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Isolation of exosomes. (A) Representative transmission electron microscopy images of exosome samples. Exosomes were isolated by ultracentrifugation and ExoQuick, and analyzed by electron microscopy. Scale bar = 100 nm. (B) PCR and western blotting analysis of isolated exosomes. Virus particles of KSHV and EBV and cell lysate of Bjab cells were also examined. KSHV KS330233, EBV W and beta-globin genes were amplified with PCR (upper panels). A faint band of KSHV is found in the exosome sample from BCBL-1 in PCR. KSHV ORF45, Lyn, CD63, HSP70, and Lyn were detected in isolated exosome samples with western blotting analysis (lower panels). A faint band of KSHV ORF45 is observed in the exosome sample from BCBL-1.
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Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about t...
Citations
... For instance, exosomes appear to promote tumor growth and metastasis by delivering molecules, such as TGFβ [29], EGFR [30], and miRNAs [31,32]. In addition, recent studies using virus-infected cells have demonstrated the incorporation of viral proteins, as well as viral and cellular RNAs, into EVs, suggesting the possibility that EVs could be involved in viral propagation, pathogenesis, and immune evasion [33][34][35][36][37][38][39][40][41][42]. Several studies reported that EVs could transfer the full-length genome of certain viruses including flaviviruses and that virus dissemination might be mediated by EVs in addition to the classical receptor-mediated infection [13][14][15][16][17][19][20][21]. ...
Extracellular vesicles (EVs) such as exosomes have been shown to play physiological roles in cell-to-cell communication by delivering various proteins and nucleic acids. In addition, several studies revealed that the EVs derived from the cells that are infected with certain viruses could transfer the full-length viral genomes, resulting in EVs-mediated virus propagation. However, the possibility cannot be excluded that the prepared EVs were contaminated with infectious viral particles. In this study, the cells that harbor subgenomic replicon derived from the Japanese encephalitis virus and dengue virus without producing any replication-competent viruses were employed as the EV donor. It was demonstrated that the EVs in the culture supernatants of those cells were able to transfer the replicon genome to other cells of various types. It was also shown that the EVs were incorporated by the recipient cells primarily through macropinocytosis after interaction with CD33 and Tim-1/Tim-4 on HeLa and K562 cells, respectively. Since the methods used in this study are free from contamination with infectious viral particles, it is unequivocally indicated that the flavivirus genome can be transferred by EVs from cell to cell, suggesting that this pathway, in addition to the classical receptor-mediated infection, may play some roles in the viral propagation and pathogenesis.
... MiRNAs can be coupled into exosomes and carried by exosomes to aid biological functions 25 . A number of studies have demonstrated that human immune cells are capable of recognizing virus-infected cells and produce exosomal miRNAs in response to viral infection [26][27][28] . In a cell model study, in pigment epithelial cells, B cell-derived miR-155 regulated biological function through exosome transmission 29 . ...
Notwithstanding recent advances in direct antiviral specialists (DAAs) for hepatitis C infection (HCV), it is yet a pervasive overall issue in patients with rheumatoid arthritis (RA). Exosomal microRNAs (miRNAs) is associated with HCV infection. However, it remains unknown how miRNAs respond following biologic disease-modifying antirheumatic drug (bDMARD) and targeted synthetic DMARD (tsDMARD) treatment in HCV patients with RA. We prospectively recruited RA patients taking anti-tumor necrosis factor-α (TNF-α) inhibitors rituximab (RTX) and tofacitinib. The serum hepatitis C viral load was measured using real-time quantitative reverse transcriptase PCR before and 6 months after bDMARD and tsDMARD therapy. HCV RNA replication activity was measured using an HCV-tricistronic replicon reporter system, and quantitative analysis of hsa-mir-122-5p and hsa-mir-155-5p in patients was performed using quantitative PCR. HCV RNA replication in hepatocytes was not affected by tofacitinib or TNF-α inhibitor treatment. Hsa-mir-155-5p and hsa-mir-122-5p were significantly expanded in RA patients with HCV as compared with those without HCV. We observed a dramatic increase in hsa-mir-122-5p and a decrease in hsa-mir-155-5p expression levels in patients taking RTX in comparison with other treatments. Finally, a reduction in hsa-mir-122-5p and an increase in hsa-mir-155-5p were observed in a time-dependent manner after tofacitinib and DAA therapy in RA-HCV patients. These results showed that hsa-mir-155-5p and hsa-mir-122-5p were significantly increased in RA-HCV patients as compared with those without HCV after taking tofacitinib. Hsa-mir-155-5p and hsa-mir-122-5p may be potential biomarkers for treatment efficacy in RA patients with HCV.
