical analyses was done using ANOVA followed by Ttests (p�0.05, n = 6; statistical groupings indicated with a letter) (See also S20 Fig). https://doi.org/10.1371/journal.ppat.1007536.g013 Secreted vesicles from an AIDS-lymphoma reprogram cell physiology contained in KSHV-EV also alter chromatin accessibility stably and irreversibly is a fascinating hypothesis and the subject of future studies.
The gradual and long-lasting reprogramming of transcriptional networks is consistent with the mechanism of action for miRNAs, which have their most physiological impact in development rather than acute signaling. EBV and KSHV express miRNAs and in infected cells these miRNAs account for as much as 50% of the miRNA pool. KSHV and EBV incorporate the viral miRNAs into EV [7,[66][67][68]. Both viruses substantially modulate the protein composition of EV [69]. In addition, EBV incorporates the LMP-1 oncogene into EV [70,71], whereas no KSHV proteins were detected in EV thus far. This phenotype is consistent with the idea that EV are pivotal for establishing local tissue homeostasis and provides a molecular mechanism for it. Further studies are needed, but for the first time there now exists a highly reproducible, physiologically relevant experimental design to study long-term EV-EC interactions.
In conclusion, our findings point toward a novel means of cellular reprogramming by viruses. They pinpoint novel, actionable pathways for intervention and biomarker development. KSHV is able to infect EC, but the larger importance of EV stems from the fact that these vesicles can carry viral components to distant locations and transfer them into cells that the virus cannot enter. This may explain some of the phenotypes that viruses, including HIV, have on uninfected cells and it may explain why clinical sequalae persist long after the virus has been cleared or entered molecular latency.

EV isolation
Total EV were isolated using approximately 400 mL of cell culture supernatant. Cells were pelleted at 4˚C at 800x g for 10 minutes. Supernatant was then passed through a 0.22 μm Nalgene Rapid Flow Filter (Thermo Fisher). Filtered supernatants were aliquoted into individual 50 mL conical tubes (Corning). EV were precipitated with 40 mg/mL PEG-8000 and incubation at 4˚C for >8 hours. Precipitates were then spun down at 4˚C at 1,200x g for 60 minutes. Pellets were resuspended in 500 μL of ice-cold 1X PBS (Gibco). Removal of non-associated molecules were done by (i) ultracentrifugation or (ii) column chromatography. (i) For ultracentrifugation, the volume was increased to~4 mL with 1X PBS and centrifuged at 4˚C at 120,000x g for 60 minutes using a Beckmann SW32 rotor. The pellet was then resuspended in 4 mL of fresh 1X PBS and centrifuged again. A total number of three washes was done. The final pellet was resuspended in 100 μL of fresh, ice-cold 1X PBS. (ii) For column chromatography, GE Sephadex G-200 was equilibrated with ice-cold 1X PBS for a total of 4 compacted bead volumes (4 mL). The resuspended EV were added to the equilibrated column and allowed to flow through the column by gravity. EV were collected in the first 1 mL of fresh, cold 1X PBS. EV were also isolated from isolated from 50 mL plasma from health donors or 10 mL PEL. Briefly, blood was processed and erythrocytes, leukocytes, platelets, and plasma were separated using Ficoll reagent (GE 17-1440-02) as above.

EV purification using CD63, CD9, and CD81 Dynabeads
Samples were enriched for CD63, CD9, and CD81 positive EV using magnetic beads (Thermo- Fisher 1060D, 10620D, and 10622D, respectively). Briefly, the total EV isolated as above were added to 80 μL of equilibrated, antibody-coated magnetic beads. Non-specific IgG-coated beads were used as a control. EV were bound to beads overnight at 4˚C and beads were washed 3X with 1X PBS. EV were eluted in 100 μL of elution buffer (Invitrogen) or 0.2 M Glycine pH = 2.0 for further analysis. Cell were authenticated by targeted amplification of STR typing loci using Ion Torrent Precision ID GlobalFiler NGS STR Panel and compared against the STR database of the German Collection of Microorganisms and Cell Cultures GmbH.

