Highly restrictive and directional penetration of the blood cerebral spinal fluid barrier by JCPyV.

The human polyomavirus JCPyV is an opportunistic pathogen that infects greater than 60% of the world's population. The virus establishes a persistent and asymptomatic infection in the urogenital system but can cause a fatal demyelinating disease in immunosuppressed or immunomodulated patients following invasion of the CNS. The mechanisms responsible for JCPyV invasion into CNS tissues are not known but direct invasion from the blood to the cerebral spinal fluid via the choroid plexus has been hypothesized. To study the potential of the choroid plexus as a site of neuroinvasion, we used an adult human choroid plexus epithelial cell line to model the blood-cerebrospinal fluid (B-CSF) barrier in a transwell system. We found that these cells formed a highly restrictive barrier to virus penetration either as free virus or as virus associated with extracellular vesicles (EVJC+). The restriction was not absolute and small amounts of virus or EVJC+ penetrated and were able to establish foci of infection in primary astrocytes. Disruption of the barrier with capsaicin did not increase virus or EVJC+ penetration leading us to hypothesize that virus and EVJC+ were highly cell-associated and crossed the barrier by an active process. An inhibitor of macropinocytosis increased virus penetration from the basolateral (blood side) to the apical side (CSF side). In contrast, inhibitors of clathrin and raft dependent transcytosis reduced virus transport from the basolateral to the apical side of the barrier. None of the drugs inhibited apical to basolateral transport suggesting directionality. Pretreatment with cyclosporin A, an inhibitor of P-gp, MRP2 and BCRP multidrug resistance transporters, restored viral penetration in cells treated with raft and clathrin dependent transcytosis inhibitors. Because choroid plexus epithelial cells are known to be susceptible to JCPyV infection both in vitro and in vivo we also examined the release of infectious virus from the barrier. We found that virus was preferentially released from the cells into the apical (CSF) chamber. These data show clearly that there are two mechanisms of penetration, direct transcytosis which is capable of seeding the CSF with small amounts of virus, and infection followed by directional release of infectious virions into the CSF compartment.

Picornavirus security proteins promote the release of extracellular vesicle enclosed viruses via the modulation of host kinases.

The discovery that extracellular vesicles (EVs) serve as carriers of virus particles calls for a reevaluation of the release strategies of non-enveloped viruses. Little is currently known about the molecular mechanisms that determine the release and composition of EVs produced by virus-infected cells, as well as conservation of these mechanisms among viruses. We previously described an important role for the Leader protein of the picornavirus encephalomyocarditis virus (EMCV) in the induction of virus-carrying EV subsets with distinct molecular and physical properties. EMCV L acts as a 'viral security protein' by suppressing host antiviral stress and type-I interferon (IFN) responses. Here, we tested the ability of functionally related picornavirus proteins of Theilers murine encephalitis virus (TMEV L), Saffold virus (SAFV L), and coxsackievirus B3 (CVB3 2Apro), to rescue EV and EV-enclosed virus release when introduced in Leader-deficient EMCV. We show that all viral security proteins tested were able to promote virus packaging in EVs, but that only the expression of EMCV L and CVB3 2Apro increased overall EV production. We provide evidence that one of the main antiviral pathways counteracted by this class of picornaviral proteins, i.e. the inhibition of PKR-mediated stress responses, affected EV and EV-enclosed virus release during infection. Moreover, we show that the enhanced capacity of the viral proteins EMCV L and CVB3 2Apro to promote EV-enclosed virus release is linked to their ability to simultaneously promote the activation of the stress kinase P38 MAPK. Taken together, we demonstrate that cellular stress pathways involving the kinases PKR and P38 are modulated by the activity of non-structural viral proteins to increase the release EV-enclosed viruses during picornavirus infections. These data shed new light on the molecular regulation of EV production in response to virus infection.

Exploiting hosts and vectors: viral strategies for facilitating transmission.