... Upregulated by EBV and KSHV in lymphomas, as well as in exosomes from them [104]. ...
Myalgic Encephalomyelitis / Chronic Fatigue Syndrome is a debilitating, multisystem disease of unknown mechanism, with a currently ongoing search for its endocrine mediators. Circulating microRNAs (miRNA) are a promising candidate for such a mediator and have been reported as significantly different in the patient population versus healthy controls by multiple studies. None of these studies, however, agree with each other on which specific miRNA are under- or over-expressed. This discrepancy is the subject of the computational study presented here, in which a deep dive into the predicted gene targets and their functional interactions is conducted, revealing that the aberrant circulating miRNAs in ME/CFS, although different between patients, seem to mainly target the same specific set of genes (p ≈ 0.0018), which are very functionally related to each other (p ≲ 0.0001). Further analysis of these functional relations, based on directional pathway information, points to impairments in exercise hyperemia, angiogenic adaptations to hypoxia, antioxidant defenses, and TGF-β signaling, as well as a shift towards mitochondrial fission, corroborating and explaining previous direct observations in ME/CFS. Many transcription factors and epigenetic modulators are implicated as well, with currently uncertain downstream combinatory effects. As the results show significant similarity to previous research on latent herpesvirus involvement in ME/CFS, the possibility of a herpesvirus origin of these miRNA changes is also explored through further computational analysis and literature review, showing that 8 out of the 10 most central miRNAs analyzed are known to be upregulated by various herpesviruses. In total, the results establish an appreciable and possibly central role for circulating microRNAs in ME/CFS etiology that merits further experimental research.
... Human herpesvirus, particularly KSV and EBV, have been described to be capable of recruiting the exosome pathway to release several components that include miRNAs, mRNAs, small non-coding RNAs, and proteins [123][124][125][126]. For instance, cells infected with EBV release exosomes that contain viral miRNAs believed to be smaller products of the transcripts of BamH1 EBV. ...
Extracellular vesicles (EVs) comprise a broad range of secreted cell-derived membrane vesicles. Beyond their more well-characterized role in cell communication, in recent years, EVs have also been shown to play important roles during infection. Viruses can hijack the biogenesis of exosomes (which are small EVs) to promote viral spreading. Additionally, these exosomes are also important mediators in inflammation and immune responses during both bacterial and viral infections. This review summarizes these mechanisms while also describing the impact of bacterial EVs in regulating immune responses. Finally, the review also focuses on the potential and challenges of using EVs, in particular, to tackle infectious diseases.
... EBVaGC and nasopharyngeal carcinoma can express higher BART clusters [24,25]. miRNAs have also been found to be highly expressed in exosomes, which are involved in intercellular communication [26][27][28][29], inducing cell apoptosis and epithelial-mesenchymal transformation, and promoting the spread and metastasis of GC [30]. ...
Aim:
EBV encodes at least 44 miRNAs involved in immune regulation and disease progression. Exosomes can be used as carriers of EBV-miRNA-BART intercellular transmission and affect the biological behavior of cells. We characterized exosomes and established a co-culture experiment of exosomes to explore the mechanism of miR-BART1-3p transmission through the exosome pathway and its influence on tumor cell proliferation and invasion.
Materials and methods:
Exosomes of EBV-positive and EBV-negative gastric cancer cells were characterized by transmission electron microscopy. NanoSight and Western blotting, and miRNA expression profiles in exosomes were sequenced with high throughput. Exosomes with high or low expression of miR-BART1-3p were co-cultured with AGS cells to study the effects on proliferation, invasion, and migration of gastric cancer cells. The target genes of EBV-miR-BART1-3p were screened and predicted by PITA, miRanda, RNAhybrid, virBase, and DIANA-TarBase v.8 databases, and the expression of the target genes after co-culture was detected by qPCR.
