Human tumor virus utilizes exosomes for intercellular communication.
Ontology highlight
ABSTRACT:
SUBMITTER: Meckes DG
PROVIDER: S-EPMC2996715 | biostudies-other | 2010 Nov
REPOSITORIES: biostudies-other
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Project description:UnlabelledIntercellular nanotube connections have been identified as an alternative pathway for cellular spreading of certain viruses. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nanotubes were observed connecting two distant cells with contiguous membranes, with the core infectious viral machinery (viral RNA, certain replicases, and certain structural proteins) present in/on the intercellular nanotubes. Live-cell movies tracked the intercellular transport of a recombinant PRRSV that expressed green fluorescent protein (GFP)-tagged nsp2. In MARC-145 cells expressing PRRSV receptors, GFP-nsp2 moved from one cell to another through nanotubes in the presence of virus-neutralizing antibodies. Intercellular transport of viral proteins did not require the PRRSV receptor as it was observed in receptor-negative HEK-293T cells after transfection with an infectious clone of GFP-PRRSV. In addition, GFP-nsp2 was detected in HEK-293T cells cocultured with recombinant PRRSV-infected MARC-145 cells. The intercellular nanotubes contained filamentous actin (F-actin) with myosin-associated motor proteins. The F-actin and myosin IIA were identified as coprecipitates with PRRSV nsp1β, nsp2, nsp2TF, nsp4, nsp7-nsp8, GP5, and N proteins. Drugs inhibiting actin polymerization or myosin IIA activation prevented nanotube formation and viral clusters in virus-infected cells. These data lead us to propose that PRRSV utilizes the host cell cytoskeletal machinery inside nanotubes for efficient cell-to-cell spread. This form of virus transport represents an alternative pathway for virus spread, which is resistant to the host humoral immune response.ImportanceExtracellular virus particles transmit infection between organisms, but within infected hosts intercellular infection can be spread by additional mechanisms. In this study, we describe an alternative pathway for intercellular transmission of PRRSV in which the virus uses nanotube connections to transport infectious viral RNA, certain replicases, and certain structural proteins to neighboring cells. This process involves interaction of viral proteins with cytoskeletal proteins that form the nanotube connections. Intercellular viral spread through nanotubes allows the virus to escape the neutralizing antibody response and may contribute to the pathogenesis of viral infections. The development of strategies that interfere with this process could be critical in preventing the spread of viral infection.
| S-EPMC4859731 | biostudies-literature
Project description:BackgroundTunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication.MethodsWe designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps.ResultsThe experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles.ConclusionsThis validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.
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Project description:Exosomes are small membrane-enclosed vesicles produced by various cells and actively released into the extracellular space. They participate in intercellular communication and transfer of biologically active proteins, lipids, and nucleic acids. Accumulating evidence suggests that exosomes derived from cells infected by some viruses selectively encapsulate viral proteins, genetic materials, or even virions to mediate cell-to-cell communication and/or virus transmission. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the global swine industry since the late 1980s. Recent studies have shown that major proteins secreted from PRRSV-infected cells are exosomal proteins and that the serum-derived exosomes from PRRSV-infected pigs contain viral proteins. However, the role of exosomes in PRRSV infection remains unclear. In this study, purified exosomes isolated from PRRSV-infected cells were shown with reverse transcription-PCR and mass spectrometry to contain viral genomic RNA and partial viral proteins. Furthermore, exosomes from PRRSV-infected cells established productive infection in both PRRSV-susceptible and -nonsusceptible cells. More importantly, exosome-mediated infection was not completely blocked by PRRSV-specific neutralizing antibodies. In summary, this study demonstrated that exosomes can mediate PRRSV transmission and are even resistant to antibody neutralization, identifying a potential immune evasion mechanism utilized by PRRSV.IMPORTANCE Exosomes have recently been characterized as bioactive vesicles that function to promote intercellular communication. The exosomes from virally infected cells containing altered compositions confer numerous novel functionalities. A study of the secretome of cells infected with PRRSV indicated that the exosomal pathway is strongly activated by PRRSV infection. Here, we demonstrate that PRRSV can utilize host exosomes to infect naive healthy cells. Furthermore, exosome-mediated viral transmission is largely resistant to PRRSV-specific neutralizing antibodies. Our study provides novel insights into an alternative mechanism of PRRSV transmission that can compromise the host's anti-PRRSV immune response.
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Project description:Despite successful control of viremia by combined antiretroviral therapy, brain infection and its resulting neurocognitive impairment remain a prevalent comorbidity in HIV infected individuals. HIV invades the brain early in the course of infection via penetration through the blood-brain barrier (BBB). While the impact of HIV on BBB astrocytes and endothelial cells is relatively well studied, the role of pericytes in BBB regulation during HIV infection remains unclear; however, it is known that a selective population of pericytes is prone to infection. In the present study, we hypothesize that injury signals are propagated from infected pericytes to neighboring cells via gap junction (GJ)-mediated intercellular communication. Among a variety of studied GJ proteins, HIV infection of human brain pericytes specifically increased expression of connexin 43 as determined by immunoblotting and immunostaining. This effect was confirmed in the brains of mice infected with EcoHIV, a mouse-specific HIV strain. In addition, HIV infection enhanced functional GJ-mediated intercellular communication in pericytes. The importance of this process was confirmed in experiments in which inhibition of GJs by carbenoxolone attenuated HIV infection. In addition to GJs, an extracellular ATP release assay revealed that HIV may also play a role in opening of connexin (Cx)-containing hemichannels (HCs). Overall, these findings indicate an important role of GJs in the propagation of HIV infection in human brain pericytes that may contribute to BBB dysfunction in brain infection and the pathogenesis of NeuroAIDS.
