scholarly journals Purification Methods and the Presence of RNA in Virus Particles and Extracellular Vesicles

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 917
Author(s):  
Yijun Zhou ◽  
Ryan P. McNamara ◽  
Dirk P. Dittmer
Keyword(s):  
Extracellular Vesicles ◽  
Differential Centrifugation ◽  
Heterogeneous Mixture ◽  
Virus Infections ◽  
Defective Interfering Particles ◽  
Virus Particles ◽  
Non Coding Rna ◽  
Input Material ◽  
Purification Methods ◽  
Mirna Abundance

The fields of extracellular vesicles (EV) and virus infections are marred in a debate on whether a particular mRNA or non-coding RNA (i.e., miRNA) is packaged into a virus particle or copurifying EV and similarly, whether a particular mRNA or non-coding RNA is contained in meaningful numbers within an EV. Key in settling this debate, is whether the purification methods are adequate to separate virus particles, EV and contaminant soluble RNA and RNA:protein complexes. Differential centrifugation/ultracentrifugation and precipitating agents like polyethylene glycol are widely utilized for both EV and virus purifications. EV are known to co-sediment with virions and other particulates, such as defective interfering particles and protein aggregates. Here, we discuss how encased RNAs from a heterogeneous mixture of particles can be distinguished by different purification methods. This is particularly important for subsequent interpretation of whether the RNA associated phenotype is contributed solely by virus or EV particles or a mixture of both. We also discuss the discrepancy of miRNA abundance in EV from different input material.

scholarly journals The Role of Extracellular Vesicles as Shuttles of RNA and Their Clinical Significance as Biomarkers in Hepatocellular Carcinoma

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 902
Author(s):  
Eva Costanzi ◽  
Carolina Simioni ◽  
Gabriele Varano ◽  
Cinzia Brenna ◽  
Ilaria Conti ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Extracellular Vesicles ◽  
Tumor Metastasis ◽  
Biological Processes ◽  
Rna Molecules ◽  
Expression Of Genes ◽  
Non Coding Rna ◽  
Donor Cells ◽  
Relevant Role

Extracellular vesicles (EVs) have attracted interest as mediators of intercellular communication following the discovery that EVs contain RNA molecules, including non-coding RNA (ncRNA). Growing evidence for the enrichment of peculiar RNA species in specific EV subtypes has been demonstrated. ncRNAs, transferred from donor cells to recipient cells, confer to EVs the feature to regulate the expression of genes involved in differentiation, proliferation, apoptosis, and other biological processes. These multiple actions require accuracy in the isolation of RNA content from EVs and the methodologies used play a relevant role. In liver, EVs play a crucial role in regulating cell–cell communications and several pathophysiological events in the heterogeneous liver class of cells via horizontal transfer of their cargo. This review aims to discuss the rising role of EVs and their ncRNAs content in regulating specific aspects of hepatocellular carcinoma development, including tumorigenesis, angiogenesis, and tumor metastasis. We analyze the progress in EV-ncRNAs’ potential clinical applications as important diagnostic and prognostic biomarkers for liver conditions.

scholarly journals Different Cross-Reactivities of IgM Responses in Dengue, Zika and Tick-Borne Encephalitis Virus Infections

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 596
Author(s):  
Karin Stiasny ◽  
Stefan Malafa ◽  
Stephan W. Aberle ◽  
Iris Medits ◽  
Georgios Tsouchnikas ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Yellow Fever ◽  
Vaccination Coverage ◽  
Virus Infections ◽  
Recent Infection ◽  
Specific Diagnosis ◽  
Virus Particles ◽  
Tick Borne Encephalitis ◽  
Structural Peculiarities ◽  
Tick Borne Encephalitis Virus

Flaviviruses circulate worldwide and cause a number of medically relevant human diseases, such as dengue, Zika, yellow fever, and tick-borne encephalitis (TBE). Serology plays an important role in the diagnosis of flavivirus infections, but can be impeded by antigenic cross-reactivities among flaviviruses. Therefore, serological diagnosis of a recent infection can be insufficiently specific, especially in areas where flaviviruses co-circulate and/or vaccination coverage against certain flaviviruses is high. In this study, we developed a new IgM assay format, which is well suited for the specific diagnosis of TBE, Zika and dengue virus infections. In the case of TBE and Zika, the IgM response proved to be highly specific for the infecting virus. In contrast, primary dengue virus infections induced substantial amounts of cross-reactive IgM antibodies, which is most likely explained by structural peculiarities of dengue virus particles. Despite the presence of cross-reactive IgM, the standardized nature and the quantitative read-out of the assay even allowed the serotype-specific diagnosis of recent dengue virus infections in most instances.

scholarly journals A non-enveloped arbovirus released in lysosome-derived extracellular vesicles induces super-infection exclusion

2020 ◽  
Author(s):  
Thomas Labadie ◽  
Polly Roy
Keyword(s):  
Extracellular Vesicles ◽  
Defense Mechanism ◽  
Degradation Process ◽  
Free Virus ◽  
Enveloped Viruses ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Super Infection