Results:
The exosomes secreted by EBV-positive and negative gastric cancer cells range in diameter from 30 nm to 150 nm and express the exosomal signature proteins CD9 and CD63. Small RNA sequencing showed that exosomes expressed some human miRNAs, among which hsa-miR-23b-3p, hsa-miR-320a-3p, and hsa-miR-4521 were highly expressed in AGS-exo; hsa-miR-21-5p, hsa-miR-148a-3p, and hsa-miR-7-5p were highly expressed in SNU-719-exo. All EBV miRNAs were expressed in SNU-719 cells and their exosomes, among which EBV-miR-BART1-5p, EBV-miR-BART22, and EBV-miR-BART16 were the highest in SNU-719 cells; EBV-miR-BART1-5p, EBV-miR-BART10-3p, and EBV-miR-BART16 were the highest in SNU-719-exo. After miR-BART1-3p silencing in gastric cancer cells, the proliferation, healing, migration, and invasion of tumor cells were significantly improved. Laser confocal microscopy showed that exosomes could carry miRNA into recipient cells. After co-culture with miR-BART1-3p silenced exosomes, the proliferation, healing, migration, and invasion of gastric cancer cells were significantly improved. The target gene of miR-BART1-3p was FAM168A, MACC1, CPEB3, ANKRD28, and USP37 after screening by a targeted database. CPEB3 was not expressed in all exosome co-cultured cells, while ANKRD28, USP37, MACC1, and FAM168A were all expressed to varying degrees. USP37 and MACC1 were down-regulated after up-regulation of miR-BART1-3p, which may be the key target genes for miR-BART1-3p to regulate the proliferation of gastric cancer cells through exosomes.
Conclusions:
miR-BART1-3p can affect the growth of tumor cells through the exosome pathway. The proliferation, healing, migration, and invasion of gastric cancer cells were significantly improved after co-culture with exosomes of miR-BART1-3p silenced expression. USP37 and MACC1 may be potential target genes of miR-BART1-3p in regulating cell proliferation.
... A study of lymphoma cell lines showed that 50% of the miRNAs in exosomes were derived from KSHVinfected cells. In fact, among KSHV-encoded miRNAs, many contained short sequences of CCCT and CCCG motifs that control the loading of miRNAs into exosomes [139]. Interestingly, the cases in which viral miRNA and host RNA interact to enhance miRNA degradation emphasize the continuous coevolution that fine-tunes the regulation of miRNA by external agents such as human DNA viruses [140]. ...
Mature microRNAs (miRNAs) are single-stranded non-coding RNA (ncRNA) molecules that act in post-transcriptional regulation in animals and plants. A mature miRNA is the end product of consecutive, highly regulated processing steps of the primary miRNA transcript. Following base-paring of the mature miRNA with its mRNA target, translation is inhibited, and the targeted mRNA is degraded. There are hundreds of miRNAs in each cell that work together to regulate cellular key processes, including development, differentiation, cell cycle, apoptosis, inflammation, viral infection, and more. In this review, we present an overlooked layer of cellular regulation that addresses cell dynamics affecting miRNA accessibility. We discuss the regulation of miRNA local storage and translocation among cell compartments. The local amounts of the miRNAs and their targets dictate their actual availability, which determines the ability to fine-tune cell responses to abrupt or chronic changes. We emphasize that changes in miRNA storage and compactization occur under induced stress and changing conditions. Furthermore, we demonstrate shared principles on cell physiology, governed by miRNA under oxidative stress, tumorigenesis, viral infection, or synaptic plasticity. The evidence presented in this review article highlights the importance of spatial and temporal miRNA regulation for cell physiology. We argue that limiting the research to mature miRNAs within the cytosol undermines our understanding of the efficacy of miRNAs to regulate cell fate under stress conditions.