Viruses have developed various strategies to ensure their survival and transmission. One intriguing strategy involves manipulating the behavior of infected arthropod vectors and hosts. Through intricate interactions, viruses can modify vector behavior, aiding in crossing barriers and improving transmission to new hosts. This manipulation may include altering vector feeding preferences, thus promoting virus transmission to susceptible individuals. In addition, viruses employ diverse dissemination methods, including cell-to-cell and intercellular transmission via extracellular vesicles. These strategies allow viruses to establish themselves in favorable environments, optimize replication, and increase the likelihood of spreading to other individuals. Understanding these complex viral strategies offers valuable insights into their biology, transmission dynamics, and potential interventions for controlling infections. Unraveling interactions between viruses, hosts, and vectors enables the development of targeted approaches to effectively mitigate viral diseases and prevent transmission.

Comprehensive proteomic analysis of HCoV-OC43 virions and virus-modulated extracellular vesicles.

Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles. IMPORTANCE: In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.

Suppression of the host antiviral response by non-infectious varicella zoster virus extracellular vesicles.

Varicella zoster virus (VZV) reactivates from ganglionic sensory neurons to produce herpes zoster (shingles) in a unilateral dermatomal distribution, typically in the thoracic region. Reactivation not only heightens the risk of stroke and other neurological complications but also increases susceptibility to co-infections with various viral and bacterial pathogens at sites distant from the original infection. The mechanism by which VZV results in complications remote from the initial foci remains unclear. Small extracellular vesicles (sEVs) are membranous signaling structures that can deliver proteins and nucleic acids to modify the function of distal cells and tissues during normal physiological conditions. Although viruses have been documented to exploit the sEV machinery to propagate infection, the role of non-infectious sEVs released from VZV-infected neurons in viral spread and disease has not been studied. Using multi-omic approaches, we characterized the content of sEVs released from VZV-infected human sensory neurons (VZV sEVs). One viral protein was detected (immediate-early 62), as well as numerous immunosuppressive and vascular disease-associated host proteins and miRNAs that were absent in sEVs from uninfected neurons. Notably, VZV sEVs are non-infectious yet transcriptionally altered primary human cells, suppressing the antiviral type 1 interferon response and promoting neuroinvasion of a secondary pathogen in vivo. These results challenge our understanding of VZV infection, proposing that the virus may contribute to distant pathologies through non-infectious sEVs beyond the primary infection site. Furthermore, this study provides a previously undescribed immune-evasion mechanism induced by VZV that highlights the significance of non-infectious sEVs in early VZV pathogenesis. IMPORTANCE: Varicella zoster virus (VZV) is a ubiquitous human virus that predominantly spreads by direct cell-cell contact and requires efficient and immediate host immune evasion strategies to spread. The mechanisms of immune evasion prior to virion entry have not been fully elucidated and represent a critical gap in our complete understanding of VZV pathogenesis. This study describes a previously unreported antiviral evasion strategy employed by VZV through the exploitation of the infected host cell's small extracellular vesicle (sEV) machinery. These findings suggest that non-infectious VZV sEVs could travel throughout the body, affecting cells remote from the site of infection and challenging the current understanding of VZV clinical disease, which has focused on local effects and direct infection. The significance of these sEVs in early VZV pathogenesis highlights the importance of further investigating their role in viral spread and secondary disease development to reduce systemic complications following VZV infections.

SARS-CoV-2 envelope protein-derived extracellular vesicles act as potential media for viral spillover.

Extracellular vesicles (EVs) are shown to be a novel viral transmission model capable of increasing a virus's tropism. According to our earlier research, cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or transfected with envelope protein plasmids generate a novel type of EVs that are micrometer-sized and able to encase virus particles. Here, we showed the capacity of these EVs to invade various animals both in vitro and in vivo independent of the angiotensin-converting enzyme 2 receptor. First, via macropinocytosis, intact EVs produced from Vero E6 (monkey) cells were able to enter cells from a variety of animals, including cats, dogs, bats, hamsters, and minks, and vice versa. Second, when given to zebrafish with cutaneous wounds, the EVs showed favorable stability in aqueous environments and entered the fish. Moreover, infection of wild-type (WT) mice with heterogeneous EVs carrying SARS-CoV-2 particles led to a strong cytokine response and a notable amount of lung damage. Conversely, free viral particles did not infect WT mice. These results highlight the variety of processes behind viral transmission and cross-species evolution by indicating that EVs may be possible vehicles for SARS-CoV-2 spillover and raising risk concerns over EVs' potential for viral gene transfer.

