Exosomes and viruses

I propose here to postulate, in the light of the latest data on these molecular mechanisms, that the viral processes are in reality in most cases, under the control of the body, and that exosomes and viruses are all variations of an universal mailing system shared by all of nature for billions of years.
I gathered evidences from cell biology demonstrating our ignorance of the fundamental nature of retroviruses or viruses in general, and exosomes in particular. This is what I will try to show by using studies that have been done on the dynamics and behavior of the different structures or molecules that will serve my purpose (prion, various vesicles, within the cell or between cells.

Among viruses, those that interest us are the enveloping viruses because they cover their protein capsid (if they have one) and their genetic material by one (or more) lipid bilayer. They can derive either from nuclear membranes like the envelopes of Herpesviridae or from plasma membranes for Orthomyxoviridae. The genetic material of these viruses can be of any type: RNA, DNA, single or double stranded.

In theory, these vesicles are not related despite their similarity because unlike viruses they do not reproduce. However, contemporary virology has moved away from this strict definition by its wide use of the terms non-infectious and defective virus. Therefore, extracellular vesicles generated by retrovirus-infected cells that carry viral proteins and even fragments of viral genomes essentially fall into this category of non-infectious viruses.

The vesicles are surrounded by a lipid membrane that also contains cell membrane proteins. Like those of viral envelopes, these proteins can determine adhesion to the plasma membrane of specific target cells. In addition to DNA polymerase activity, tumor vesicles also showed endogenous reverse transcriptase activity more commonly associated with viruses.

Despite the important differences in membrane morphology, recent research has revealed a variety of similar mechanisms in bacteria, pointing to an identity of form and function of vesicular formations between the three domains of life (bacteria, archaea and eukaryotes or cells with nuclei including animal, plant or fungal organisms, unicellular or multicellular). I will only mention them here in the spirit of completeness.

Bacteria have on their inner membrane a layer of peptidoglycan, a rigid substance giving very particular conditions for the budding of vesicles on the surface. The vast majority of bacteria also have a layer of external lipopolysaccharide (LPS) and underneath a little peptidoglycan located in the periplasmic space. They are generally referred to as Gram-negative or bidermic bacteria.
In contrast, most bacteria in the phylum Firmicutes have a single membrane covered by a thick layer of peptidoglycan. The phylum Actinobacteria is quite distinct from the classical types, their thin peptidoglycan layer is directly covered by a thick polysaccharide layer. Many bacteria also contain a protein S-layer.

Several types of extracellular vesicles have been described¹, the most studied being formed by budding from the LPS-containing outer membrane (OM) and containing mainly periplasmic components, however, some formed from the outer and inner membranes of didermatid bacteria have been identified in several species, and contained elements of the whole bacterium. Finally, vesicles have been observed in Gram+ bacteria with thick envelopes, crossing the thickness according to a mechanism that is still unknown and thought to be impossible.

The same functions are found as in eukaryotes and viruses: resistance of vesicles to enzymatic attack, allowing the delivery of contents at long distance, with the same specificity towards a target cell of the host organism, and to foil the immune system of the latter² for example, by carrying specific antigens serving as decoys³. They can also help bacterial colonization by selectively killing or promoting the growth of other bacterial species⁴, by exchanging genetic material (transformasome). And as in the animal case, increased vesiculation may aid in removal of toxic by-products after exposure to stress⁵.

Vesicles are also associated with long filamentous structures connecting cells in all three domains of life.

a) Tunneling NanoTubes connecting eukaryotic (human) cells, with labeled vesicles indicated by arrow
b) ‘Nanotubes’ produced by the bacteria S. oneidensis form outer membrane extensions with regular constrictions forming vesicles
c) ‘Nanopods’ produced by the archaeon T. prieurii.

These nanotubes (membranous structures 50-200 nm in diameter and up to several cell diameters in length) often contain membrane-surrounded vesicle arrays, suggesting that they may be involved in vesicle production, particularly by some cancer cells.
Although studies are already piling up, we still know a lot more about eukaryotes.

Due to the small size and heterogeneity of vesicles, their detection and classification is difficult. Although different types of vesicles have been identified, widely used terms such as exosomes ectosomes and microparticles are often inconsistent, especially in the older literature.
In addition, the extent to which the various morphologies contribute to the processes studied is largely unknown. For practical reasons, the various terms are to be considered here roughly interchangeable. Electron microscopy remains the gold standard technique for determining vesicle morphology and size.

Exosomes can cross biological barriers (such as the blood-brain barrier) and penetrate cells with a high degree of specificity. Hence their significant interest as drug delivery agents and diagnostic biomarkers.

In general, their diameter does not exceed 120 nm, which is smaller than the maximum theoretical resolution of a conventional optical microscope. The fate and interactions of exosomes inside cells are therefore difficult to study, which limits investigation in the field⁶.