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Project description:Hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) is a candidate of radiosensitizers which enables both anti-tumour and anti-metastasis effects in X-ray therapy. The curative effects under such 4-MU administration have been investigated in vitro; however, the radiosensitizing mechanisms remain unclear. Here, we investigated the radiosensitizing effects under 4-MU treatment from cell experiments and model estimations. We generated experimental surviving fractions of human fibrosarcoma cells (HT1080) after 4-MU treatment combined with X-ray irradiation. Meanwhilst, we also modelled the pharmacological effects of 4-MU treatment and theoretically analyzed the synergetic effects between 4-MU treatment and X-ray irradiation. The results show that the enhancement of cell killing by 4-MU treatment is the greatest in the intermediate dose range of around 4 Gy, which can be reproduced by considering intercellular communication (so called non-targeted effects) through the model analysis. As supposed to be the involvement of intercellular communication in radiosensitization, the oxidative stress level associated with reactive oxygen species (ROS), which leads to DNA damage induction, is significantly higher by the combination of 4-MU treatment and irradiation than only by X-ray irradiation, and the radiosensitization by 4-MU can be suppressed by the ROS inhibitors. These findings suggest that the synergetic effects between 4-MU treatment and irradiation are predominantly attributed to intercellular communication and provide more efficient tumour control than conventional X-ray therapy.
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Project description:Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread.
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Project description:Hypertrophic Cardiomyopathy (HCM) is a common inherited disorder characterized by unexplained left ventricular hypertrophy with or without left ventricular outflow tract (LVOT) obstruction. Single-nuclei RNA-sequencing (snRNA-seq) of both obstructive and nonobstructive HCM patient samples has revealed alterations in communication between various cell types, but no direct and integrated comparison between the two HCM phenotypes has been reported. We performed a bioinformatic analysis of HCM snRNA-seq datasets from obstructive and nonobstructive patient samples to identify differentially expressed genes and distinctive patterns of intercellular communication. Differential gene expression analysis revealed 37 differentially expressed genes, predominantly in cardiomyocytes but also in other cell types, relevant to aging, muscle contraction, cell motility, and the extracellular matrix. Intercellular communication was generally reduced in HCM, affecting the extracellular matrix, growth factor binding, integrin binding, PDGF binding, and SMAD binding, but with increases in adenylate cyclase binding, calcium channel inhibitor activity, and serine-threonine kinase activity in nonobstructive HCM. Increases in neuron to leukocyte and dendritic cell communication, in fibroblast to leukocyte and dendritic cell communication, and in endothelial cell communication to other cell types, largely through changes in the expression of integrin-β1 and its cognate ligands, were also noted. These findings indicate both common and distinct physiological mechanisms affecting the pathogenesis of obstructive and nonobstructive HCM and provide opportunities for the personalized management of different HCM phenotypes.
| S-EPMC8780670 | biostudies-literature
Project description:HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Several recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, how the intercellular Nef transfer occurs is in dispute. In the current study, we attempted to address this important issue using several complementary strategies, a panel of exosomal markers, and human CD4+ T lymphocyte cell line Jurkat and a commonly used cell line 293T. First, we showed that Nef was transferred from Nef-expressing or HIV-infected Jurkat to naïve Jurkat and other non-Jurkat cells and that the transfer required the membrane targeting function of Nef and was cell density-dependent. Then, we showed that Nef transfer was cell-cell contact-dependent, as exposure to culture supernatants or exosomes from HIV-infected Jurkat or Nef-expressing Jurkat and 293T led to little Nef detection in the target cells Jurkat. Thirdly, we demonstrated that Nef was only detected to be associated with HIV virions but not with acetylcholinesterase (AChE+) exosomes from HIV-infected Jurkat and not in the exosomes from Nef-expressing Jurkat. In comparison, when it was over-expressed in 293T, Nef was detected in detergent-insoluble AChE+/CD81 low/TSG101 low exosomes, but not in detergent-soluble AChE-/CD81 high/TSG101 high exosomes. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out 293T. Taken together, these results show that exosomes are unlikely involved in intercellular Nef transfer. In addition, this study reveals existence of two types of exosomes: AChE+/CD81 low/TSG101 low exosomes and AChE-/CD81 high/TSG101 high exosomes.
| S-EPMC4412529 | biostudies-literature
Project description:Exosomes are nanoparticles secreted by all cell types and are a large component of the broader class of nanoparticles termed extracellular vesicles (EVs). Once secreted, exosomes gain access to the interstitial space and ultimately the circulation, where they exert local paracrine or distal systemic effects. Because of this, exosomes are important components of an intercellular and intraorgan communication system capable of carrying biologic signals from one cell type or tissue to another. The exosomal cargo consists of proteins, lipids, miRNAs, and other RNA species, and many of the biologic effects of exosomes have been attributed to miRNAs. Exosomal miRNAs have also been used as disease biomarkers. The field of exosome biology and metabolism is rapidly expanding, with new discoveries and reports appearing on a regular basis, and it is possible that potential therapeutic approaches for the use of exosomes or miRNAs in metabolic diseases will be initiated in the near future.
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