Extracellular vesicles in the pathogenesis of neurotropic viruses.

Neurotropic viruses, characterized by their capacity to invade the central nervous system, present a considerable challenge to public health and are responsible for a diverse range of neurological disorders. This group includes a diverse array of viruses, such as herpes simplex virus, varicella zoster virus, poliovirus, enterovirus and Japanese encephalitis virus, among others. Some of these viruses exhibit high neuroinvasiveness and neurovirulence, while others demonstrate weaker neuroinvasive and neurovirulent properties. The clinical manifestations of infections caused by neurotropic viruses can vary significantly, ranging from mild symptoms to severe life-threatening conditions. Extracellular vesicles (EVs) have garnered considerable attention due to their pivotal role in intracellular communication, which modulates the biological activity of target cells via the transport of biomolecules in both health and disease. Investigating EVs in the context of virus infection is crucial for elucidating their potential role contribution to viral pathogenesis. This is because EVs derived from virus-infected cells frequently transfer viral components to uninfected cells. Importantly, EVs released by virus-infected cells have the capacity to traverse the blood-brain barrier (BBB), thereby impacting neuronal activity and inducing neuroinflammation. In this review, we explore the roles of EVs during neurotropic virus infections in either enhancing or inhibiting viral pathogenesis. We will delve into our current comprehension of the molecular mechanisms that underpin these roles, the potential implications for the infected host, and the prospective diagnostic applications that could arise from this understanding.

Hijacking host extracellular vesicle machinery by hepatotropic viruses: current understandings and future prospects.

Recent advances in studies exploring the roles of extracellular vesicles (EVs) in viral transmission and replication have illuminated hepatotropic viruses, such as hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV), and hepatitis E (HEV). While previous investigations have uncovered these viruses' ability to exploit cellular EV pathways for replication and transmission, most have focused on the impacts of exosomal pathways. With an improved understanding of EVs, four main subtypes, including exosomes, microvesicles, large oncosomes, and apoptotic bodies, have been categorized based on size and biogenic pathways. However, there remains a noticeable gap in comprehensive reviews summarizing recent findings and outlining future perspectives for EV studies related to hepatotropic viruses. This review aims to consolidate insights into EV pathways utilized by hepatotropic viruses, offering guidance for the future research direction in this field. By comprehending the diverse range of hepatotropic virus-associated EVs and their role in cellular communication during productive viral infections, this review may offer valuable insights for targeting therapeutics and devising strategies to combat virulent hepatotropic virus infections and the associated incidence of liver cancer.

Identification of mungbean yellow mosaic India virus and susceptibility-related metabolites in the apoplast of mung bean leaves.

KEY MESSAGE: The investigation of MYMIV-infected mung bean leaf apoplast revealed viral genome presence, increased EVs secretion, and altered stress-related metabolite composition, providing comprehensive insights into plant-virus interactions. The apoplast, an extracellular space around plant cells, plays a vital role in plant-microbe interactions, influencing signaling, defense, and nutrient transport. While the involvement of apoplast and extracellular vesicles (EVs) in RNA virus infection is documented, the role of the apoplast in plant DNA viruses remains unclear. This study explores the apoplast's role in mungbean yellow mosaic India virus (MYMIV) infection. Our findings demonstrate the presence of MYMIV genomic components in apoplastic fluid, suggesting potential begomovirus cell-to-cell movement via the apoplast. Moreover, MYMIV infection induces increased EVs secretion into the apoplast. NMR-based metabolomics reveals altered metabolic profiles in both apoplast and symplast in response to MYMIV infection, highlighting key metabolites associated with stress and defense mechanisms. The data show an elevation of α- and ß-glucose in both apoplast and symplast, suggesting a shift in glucose utilization. Interestingly, this increase in glucose does not contribute to the synthesis of phenolic compounds, potentially influencing the susceptibility of mung bean to MYMIV. Fructose levels increase in the symplast, while apoplastic sucrose levels rise significantly. Symplastic aspartate levels increase, while proline exhibits elevated concentration in the apoplast and reduced concentration in the cytosol, suggesting a role in triggering a hypersensitive response. These findings underscore the critical role of the apoplast in begomovirus infection, providing insights for targeted viral disease management strategies.