Several studies have shown that the populations of extracellular vesicles are very heterogeneous, even in pure cell culture, each cell type being able to produce different types. However, it seems that some are produced exclusively by certain cells. To further complicate matters, the content of extracellular vesicles varies depending on the source and the original isolation or enrichment technique.

Summary of different morphologies

In 2007, Lötvall demonstrates that eukaryotic extracellular vesicles (exosomes) contain large amounts of RNA of all kinds and transfer these to target cells⁷ After that, it was found that the RNA content was highly specific, and sometimes enriched several thousand times relative to their host cells, even between different subpopulations of the exosomes which suggests an active RNA packaging mechanism⁸.

Although there is intact mRNA and long non-coding RNA present in EVs, most RNAs are fragmented or small in size. As mentioned above, these RNAs encapsulated in vesicles can have a profound impact on recipient cells, transferring between different cell types a signal causing transient transformation of recipient cells e.g. leading to the production of new proteins (case of mRNA transfer), or regulation of gene expression in the case of miRNAs.

The same researcher, in 2013 found that RNAs isolated from apoptotic vesicles (forming after programmed cell death), membrane vesicles and exosomes have very different profiles. Membrane vesicles have the least amount of RNA, apoptotic vesicles the most. This could reflect an active process of communication to other surrounding cells informing about the causes and conditions of apoptotic cell death.

Vesicle Formation

The classical pathway involves the formation of intraluminal vesicles (ILVs) in MVEs (Multivesicular endosomes). In turn, the MVE membrane fuses with the plasma membrane, resulting in the release of VILs. When secreted, VILs are called exosomes. Alternatively, the direct pathway involves the release of vesicles, indistinguishable from exosomes, directly from the plasma membrane.
In most cases, the classical pathway is controlled by the proteins of the ESCRT pathway (for endosomal sorting complexes required for transport), and the Rab-GTPases proteins for addressing the cell surface. An influx of intracytoplasmic calcium coupled with cytoskeletal remodeling will lead to externalization of phosphatidylserine and budding.

In addition, ESCRT-independent pathways are probably involved in exosome formation, although they are less well understood. Indeed, depletion of key proteins in different ESCRT complexes does not abolish the formation of multivesicular bodies (temporary organelles of common vesicle formation).

These ESCRT-independent mechanisms are thought to involve HSPs (Heat Shock Proteins),lipids, and tetraspanins: the lipid composition of small vesicles is different from that of the mother cell, although it is related to cell type. As for structural lipids, small EVs are enriched in cholesterol and sphingolipids, indicating a membrane composition similar to that of the lipid rafts floating on the surface of the mother cell, and involved in a large number of interactions. In addition to structural lipids, extracellular vesicles also contain active lipids such as prostaglandins and lysophospholipids.

Redundant pathways are a common phenomenon in biology, and thus multiple non-exclusive mechanisms may be responsible for sorting various proteins for exosomal export.

Little or nothing is known about the loading of nucleic acids.
In one study, it was shown that double-stranded DNA would rather be adsorbed on the outside of vesicles and large vesicles carry most of the tumor double-stranded DNA circulating in the plasma of prostate cancer patients. Because of their oncogenic potential much data on extracellular vesicles in the context of tumors have been collected, but this also reflects the fact that the spread or functions of endogenous exosomes in vivo or data on adult tissues under healthy i.e. homeostatic equilibrium conditions are pretty much unknown, in comparison.

The existence of sorting mechanisms is assumed but not known. For example, Villarroya-Beltri et al⁹ found sequence motifs that are highly enriched in exosome-associated miRNAs (an important class with a ubiquitous role as a negative regulator of gene expression), compared to cellular miRNAs. It also determines that a specific protein ¹⁰ binds to these EXOmotifs to induce RNA loading.

Sequence motifs on messenger RNAs lead to vesicle enrichment via a mechanism involving interactions between mRNAs and microRNAs, sometimes involving specific transcription factors binding degenerate consensus sequences in the upstream untranslated region (UTR) of the RNA¹¹.

The general rules governing RNA loading efficiency are not well understood. Therapeutic uses are limited to applying an empirical method that works more or less, without knowing why, nor what will be the fate of said mRNA once the target cell is reached. But there is no longer any doubt that certain species of molecules are recruited in a selective and controlled manner.

Virus Formation

Conceptually, virus budding can be divided into two steps:

1. membrane deformation, when the membrane is wrapped around the assembling virion,
2. membrane fission, when the neck of the bud is severed.
   The structural proteins of enveloped viruses generally bind to membranes and form spherical or helical assemblies. Thus, assembly and budding are often inextricably linked processes. These fusion proteins undergo dramatic conformational changes, converting the free energy released by fusion protein folding into energy that is used to fuse viral and cellular membranes.