AbstractRecent developments on extracellular vesicles (EVs) containing multiple virus particles challenge the rigid definition of non-enveloped viruses. However, how non-enveloped viruses hijack cell machinery to promote non-lytic release in EVs, and their functional roles, remain to be clarified. Here we used Bluetongue virus (BTV) as a model of a non-enveloped arthropod-borne virus and observed that the majority of viruses are released in EVs, both in vitro and in the blood of infected animals. Based on the cellular proteins detected in these EVs, and use of inhibitors targeting the cellular degradation process, we demonstrated that these extracellular vesicles are derived from secretory lysosomes, in which the acidic pH is neutralized upon the infection. Moreover, we report that secreted EVs are more efficient than free-viruses for initiating infections, but that they trigger super-infection exclusion that only free-viruses can overcome.Author summaryRecent discoveries of non-enveloped virus secreted in EVs opened the door to new developments in our understanding of the transmission and pathogenicity of these viruses. In particular, how these viruses hijack the host cellular secretion machinery, and the role of these EVs compared with free-virus particles remained to be explored. Here, we tackled these two aspects, by studying BTV, an emerging arthropod-borne virus causing epidemics worldwide. We showed that this virus is mainly released in EVs, in vivo and in the blood of infected animals, and that inhibition of the cell degradation machinery decreases the release of infectious EVs, but not free-virus particles. We found that BTV must neutralize the pH of lysosomes, which are important organelles of the cell degradation machinery, for efficient virus release in EVs. Our results highlight unique features for a virus released in EVs, explaining how BTV transits in lysosomes without being degraded. Interestingly, we observed that EVs are more infectious than free-virus particles, but only free-viruses are able to overcome the super-infection exclusion, which is a common cellular defense mechanism. In conclusion, our study stresses the dual role played by both forms, free and vesicular, in the virus life cycle.

scholarly journals Origins and Function of VL30 lncRNA Packaging in Small Extracellular Vesicles: Implications for Cellular Physiology and Pathology

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1742
Author(s):  
Stefania Mantziou ◽  
Georgios S. Markopoulos
Keyword(s):  
Extracellular Vesicles ◽  
Active Regions ◽  
Regulatory Elements ◽  
Binding Motif ◽  
Regulatory Sequences ◽  
Chromosomal Distribution ◽  
Non Coding Rna ◽  
Rna Biology ◽  
And Function

Long non-coding RNAs (lncRNAs) have emerged during the post-genomic era as significant epigenetic regulators. Viral-like 30 elements (VL30s) are a family of mouse retrotransposons that are transcribed into functional lncRNAs. Recent data suggest that VL30 RNAs are efficiently packaged in small extracellular vesicles (SEVs) through an SEV enrichment sequence. We analysed VL30 elements for the presence of the distinct 26 nt SEV enrichment motif and found that SEV enrichment is an inherent hallmark of the VL30 family, contained in 36 full-length elements, with a widespread chromosomal distribution. Among them, 25 elements represent active, present-day integrations and contain an abundance of regulatory sequences. Phylogenetic analysis revealed a recent spread of SEV-VL30s from 4.4 million years ago till today. Importantly, 39 elements contain an SFPQ-binding motif, associated with the transcriptional induction of oncogenes. Most SEV-VL30s reside in transcriptionally active regions, as characterised by their distribution adjacent to candidate cis-regulatory elements (cCREs). Network analysis of SEV-VL30-associated genes suggests a distinct transcriptional footprint associated with embryonal abnormalities and neoplasia. Given the established role of VL30s in oncogenesis, we conclude that their potential to spread through SEVs represents a novel mechanism for non-coding RNA biology with numerous implications for cellular homeostasis and disease.

Short non-coding RNA sequencing of glioblastoma extracellular vesicles

2020 ◽  
Vol 146 (2) ◽  
pp. 253-263 ◽  
Author(s):  
Tristan de Mooij ◽  
Timothy E. Peterson ◽  
Jared Evans ◽  
Brandon McCutcheon ◽  
Ian F. Parney
Keyword(s):  
Rna Sequencing ◽  
Extracellular Vesicles ◽  
Non Coding Rna

scholarly journals Analysis of cancer-related mutations in extracellular vesicles RNA by Droplet Digital™ PCR

BioTechniques ◽  
2020 ◽  
Vol 69 (2) ◽  
pp. 99-107
Author(s):  
Soo Ann Yap ◽  
Agnieszka Münster-Wandowski ◽  
Anika Nonnenmacher ◽  
Ulrich Keilholz ◽  
Sandra Liebs
Keyword(s):  
Colorectal Cancer ◽  
Tumor Progression ◽  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Mek Inhibitor ◽  
Diagnostic Information ◽  
Braf V600e ◽  
Plasma Samples ◽  
Differential Centrifugation ◽  
Potential Biomarkers

Extracellular vesicles (EVs) are taking their place as potential biomarkers in the field of liquid biopsy. In this study, EVs were isolated from plasma samples of 31 patients with colorectal cancer and melanoma via differential centrifugation and Droplet Digital™ PCR (Bio-Rad, CA, USA) was used to profile BRAF V600E/K, KRAS G12A/C/D/V and KRAS G13D mutations from EV-derived cDNA. The concordance rates with corresponding tissue were 54% and 44% in the colorectal cancer and melanoma cohort, respectively. Two patients displayed mutations in EVs not previously detected in tissue as evidence for emerging molecular resistance to anti-EGFR and BRAF/MEK inhibitor therapy prior to radiological evidence of tumor progression. We concluded that EV-derived nucleic acids may provide clinically relevant diagnostic information and mirror evolution of the disease.