Shedding Light on Viral Shedding: Novel Insights into Nuclear Assembly, Cytoplasmic Transformation and Extracellular Vesicle Release of the BK Virus.

Despite the high prevalence of BK polyomavirus (BKPyV) and the associated risk for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant (KTX) recipients, many details on viral processes such as replication, maturation, assembly and virion release from host cells have not been fully elucidated. VP1 is a polyomavirus-specific protein that is expressed in the late phase of its replicative cycle with important functions in virion assembly and infectious particle release. This study investigated the localization and time-dependent changes in the distribution of VP1-positive viral particles and their association within the spectrum of differing cell morphologies that are observed in the urine of KTX patients upon active BKPyV infection. We found highly differing recognition patterns of two anti-VP1 antibodies with respect to intracellular and extracellular VP1 localization, pointing towards independent binding sites that were seemingly associated with differing stages of virion maturation. Cells originating from single clones were stably cultured out of the urine sediment of KTX recipients with suspected BKPyVAN. The cell morphology, polyploidy, virus replication and protein production were investigated by confocal microscopy using both a monoclonal (mAb 4942) and a polyclonal rabbit anti-VP1-specific antibody (RantiVP1 Ab). Immunoblotting was performed to investigate changes in the VP1 protein. Both antibodies visualized VP1 and the mAb 4942 recognized VP1 in cytoplasmic vesicles exhibiting idiomorphic sizes when released from the cells. In contrast, the polyclonal antibody detected VP1 within the nucleus and in cytoplasm in colocalization with the endoplasmic reticulum marker CNX. At the nuclear rim, VP1 was recognized by both antibodies. Immunoblotting revealed two smaller versions of VP1 in urinary decoy cell extracts, potentially from different translation start sites as evaluated by in silico analysis. Oxford Nanopore sequencing showed integration of BKPyV DNA in chromosomes 3, 4 and 7 in one of the five tested primary cell lines which produced high viral copies throughout four passages before transcending into senescence. The different staining with two VP1-specific antibodies emphasizes the modification of VP1 during the process of virus maturation and cellular exit. The integration of BKPyV into the human genome leads to high virus production; however, this alone does not transform the cell line into a permanently cycling and indefinitely replicating one.

The Association of Human Astrovirus with Extracellular Vesicles Facilitates Cell Infection and Protects the Virus from Neutralizing Antibodies.

Viral gastroenteritis has a global distribution and represents a high risk for vulnerable population and children under 5 years due to acute diarrhea, fever and dehydration. Human astroviruses (HAstV) have been identified as the third most important cause of viral gastroenteritis in pediatric and immunocompromised patients. Furthermore, HAstV has been reported in biopsies taken from patients with encephalitis, meningitis and acute respiratory infection, yet it is not clear how the virus reaches these organs. In this work we have tested the possibility that the released astrovirus particles could be associated with extracellular vesicles. Comparison between vesicles purified from HAstV Yuc8 infected and mock-infected cells showed that infection enhances production of vesicles larger than 150 nm. These vesicles contain CD63 and Alix, two markers of vesicular structures. Almost 70% of the extracellular virus present in clarified supernatant at 18 h postinfection was found associated with vesicular membranes, and this association facilitates cell infection in the absence of trypsin activation and protects virions from neutralizing antibodies. Our findings suggest a new pathway for HAstV spread and might represent an explanation for the extra-intestinal presence of some astrovirus strains. IMPORTANCE Astroviruses are an important cause of diarrhea in vulnerable population, particularly children; recently some reports have found these viruses in extra-intestinal organs, including the central nervous system, causing unexpected clinical disease. In this work, we found that human astrovirus strain Yuc8 associates with extracellular vesicles, possibly during or after their cell egress. The association with vesicles doubled astrovirus infectivity in less susceptible cells and rendered virus particles insensitive to neutralization by antibodies. These data suggest that extracellular vesicles could represent a novel pathway for astrovirus to disseminate outside the gastrointestinal tract.

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.

JC Virus infected choroid plexus epithelial cells produce extracellular vesicles that infect glial cells independently of the virus attachment receptor.