Budding is usually tightly coupled to virion assembly, so most viruses use their structural proteins to recruit the ESCRT pathway. It appears that they also use host factors¹², for example, in the GAG polyprotein (the main structural element of retroviruses), in HIV-1, the energy released is not required for Gag polymerization but for detaching nascent virions from the plasma membrane.
Subsequent studies identified two different short peptide motifs in p6 Gag that contributed to the efficiency of HIV-1 budding. In parallel, others have identified distinct short peptide motifs in the structural proteins of other viruses, called late assembly domains of which at least five distinct but interchangeable classes have subsequently been identified, often leading to the identification of analogous motifs within cellular proteins, recruiting ESCRT factors, an interaction verified experimentally.

Funny system where thieves and bankers agree on a common system of semaphores to keep each other in sight…In other cases, budding seems more complex or entirely independent of known cellular pathways.

Exosomes in the Facilitation of the Viral Process

Exosomes and viruses share, in addition to budding, probably the mechanisms of specific cargo packaging and membrane budding for cell release. Most surprisingly, different viruses with very different evolutionary pathways seem to converge in their use of the endocytic pathway for entry and exit of their host cells.

Similarly, the hepatitis C virus has been shown to incorporate its entire RNA genome into exosomes without surface proteins, leaving them infectious. Most of these data on the function of extracellular vesicles such as exosomes, however, are collected from transformed cancer cells and depend on the use of a heterogeneous population of vesicles purified from supernatant or liquid cell cultures.

Therefore, the spread of endogenous exosomes in vivo is largely unknown, and data on their biogenesis and role in normal development of tissues and adult tissues under homeostatic conditions are clearly lagging and understanding their transfer and fate in recipient cells is crucial.

The Trojan exosome hypothesis, which has since been proven, proposed that HIV evolved to use the exosomal system (more precisely ESCRT and Rab GTPases), in the absence of a receptor-based system. It is known that this is not unique to HIV, but common to both enveloped and non-enveloped viruses. This mechanism was demonstrated using HIV-infected dendritic cells, which were able to transfer the virus to closely associated uninfected T cells via exosomes… Furthermore, infectivity is reduced in the absence of exosomes, assuming that both are integral parts of the same mechanism.

It seems sound to me to consider the possibility that the viral process is only one aspect of intercellular communication, including between different organisms, although in ecological ways that are not yet understood.

Through microRNAs, some viruses can modulate cellular processes as diverse as immune evasion, apoptosis, proliferation, and even viral infectivity. These viral miRNAs, in conjunction with endogenous miRNAs, are thought to play a role in modulating the expression of target genes in recipient cells. In addition to RNAs, infected cells may also excrete specific viral proteins via exosomes, thereby modulating cellular processes in surrounding cells (cytokines, interferons).

For example: exosomes released from HTLV-1 (human T-lymphotropic virus 1) infected cells contain not only viral mRNA transcripts, such as those of Tax, HBZ and Env, but also the biologically active trans-activator protein Tax. In addition, the HTLV-1 Tax protein was shown in exosomes isolated from cerebrospinal fluid of patients with HTLV-1-associated tropical spastic paraparesis myelopathy, suggesting that HTLV-1 may modulate its microenvironment by selective secretion of specific viral cargoes.

**Substantial evidence indicates that many different types of pathogens, including bacteria, viruses, and protozoa ¹³ (and prions, see below) can exploit the exosomal pathway, for secretion or movement.

These parasites can also influence exported cellular products. A body of evidence indicates that exosomes in virus-infected cells can induce processes as diverse as immune evasion, apoptosis, angiogenesis, proliferation, trans-cellular spread and cytokine modulation. The molecular details of how these processes are triggered are poorly understood, and they differ between tissues, cell states, viruses…

Another well-known mechanism by which some viruses can evade immune responses is by down-regulating viral lytic gene expression and persisting in infected cells in a latent state, where the absence of viral antigen in infected cells means that the immune response cannot be triggered, but also simply that the virus is paused.

For example, herpes simplex type 1 (HSV-1) replicates in mucosal epithelial cells during primary infection and then enters the sensory neurons where it establishes a lifetime latency. During the latent state, although no viral protein is expressed, numerous miRNAs (viral microRNAs) have been detected, and some of these miRNAs appear to play a central role in suppressing viral gene expression and maintaining latency. And transmit antiviral factors, to the same effect.

There is some evidence that EVs, although less efficient than virions themselves, can transfer cytosolic proteins involved in antiviral responses, such as APOBEC3G and cGAMP (33-36), to recipient cells. In addition to miRNAs, extracellular vesicles also contain a wide variety of other small noncoding RNAs, such as fragments of protein-coding regions and repeat sequences, which could also act as regulatory RNAs by influencing gene expression.