... Therefore, miRNAs are considered one of the most important cargos carried by exosomes derived from KSHV-infected cells, including KSHV-encoded miRNAs and host miRNAs [51]. Hoshina et.al's study showed that almost half the miRNAs (48%) inside the exosomes are KSHV-encoded miRNAs [161], which the highest expression is related to miRK12-3-5p and among the human miRNAs, miR-17-92 cluster, mir-10b-5p and mir-143-3p were reported as the most abundant in exosomes [51,161]. Some investigations using various methods such as detailed bioinformatics analysis of the genome (Gene ontology) have shown that these exosomal miRNA and oncogenic exosomes can mediate virus pathogenicity by affecting different signal pathways, the metabolic activity of the target cells and other factors such as cytokines and complement system, leading to the development of virus-associated tumors [51,158,160]. ...
... Therefore, miRNAs are considered one of the most important cargos carried by exosomes derived from KSHV-infected cells, including KSHV-encoded miRNAs and host miRNAs [51]. Hoshina et.al's study showed that almost half the miRNAs (48%) inside the exosomes are KSHV-encoded miRNAs [161], which the highest expression is related to miRK12-3-5p and among the human miRNAs, miR-17-92 cluster, mir-10b-5p and mir-143-3p were reported as the most abundant in exosomes [51,161]. Some investigations using various methods such as detailed bioinformatics analysis of the genome (Gene ontology) have shown that these exosomal miRNA and oncogenic exosomes can mediate virus pathogenicity by affecting different signal pathways, the metabolic activity of the target cells and other factors such as cytokines and complement system, leading to the development of virus-associated tumors [51,158,160]. ...
Background:
Exosomes are the smallest group of extracellular vesicles in size from 30 to 150 nm, surrounded by a lipid bilayer membrane, and originate from multivesicular bodies secreted by different types of cells, such as virus-infected cells. The critical role of exosomes is information transfer among cells, representing a unique way for intercellular communication via a load of many kinds of molecules, including various signaling proteins and nucleic acids. In this review, we aimed to comprehensively investigate the role of exosomes in promoting human oncogenic viruses-associated cancers.
Methods:
Our search was conducted for published researches between 2000 and 2022 by using several international databases includeing Scopus, PubMed, and Web of Science as well as Google scholar. We also reviewed additional evidence from relevant published articles.
Results:
It has been shown that exosomes can create the conditions for viral spread in viral infections. Exosome secretion in a human tumor virus can switch on the cell signaling pathways by transferring exosome-encapsulated molecules, including viral oncoproteins, signal transduction molecules, and virus-encoded miRNAs, into various cells.
Conclusion:
Given the role of exosomes in viruses-associated cancers, they can also be considered as molecular targets in diagnosis and treatment.
... These characteristics demonstrate a remarkable potential for microRNAs to be used as biomarkers for disease. miRNAs have been successfully used to classify cancer, identify cancer tissue origin, determine prognosis and disease progression, predict resistance to chemotherapy, monitor therapy, and screen for disease [13][14][15][16][17]. HHV8 infection or HHV8-related malignancies can induce a unique signature of human and viral intracellular and extracellular miRNAs [18], and this has been confirmed in vitro [19]. These data, although highly suggestive, cannot be extrapolated to epidemic KS due to the additive effects of HIV infection on cellular miRNA signaling. ...
... Exosomes help to protect circulating miRNAs from degradation by RNases in the extracellular environment [34]. Host-encoded miRNAs have previously been found in exosomes released from KSHV-infected lymphoma cell lines [19]. Indeed, some of the dysregulated miRNAs in patients with epidemic KS in the current study have been reported to be expressed in KS endothelial tumor cells. ...
Background
Human herpesvirus 8 (HHV-8) causes Kaposi's sarcoma (KS). Kaposi sarcoma in HIV/AIDS patients is referred to as epidemic KS and is the most common HIV-related malignancy worldwide. The lack of a diagnostic assay to detect latent and early-stage disease has increased disease morbidity and mortality. Serum miRNAs have previously been used as potential biomarkers of normal physiology and disease. In the current study, we profiled unique serum miRNAs in patients with epidemic KS to generate baseline data to aid in developing a miRNA-based noninvasive biomarker assay for epidemic KS.