The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy (PML) in immunosuppressed and immunomodulated patients. Initial infection with JCPyV is common and the virus establishes a long-term persistent infection in the urogenital system of 50-70% of the human population worldwide. A major gap in the field is that we do not know how the virus traffics from the periphery to the brain to cause disease. Our recent discovery that human choroid plexus epithelial cells are fully susceptible to virus infection together with reports of JCPyV infection of choroid plexus in vivo has led us to hypothesize that the choroid plexus plays a fundamental role in this process. The choroid plexus is known to relay information between the blood and the brain by the release of extracellular vesicles. This is particularly important because human macroglia (oligodendrocytes and astrocytes), the major targets of virus infection in the central nervous system (CNS), do not express the known attachment receptors for the virus and do not bind virus in human tissue sections. In this report we show that JCPyV infected choroid plexus epithelial cells produce extracellular vesicles that contain JCPyV and readily transmit the infection to human glial cells. Transmission of the virus by extracellular vesicles is independent of the known virus attachment receptors and is not neutralized by antisera directed at the virus. We also show that extracellular vesicles containing virus are taken into target glial cells by both clathrin dependent endocytosis and macropinocytosis. Our data support the hypothesis that the choroid plexus plays a fundamental role in the dissemination of virus to brain parenchyma.

Local oncolytic adenovirotherapy produces an abscopal effect via tumor-derived extracellular vesicles.

Extracellular vesicles (EVs) play important roles in various intercellular communication processes. The abscopal effect is an interesting phenomenon in cancer treatment, in which immune activation is generally considered a main factor. We previously developed a telomerase-specific oncolytic adenovirus, Telomelysin (OBP-301), and occasionally observed therapeutic effects on distal tumors after local treatment in immunodeficient mice. In this study, we hypothesized that EVs may be involved in the abscopal effect of OBP-301. EVs isolated from the supernatant of HCT116 human colon carcinoma cells treated with OBP-301 were confirmed to contain OBP-301, and they showed cytotoxic activity (apoptosis and autophagy) similar to OBP-301. In bilateral subcutaneous HCT116 and CT26 tumor models, intratumoral administration of OBP-301 produced potent antitumor effects on tumors that were not directly treated with OBP-301, involving direct mediation by tumor-derived EVs containing OBP-301. This indicates that immune activation is not the main factor in this abscopal effect. Moreover, tumor-derived EVs exhibited high tumor tropism in orthotopic HCT116 rectal tumors, in which adenovirus E1A and adenovirus type 5 proteins were observed in metastatic liver tumors after localized rectal tumor treatment. In conclusion, local treatment with OBP-301 has the potential to produce abscopal effects via tumor-derived EVs.

Therapeutic Application of Extracellular Vesicles-Capsulated Adeno-Associated Virus Vector via nSMase2/Smpd3, Satellite, and Immune Cells in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is caused by loss-of-function mutations in the dystrophin gene on chromosome Xp21. Disruption of the dystrophin-glycoprotein complex (DGC) on the cell membrane causes cytosolic Ca2+ influx, resulting in protease activation, mitochondrial dysfunction, and progressive myofiber degeneration, leading to muscle wasting and fragility. In addition to the function of dystrophin in the structural integrity of myofibers, a novel function of asymmetric cell division in muscular stem cells (satellite cells) has been reported. Therefore, it has been suggested that myofiber instability may not be the only cause of dystrophic degeneration, but rather that the phenotype might be caused by multiple factors, including stem cell and myofiber functions. Furthermore, it has been focused functional regulation of satellite cells by intracellular communication of extracellular vesicles (EVs) in DMD pathology. Recently, a novel molecular mechanism of DMD pathogenesis-circulating RNA molecules-has been revealed through the study of target pathways modulated by the Neutral sphingomyelinase2/Neutral sphingomyelinase3 (nSMase2/Smpd3) protein. In addition, adeno-associated virus (AAV) has been clinically applied for DMD therapy owing to the safety and long-term expression of transduction genes. Furthermore, the EV-capsulated AAV vector (EV-AAV) has been shown to be a useful tool for the intervention of DMD, because of the high efficacy of the transgene and avoidance of neutralizing antibodies. Thus, we review application of AAV and EV-AAV vectors for DMD as novel therapeutic strategy.

Extracellular Vesicle TGF-ß1 Is Linked to Cardiopulmonary Dysfunction in Human Immunodeficiency Virus.