Extracellular vesicles: their role in cancer biology and epithelial–mesenchymal transition

2016 ◽  
Vol 474 (1) ◽  
pp. 21-45 ◽  
Author(s):  
Shashi K. Gopal ◽  
David W. Greening ◽  
Alin Rai ◽  
Maoshan Chen ◽  
Rong Xu ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Cancer Biology ◽  
Epithelial Mesenchymal Transition ◽  
Current Knowledge ◽  
Cell Communication ◽  
Metastatic Potential ◽  
Messenger Rnas ◽  
Mesenchymal Transition ◽  
Non Coding Rna ◽  
Long Non Coding Rna

Cell–cell communication is critical across an assortment of physiological and pathological processes. Extracellular vesicles (EVs) represent an integral facet of intercellular communication largely through the transfer of functional cargo such as proteins, messenger RNAs (mRNAs), microRNA (miRNAs), DNAs and lipids. EVs, especially exosomes and shed microvesicles, represent an important delivery medium in the tumour micro-environment through the reciprocal dissemination of signals between cancer and resident stromal cells to facilitate tumorigenesis and metastasis. An important step of the metastatic cascade is the reprogramming of cancer cells from an epithelial to mesenchymal phenotype (epithelial–mesenchymal transition, EMT), which is associated with increased aggressiveness, invasiveness and metastatic potential. There is now increasing evidence demonstrating that EVs released by cells undergoing EMT are reprogrammed (protein and RNA content) during this process. This review summarises current knowledge of EV-mediated functional transfer of proteins and RNA species (mRNA, miRNA, long non-coding RNA) between cells in cancer biology and the EMT process. An in-depth understanding of EVs associated with EMT, with emphasis on molecular composition (proteins and RNA species), will provide fundamental insights into cancer biology.

scholarly journals Critical Roles of Tumor Extracellular Vesicles in the Microenvironment of Thoracic Cancers

2020 ◽  
Vol 21 (17) ◽  
pp. 6024
Author(s):  
Lyna Kara-Terki ◽  
Lucas Treps ◽  
Christophe Blanquart ◽  
Delphine Fradin
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Extracellular Vesicles ◽  
Intercellular Communication ◽  
Special Focus ◽  
Pleural Mesothelioma ◽  
Lung Cancers ◽  
Non Coding Rna ◽  
Exosomal Mirnas ◽  
Tumor Growth And Metastasis

Extracellular vesicles (EVs), such as exosomes, are critical mediators of intercellular communication between tumor cells and other cells located in the microenvironment but also in more distant sites. Exosomes are small EVs that can carry a variety of molecules, such as lipids, proteins, and non-coding RNA, especially microRNAs (miRNAs). In thoracic cancers, including lung cancers and malignant pleural mesothelioma, EVs contribute to the immune-suppressive tumor microenvironment and to tumor growth and metastasis. In this review, we discuss the recent understanding of how exosomes behave in thoracic cancers and how and why they are promising liquid biomarkers for diagnosis, prognosis, and therapy, with a special focus on exosomal miRNAs.

scholarly journals Tobacco mosaic virus and the study of early events in virus infections

1999 ◽  
Vol 354 (1383) ◽  
pp. 603-611 ◽  
Author(s):  
John G. Shaw
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Host Cells ◽  
Progeny Virus ◽  
Virus Infections ◽  
Relevant Research ◽  
Protein Subunits ◽  
Virus Particles ◽  
Initial Interaction ◽  
Positive Strand Rna

In order to establish infections, viruses must be delivered to the cells of potential hosts and must then engage in activities that enable their genomes to be expressed and replicated. With most viruses, the events that precede the onset of production of progeny virus particles are referred to as the early events and, in the case of positive–strand RNA viruses, they include the initial interaction with, and the entry of, host cells and the release (uncoating) of the genome from the virus particles. Though the early events remain one of the more poorly understood areas of plant virology, the virus with which most of the relevant research has been performed is tobacco mosaic virus (TMV). In spite of this effort, there remains much uncertainty about the form or constituent of the virus that actually enters the initially invaded cell in a plant and about the mechanism(s) that trigger the subsequent uncoating (virion disassembly) reactions. A variety of approaches have been used in attempts to determine the fate of TMV particles that are involved in the establishment of an infection and these are briefly described in this review. In some recent work, it has been proposed that the uncoating process involves the bidirectional release of coat protein subunits from the viral RNA and that these activities may be mediated by cotranslational and coreplicational disassembly mechanisms.

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