Lessons for SARS-CoV-2 study (COVID-19 disease) from its exosome relatives

Our first modern global pandemic is caused by a nanosized lipid vesicle, called SARS-CoV-2. Its molecular structure and biogenesis have remarkable similarities with Extracellular Vesicles (EVs, most notably exosomes) that are constantly shed by all cells during their life. Their resemblance may not be a coincidence. Growing body of evidence has shown that EVs have significant roles in various biological processes, including viral infection, transmission and anti-viral response. Drawing comparison with the virus might shed light on how we could fight the COVID-19 disease. This may include novel EV research and diagnostics technologies as well as novel EV-based treatments.


Introduction
SARS-CoV-2 has shortly become one of the most studied viruses following the COVID-19 outbreak in Wuhan in December 2019. These nanosized lipid vesicles have put at risk the global healthcare system and the world economy. SARS-CoV-2 have striking similarities with EVs, most notably exosomes, since they share many features (diameter, density, structure, cargo type, cargo origin, cell entry mechanism and potentially exit mechanism) despite having different functions.
EVs are heterogeneous vesicles naturally released by a cell during its life and usually carry biologically active molecules that can deliver their messages to local or distant targets.
More generally, EVs and enveloped viruses have been hypothesized to be, in fact, "close relatives" (Hoen et al., 2016) and they might have common ancestry roots.
There are two possible assumptions: either enveloped viruses, such as SARS-CoV-2, developed from primitive lipid vesicles by encapsulating nucleic acids and incorporating specific membrane molecules for which cells have receptors; or EVs evolved as defective viruses that lost the machinery for nucleic acid replication and membrane molecules that mediate viral infection (Margolis & Sadovsky, 2019). SARS-CoV-2 is part of the coronavirus family, whose birth estimates vary widely, from 10,000 years ago to 300 million years ago. We are now aware of dozens of strains (Graham et al., 2013) (Gorbalenya et al., 2020), seven of which infect humans. Among the four that cause common colds, two (OC43 and HKU1) would come from rodents, and the other two (229E and NL63) from bats. The three that cause severe disease -SARS-CoV, MERS-CoV and SARS-CoV-2all are believed to come from bats (Cyranoski, 2020). For SARS-CoV, RNA-binding proteins were detected, among other host proteins, suggesting host RNA were also present since they are packed together (Neuman et al., 2008). Therefore, it is expected to find host cell molecules in SARS-CoV-2 as well although their identity is currently unknown.

SARS-CoV-2 and EV intracellular crosstalk
EV cargo from SARS-CoV-2 infected cells Given the vesicular machinery crosstalk mentioned above, it is reasonable to hypothesise that EVs secreted by SARS-CoV-2 infected cells contain viral proteins and RNA cargo. It is also possible that SARS-CoV-2 might tag along with EVs whose entry is cleared by the receiving cell. HIV-1 was speculated to bind to EVs via TIM-4, CD9 and/or CD81 (Sims et al., 2018).

Potential role of EVs in promoting or blocking SARS-CoV-2 infection
Interestingly, virus-infected cells tend to produce more EVs than virions, already suggesting that they It is tempting to speculate that engineered EVs might be used to compete or interfere with virus entry and infection but such approaches have not been tested.

Discussion
Helping the global research & diagnostics effort on SARS-CoV-2 with novel EV tools There are two main challenges for SARS-CoV-2 research: low-contaminant isolation & functional virion quantification in various biospecimens.
Regarding virus isolation in infected patients or cell media culture, they both contain a high amount of small EVs whose biochemical similarities (size, density) (Renner et al., 2018) make them indistinguishable from most of viruses for standard techniques (e.g. ultracentrifugation or size exclusion chromatography). They are therefore co-isolated together -along with other contaminants-, creating problems for downstream research applications such as mass spectrometry or X-ray crystallography (Mateu, 2013), (Greco et al., 2014). Besides, hybrid virion-EVs may complicate things further (Hoen et al., 2016). Novel EV immuno-isolation techniques (with one or two surface markers) could eliminate unwanted EVs or simply be repurposed to isolate the virus using its unique surface markers (e.g. Spike protein, Envelope protein and/or Membrane protein). It is interesting to note that SARS-CoV-2 may also present tetraspanins and other host molecules on its surface, therefore standard, single marker isolation approaches might not be sufficient.
Once separated, the virions could be lysed to study more stable virion proteins than fast-mutating Spike protein. This could be very valuable (Schoeman & Fielding, 2019) in designing more sustainable SARS-CoV-2 vaccines.
Regarding functional virion quantification, it may be useful to better understand virulence factors, such as disease progression, by identifying viable versus non-viable particles. Accurate isolation and quantification approaches using novel EV capture and quantification tools modified to handle viruses, could facilitate patient stratification and prognosis efforts.
There is one remaining clinical challenge for COVID-19 diagnostics which could be dealt with by using In terms of antiviral, engineered EVs can also be used for targeted drug delivery by using the same viral protein that binds cells susceptible to being infected. Alternatively, some EVs have natural affinity to inflammation sites. Platelet-derived EVs loaded with anti-inflammatory TPCA-1 were shown to calm cytokine storm, often associated with severe COVID-19 cases, in mice model with acute lung injury (Ma et al., 2020). EVs expressing specific proteins, such as ACE2, could act as virus binders and thereby reduce infectivity.
Another promising prospect of EV-based therapeutics is the administration of mesenchymal stem cells (MSCs)-derived EVs to reduce inflammation and injury in respiratory diseases. This is currently being tested in clinical trials in China for to the treatment of COVID-19 related severe pneumonia