Extracellular vesicles (EVs) have emerged as important mediators in cell-cell communication; however, their relevance in pulmonary hypertension (PH) secondary to human immunodeficiency virus (HIV) infection is yet to be explored. Considering that circulating monocytes are the source of the increased number of perivascular macrophages surrounding the remodeled vessels in PH, this study aimed to identify the role of circulating small EVs and EVs released by HIV-infected human monocyte-derived macrophages in the development of PH. We report significantly higher numbers of plasma-derived EVs carrying higher levels of TGF-ß1 (transforming growth factor-ß1) in HIV-positive individuals with PH compared with individuals without PH. Importantly, levels of these TGF-ß1-loaded, plasma-derived EVs correlated with pulmonary arterial systolic pressures and CD4 counts but did not correlate with the Dl CO or viral load. Correspondingly, enhanced TGF-ß1-dependent pulmonary endothelial injury and smooth muscle hyperplasia were observed. HIV-1 infection of monocyte-derived macrophages in the presence of cocaine resulted in an increased number of TGF-ß1-high EVs, and intravenous injection of these EVs in rats led to increased right ventricle systolic pressure accompanied by myocardial injury and increased levels of serum ET-1 (endothelin-1), TNF-α, and cardiac troponin-I. Conversely, pretreatment of rats with TGF-ß receptor 1 inhibitor prevented these EV-mediated changes. Findings define the ability of macrophage-derived small EVs to cause pulmonary vascular modeling and PH via modulation of TGF-ß signaling and suggest clinical implications of circulating TGF-ß-high EVs as a potential biomarker of HIV-associated PH.

Extracellular vesicles secreted by HBV-infected cells modulate HBV persistence in hydrodynamic HBV transfection mouse model.

Hepatitis B, a viral infection that affects the liver, is thought to affect over 257 million people worldwide, and long-term infection can lead to life-threatening issues such as cirrhosis or liver cancer. Chronic hepatitis B develops by the interaction between hepatitis B virus (HBV) and host immune response. However, questions of how HBV-infected cells thwart immune system defenses remain unanswered. Extracellular vesicles (EVs) are used for cellular communication, carrying cargoes such as RNAs, proteins, and lipids and delivering them intracellularly after being endocytosed by target cells. HBV-infected liver cells secrete several types of EVs into body fluids such as complete and incomplete virions, and exosomes. We previously demonstrated that monocytes that incorporated EVs moved to immunoregulatory phenotypes via up-regulation of PD-L1, an immunocheckpoint molecule, and down-regulation of CD69, a leukocyte activation molecule. In this study, we transfected mice with HBV using hydrodynamic injection and studied the effects of EVs secreted by HBV-infected liver cells. EVs secreted from cells with HBV replication strongly suppressed the immune response, inhibiting the eradication of HBV-replicating cells in the mice transfected with HBV. EVs were systemically incorporated in multiple organs, including liver, bone marrow (BM), and intestine. Intriguingly, the BM cells that incorporated EVs acquired intestinal tropism and the dendritic cell populations in the intestine increased. These findings suggest that the EVs secreted by HBV-infected liver cells exert immunosuppressive functions, and that an association between the liver, bone marrow, and intestinal tract exists through EVs secreted from HBV-infected cells.

Emerging roles of extracellular vesicles in COVID-19, a double-edged sword?

The sudden outbreak of SARS-CoV-2-infected disease (COVID-19), initiated from Wuhan, China, has rapidly grown into a global pandemic. Emerging evidence has implicated extracellular vesicles (EVs), a key intercellular communicator, in the pathogenesis and treatment of COVID-19. In the pathogenesis of COVID-19, cells that express ACE2 and CD9 can transfer these viral receptors to other cells via EVs, making recipient cells more susceptible for SARS-CoV-2 infection. Once infected, cells release EVs packaged with viral particles that further facilitate viral spreading and immune evasion, aggravating COVID-19 and its complications. In contrast, EVs derived from stem cells, especially mesenchymal stromal/stem cells, alleviate severe inflammation (cytokine storm) and repair damaged lung cells in COVID-19 by delivery of anti-inflammatory molecules. These therapeutic beneficial EVs can also be engineered into drug delivery platforms or vaccines to fight against COVID-19. Therefore, EVs from diverse sources exhibit distinct effects in regulating viral infection, immune response, and tissue damage/repair, functioning as a double-edged sword in COVID-19. Here, we summarize the recent progress in understanding the pathological roles of EVs in COVID-19. A comprehensive discussion of the therapeutic effects/potentials of EVs is also provided.