Methods
This was a comparative cross-sectional study involving 27 patients with epidemic KS and 27 HIV-positive adults with no prior diagnosis or clinical manifestation of KS. DNA and RNA were isolated from blood and serum collected from study participants. Nested PCR for circulating HHV-8 DNA was performed on the isolated DNA, whereas miRNA library preparation and sequencing for circulating miRNA were performed on the RNA samples. The miRge2 pipeline and EdgeR were used to analyse the sequencing data.
Results
Fifteen out of the 27 epidemic KS-positive subjects (55.6%) tested positive for HHV-8 DNA, whereas only 3 (11.1%) out of the 27 HIV-positive, KS-negative subjects tested positive for HHV-8 DNA. Additionally, we found a unique miRNA expression signature in 49 circulating miRNAs in epidemic KS subjects compared to subjects with no epidemic KS, with 41 miRNAs upregulated and 8 miRNAs downregulated. Subjects with latent KS infection had a differential upregulation of circulating miR-193a compared to HIV-positive, KS-negative subjects for whom circulating HHV-8 DNA was not detected. Further analysis of serum from epidemic KS patients revealed a miRNA signature according to KS tumor status and time since first HIV diagnosis.
Conclusions
This study reveals unique circulating miRNA profiles in the serum of patients with epidemic KS versus HIV-infected subjects with no KS, as well as in subjects with latent KS. Many of the dysregulated miRNAs in epidemic KS patients were previously reported to have crucial roles in KS infection and latency, highlighting their promising roles as potential biomarkers of latent or active KS infection.
... The following reasons for this discrepant observation were considered: first, the methodologies used to determine the miRNA expression value were different in the NGS assay and qRT-PCR. In the NGS assay, the expression value was determined including precursor miRNAs, whereas only mature miRNAs were detected by qRT-PCR (Hoshina et al. 2016;Schmittgen et al. 2008). Moreover, the normalization technique of the expression value was different between the two assays. ...
Primary human herpesvirus 6 (HHV-6) infection is sometimes accompanied by acute encephalopathy with reduced subcortical diffusion (AED) in immunocompetent children. We investigated exosomal microRNA (miRNA) expression profiles in cerebrospinal fluid (CSF) and sera of patients with HHV-6-associated AED (n = 5) and febrile seizure (FS) (n = 5) using high-throughput sequencing. A total of 176 and 663 miRNAs were identified in CSF and serum exosomes, respectively. Comparative analysis determined that some miRNAs (miR-381-3p, miR-155) were exclusively expressed in the CSF exosomes of AED but not of FS patients, suggesting their potential application as novel diagnostic biomarkers for AED.
... Moreover, the miRNAs released extracellularly are bound to the Ago proteins. These mechanisms protect the miRNAs against degradation by the nucleases [35,105,106] Whether v-miRNAs or cellular miRNAs DOI: http://dx.doi.org /10.5772/intechopen.100370 ...
MicroRNAs (miRNAs), first discovered in the year 1993 in the nematode C. elegans, are small, approximately 22-nucleotide-long, non-coding RNAs that regulate gene expression. Cellular miRNAs have been implicated in the control of many biological processes, and their dysregulation is associated with different diseases. They can be significantly up/downregulated upon infection or disease, serving as excellent biomarkers and therapeutic targets. Several human DNA viruses, including many herpesviruses, have now been reported to encode viral miRNAs. There are a variety of possible interactions and mechanisms of viral microRNAs (vmiRNAs) which are yet to be remains obscure. Viral miRNAs can function as orthologs of cellular miRNAs and regulate their expression. Additionally, viruses have also developed vmiRNA mechanisms to avoid being targeted by the host miRNAs. Herpes Simplex Viruses (HSV-1 & HSV-2) cause genital and oral herpes, establishing lifelong latent infections in their hosts, and it is one of the most prevalent sexually transmitted infections (STIs) worldwide. vmiRNAs play essential roles in Herpesvirus biology. In this chapter, we will discuss the current knowledge about miRNAs and their role in different stages of Herpesvirus infection. It will also elaborate the biomarkers, therapeutic potential of these molecules, and the prospective areas of future research.
