( Figure 1) Apoptotic bodies result from cells undergoing apoptosis and can thus contain any part of those cells, including chromatin and mitochondria. These EV have historically been classified based on their origin, content, and size. EV are divided into three major classes: (i) apoptotic bodies, (ii) shedding microvesicles, and (iii) exosomes. We will start our discussion by defining some key terms. Should we postulate similar, target-cell specific mediated entry for EV, or is the concept of a single infection event, EV bolus the wrong experimental paradigm? Viruses overcome physiological dilution by utilizing highly specific receptors and co-receptors, like cluster of differentiation 4 (CD4) and C-C chemokine receptor type 5 (CCR5) on T cells in the case of human immunodeficiency viruses (HIV), and by being able to replicate in infected cells, thereby amplifying the signal locally. Fick’s laws apply to all experiments conducted in culture dishes and represents a lower limit regarding experiments in animals, where inter-tissue diffusion, intra-tissue diffusion, clearance, and blood flow rapidly dilute bolus injections of EV. Elementary physics posits that Fick’s laws govern EV concentration, diminishing inversely to the cube of distance (1/d 3). Is it possible that EV may have contaminated the virion preparations in these studies? Has there been enough experimental evidence to rule out EV contamination?įorth, how many RNAs on average does each EV contain? How do experimental models affect the heterogeneity of results? How can EV induce a physiologically significant response while current evidence shows the encased copies of RNAs are far from enough? Upon release, EV are rapidly diluted in solution. For RNA viruses as well, RNAs other than full-length genomic RNA have been reported in mature virion preparations. For DNA viruses, mature virion preparations have been repeatedly found associated with RNA. Third, do DNA viruses package miRNAs or mRNAs into virus particles ? All viruses package their respective genomes into an inner protein shell, the capsid, in a targeted, coordinated, and well-controlled process. Second, what have we learned from different purification methods? Do we need standards and, if so, what criteria should be used to evaluate claims of RNA-EV and RNA-virion associations? Specifically, we will consider the following questions:įirst, how do RNAs co-purify with viruses and with EV by different purification methods? We will discuss questions central to the packaging and delivery of RNAs by EV from foundational knowledge learned through virus infection. Hence, we use virions as a point of comparison and frame of reference for this review. Virus particles, or virions, have been studied for over 100 years and continue to be the subject of intense interest in the fields of basic sciences and vaccine manufacturing. Virus particles have similar biophysical attributes to naturally occurring extracellular vesicles (EV). We also discuss the discrepancy of miRNA abundance in EV from different input material. 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. Here, we discuss how encased RNAs from a heterogeneous mixture of particles can be distinguished by different purification methods. EV are known to co-sediment with virions and other particulates, such as defective interfering particles and protein aggregates. Differential centrifugation/ultracentrifugation and precipitating agents like polyethylene glycol are widely utilized for both EV and virus purifications. 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. 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.
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