Extracellular vesicles regulate gap junction-mediated intercellular communication and HIV-1 infection of human neural progenitor cells.

Human immunodeficiency virus-1 (HIV-1) has been shown to cross the blood-brain barrier and cause HIV-associated neurocognitive disorders (HAND) through a process that may involve direct or indirect interactions with the central nervous system (CNS) cells and alterations of amyloid ß (Aß) homeostasis. The present study focused on the mechanisms of HIV-1 infecting human neural progenitor cells (hNPCs) and affecting NPC intercellular communications with human brain endothelial cells (HBMEC). Despite the lack of the CD4 receptor, hNPCs were effectively infected by HIV-1 via a mechanism involving the chemokine receptors, CXCR4 and CCR5. HIV-1 infection increased expression of connexin-43 (Cx43), phosphorylated Cx43 (pCx43), and pannexin 2 (Panx2) protein levels in hNPCs, suggesting alterations in gap-junction (GJ) and pannexin channel communication. Indeed, a functional GJ assay indicated an increase in communication between HIV-infected hNPCs and non-infected HBMEC. We next analyzed the impact of HBMEC-derived extracellular vesicles (EVs) and EVs carrying Aß (EV-Aß) on the expression of Cx43, pCx43, and Panx2 in HIV-1 infected and non-infected hNPCs. Exposure to EV-Aß resulted in significant reduction of Cx43 and pCx43 protein expression in non-infected hNPCs when compared to EV controls. Interestingly, EV-Aß treatment significantly increased levels of Cx43, pCx43, and Panx2 in HIV-1-infected hNPCs when compared to non-infected controls. These results were confirmed in a GJ functional assay and an ATP release assay, which is an indicator of connexin hemichannel and/or pannexin channel functions. Overall, the current study demonstrates the importance of hNPCs in HIV-1 infection and indicates that intercellular communications between infected hNPCs and HBMEC can be effectively modulated by EVs carrying Aß as their cargo.

Extracellular vesicles from HTLV-1 infected cells modulate target cells and viral spread.

BACKGROUND: The Human T-cell Lymphotropic Virus Type-1 (HTLV-1) is a blood-borne pathogen and etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 has currently infected up to 10 million globally with highly endemic areas in Japan, Africa, the Caribbean and South America. We have previously shown that Extracellular Vesicles (EVs) enhance HTLV-1 transmission by promoting cell-cell contact. RESULTS: Here, we separated EVs into subpopulations using differential ultracentrifugation (DUC) at speeds of 2 k (2000×g), 10 k (10,000×g), and 100 k (100,000×g) from infected cell supernatants. Proteomic analysis revealed that EVs contain the highest viral/host protein abundance in the 2 k subpopulation (2 k > 10 k > 100 k). The 2 k and 10 k populations contained viral proteins (i.e., p19 and Tax), and autophagy proteins (i.e., LC3 and p62) suggesting presence of autophagosomes as well as core histones. Interestingly, the use of 2 k EVs in an angiogenesis assay (mesenchymal stem cells + endothelial cells) caused deterioration of vascular-like-tubules. Cells commonly associated with the neurovascular unit (i.e., astrocytes, neurons, and macrophages) in the blood-brain barrier (BBB) showed that HTLV-1 EVs may induce expression of cytokines involved in migration (i.e., IL-8; 100 k > 2 k > 10 k) from astrocytes and monocyte-derived macrophages (i.e., IL-8; 2 k > 10 k). Finally, we found that EVs were able to promote cell-cell contact and viral transmission in monocytic cell-derived dendritic cell. The EVs from both 2 k and 10 k increased HTLV-1 spread in a humanized mouse model, as evidenced by an increase in proviral DNA and RNA in the Blood, Lymph Node, and Spleen. CONCLUSIONS: Altogether, these data suggest that various EV subpopulations induce cytokine expression, tissue damage, and viral spread.