Fluorescence and Immunofluorescence microscopy
hTERT-HUVECs were grown on a cover slip in 6 well plates in a total volume of 3 mL and treated with labeled 10 9 EV/mL for the indicated time period at 37˚C. Cells were then rinsed with PBS and fixed in 4% paraformaldehyde for 10 minutes at RT, washed 3 times with PBS and the cells permeabilized using 0.5% Triton X-100 in PBS for 10 minutes and washed 3x with PBS. For indirect immunofluorescence, coverslips were blocked in a solution of 10% goat serum (Vector Labs) in PBS with 0.2% Triton X-100 and incubated with primary antibodies: anti-IRF3 antibody (Cell Signaling, #4962, 1:100 dilution) anti-P-NF-κB p65 (S536) (clone 93H1, Cell Signaling, 1:100 dilution). Coverslips were washed three times with PBS-0.2% Triton with 2% BSA and incubated with FITC-conjugated anti-rabbit secondary antibody (#FI-1000, Vector Labs Inc. 1:500 dilution). Cells were washed three times with PBS-0.2% Triton X-100 with 2% BSA and stained with 0.2 μg/mL DAPI (Sigma) prior to mounting in VectaShield (Vector Labs). Cells were imaged on a Leica DM4000B microscope with a Q-Imaging Retiga-2000RV camera and HCX-PL-APO 506187 lens at 63x magnification. De-convoluted images (Simple PCI 6 software Metamorph v 7.8.12.0, 10 iterations RB, GB or RGB) were then opened in Imaris V 9.2.0. and background subtraction of all channels was done using recommended settings of 400 um filter width. Localizations of EV-delivered Dil and ExoGreen were done using the "Add Spots" command using spots of different sizes depending on fluorescence intensity. Regions of spot calling were standardized to linear detection ranges using absolute intensity. For nuclei staining, the "Add New Surfaces" command was used.

WNV infection and Poly I:C treatment
As a positive control for IRF3 activation, hTERT-HUVECs were infected with West Nile Virus NY99 (WNV) at a MOI of 5 (observed after 36 hours) or Poly I:C at 5 μg/mL (observed after 12 hours).

Western Blotting and Silver staining
Pellets (10 10 EV or 10 6 cells) were lysed in 100 μL NP-40 lysis buffer and run on an 8% SDS-PAGE gel, transferred to a nitrocellulose membrane (Hybond) and blocked in 5% dry milk in TBS overnight at 4˚C. Antibodies are listed in S1 Table. For detection of tetraspanins, non-reducing conditions were used. To visualize total protein by silver stain, we used the Pierce Silver Stain Kit (ThermoFisher) after which bands were excised and analyzed by mass spectrometry at the UT Southwester core (https://www.utsouthwestern.edu/research/corefacilities/proteomics-core.html).

Cell proliferation and migration
(i) Scratch assays were performed as previously described [7]. Briefly, hTERT-HUVECs were grown in a 24-well plate (Corning) prior to treatment with EV. The wound was initiated using a standard 200 μL pipette tip and the cells were then washed and replaced with fresh media containing one of the following as a chemo-attractant: 10% FBS, 10 ng/mL VEGF (Peprotech), 1 U/mL hIL-6 (Peprotech), 10 ng/mL PDGF-β (Peprotech) or 10 ng/mL SDF-1α (CXCL12) (R&D Systems). The culture was monitored over time. Images were obtained using a Leica DMIL microscope with a HI Plan 10x/0.25 PHI objective and QImaging camera (Cooled color, RTV 10 bit) paired with QCapture imaging software 3.0. Images are shown at 100x magnification and were analyzed using ImageJ software to calculate the percent wound closure at a given time point. (ii) Cell proliferation and migration was analyzed using the xCelligence RTCA DP instrument as previously described [7]. Briefly, hTERT-HUVECs were treated with EV for 24 hours and proliferation measured by conductance. For migration, both sides of the xCelligence CIM Plate 16 (Acea Biosciences) plate membrane were coated with 20 μg/mL fibronectin prior to assembly and media containing FBS or a specified cytokine was placed in the lower chamber as the chemo-attractant. Cells were plated at 15,000 cells per well of the upper chamber. Reads were taken every 2 minutes for a period of 12 hours. The cell index reflects the degree of cellular migration towards the specified chemo-attractant.

Gene expression profiling
Total RNA was isolated using TRI reagent (Molecular Research Center) as previously described (https://www.med.unc.edu/vironomics/services/protocols/), treated with Turbo DNA-free kit (Ambion, Life Technologies) and 100 ng of DNA-free RNA, as determined by Nanodrop, was used as input for High Capacity cDNA synthesis kit (Applied Biosystems, Life Technologies). Custom NF-κB and endothelial lineage real-time qPCR arrays were used previously published [72].

Electron microscopy
EV were adsorbed on a glow-charged carbon coated 400-mesh copper grids for 2 minutes and then stained with 2% (weight/volume) uranyl acetate in water. Transmission electron microscopy (TEM) images were taken using a Philips CM12 electron microscope at 80 kilovolts. Images were captured on a Gatan Orius camera (2000 x 2000 pixels) using the Digital Micrograph software (Gatan, Pleasanton, CA). Images were then cropped in Adobe Photoshop.

Statistics and Bioinformatics
(i) For continuous, variable pairwise T-tests were performed to determine statistical significance for n � 3 biological replicates. (ii) For comparison of mass spectrometry data, a hypergeometric test was used. (iii) For analysis of RNAseq data we used a custom pipeline. The decision to use our particular analysis is discussed at length at https://support.bioconductor. org/p/62684/. In brief, STAR-Aligned BAM files representing table of counts for each samples were processed using DESeq and other Bioconductor packages: https://bioconductor.org/ packages/devel/bioc/vignettes/GenomicAlignments/inst/doc/summarizeOverlaps.pdf.