Purification methods and the presence of RNA in virus particles and extracellular vesicles

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.

Researchers from the University of North Carolina School of Medicine 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. The researchers also discuss the discrepancy of miRNA abundance in EV from different input material.

Extracellular vesicles (EV) and virus particles share similar vesicular budding process and composition, including proteins, nucleic acids, and lipids

(A) Microvesicles bud off at the plasma membrane, similar to the canonical human immunodeficiency viruses (HIV) budding process. Exosomes originate from inward budding of the late endosome into multivesicular body (MVB) and later release at the plasma membrane, similar to the canonical enveloped hepatitis A viruses (HAV) budding process. (B) Extracellular vesicles may carry makers like tetraspanins, esterases, Alix, and Tsg101. The encased nucleic acids are protected from nucleases. (C). A virus particle consists of an envelope, capsid, tegument, and viral genome.