In some viral infections, such as hepatitis B and herpes as well, non-infectious subviral particles are released into the serum, in some cases without viral capsid or viral DNA, often at levels 1000-fold higher than mature infectious particles. One hypothesis is that these subviruses would lure the immune system. In addition to immune modulation, exosomes released from some virus-infected cells may promote viral infection and spread as discussed with HIV-1.
In a similar vein, T cells can produce vesicles containing the HIV CD4 receptor, allowing them to bind to viral particles as antibodies would, thereby decreasing the number of virions that would otherwise affect CD4+ T cells.
Thus, the cells are able to selectively exchange antigens with each other, certainly to direct the process of clonal selection in secondary lymphoid organs.

Conversely, in vivo, exosomes can interact with viruses and each other directly or via modulation of host responses, thus participating in a war and peace between viruses and the host.
In 2014, exosomes derived from infected cells containing Tax proteins and pro-inflammatory factors, as well as viral mRNA transcripts including Tax, HBZ and Env (viral envelope protein) were found. In addition, giving these Tax-expressing exosomes to other cells improved their cell survival to Fas antibody treatment, indicating induction of NF-kB and activation of AKT (2 anti-apoptotic factors).
Another study found similar processes for HIV. When transferred via exosomes, TAR RNA can increase the population of susceptible target cells. Inside EV target cells, full-length TAR RNA is transformed into miRNAs, which quench the mRNA encoding the Bcl-2 interacting protein (involved in induction of apoptosis).

Viruses of several different species commonly were found ¹⁴ to travel in the same exosomes throughout the body, and thus were (contrary to what was thought) in physical proximity, which would allow for increased exchange.

The mentioned article still talks about exploitation of vesicular trafficking and genetic cooperation between viral species, as if it were a criminal alliance with a will to do harm. But viruses have no more will than they have metabolism, they are only molecular robots, and they are entirely passive.
In particular the content of HIV is very limited, known at the tip of one’s fingers. One could well compare it to a computer whose program in assembler has so well elaborated to take part of the instruction set of the CISC processor (for Complex Instruction Set Complex, hard to imagine more complex than an animal cell!). And to keep this extreme syntony, in spite of this same enormous mutation rate which allows it to escape the immune system and vaccine attempts.

So there is no reason why the interaction between viral proteins of different species should produce anything other than disorder: putting a helical gear there instead of a normal straight road is not known to improve a mechanism, and a cell is several hundred times more complex than a machine, in fact scientists compare it more to a whole city than to a factory. What kind of miracle of improbability does it take to make virologists question their view?

This proximity and this traffic, is one more proof of the arranged and programmed nature of the viral phenomenon, in concert with the cellular dynamics. And not the blind product of a mutative process still much more chaotic on average than our cells. Typically, influenza, HIV and coronaviruses mutate several dozen times. And yet the information necessary for proper functioning is maintained.
So we invoke, as always… the sacrosanct natural selection. But since it is an article of faith without the slightest statistical justification or mathematical model (the complete life cycle involving the whole cell, thus exceeding our computers by several millions or billions) we don’t need to ask ourselves any questions, and we will put forward the same argument no matter what the facts are that go against the grain and the enormity of the claim.

Simply, because there can be no other explanation: it is a dogma, a religion.

Other functional studies of exosomes released from infected cells have shown that infected cells release not only infectious virions, which are capable of spreading infection, but also a variety of non-replicative particles, which are difficult to classify, as they could be considered either as defective viral particles or vesicles containing viral elements, such as viral proteins and viral regulatory double-stranded DNA.

These non-replicating particles typically include cytidine deaminase, degrading retroviral RNA by random mutation, inhibiting viral replication. Exosomes containing host miRNAs produced by virus-resistant cells can confer resistance to other cells. This has been demonstrated for trophoblasts, which are largely resistant to infection by various viruses, including HIV, likely contributing to fetal protection in vivo. Exosomes produced by these cells in vitro carry host miRNAs and deliver them to virus-susceptible cells, making them resistant to virus infection.

One can see the analogy with the aforementioned virus defectives indistinguishable from exosomes, which confer resistance AND at the same time reduce the immune response. It seems to me that these are two sides of the same phenomenon.

Exosomes and the Removal of Denatured Proteins in Neurodegenerative diseases

In humans, prion disease occurs in sporadic, familial and acquired etiologies. However, all forms of the disease are transmissible, with possible routes of infection through dietary exposure, medical procedures and blood transfusion. The isoform of the normal prion protein, PrP C, is encoded by PRNP and is expressed in all tissues of the human body, with the highest levels of expression observed in central nervous system and brain tissues.

• PrPC and PrPSc have been isolated in association with exosomes, and PrPSc-containing exosomes were infectious in both animal and human cell bioassays.
• Alzheimer’s disease (AD) is the most common form of dementia in humans and is pathologically characterized by the extracellular deposition of insoluble amyloid plaques consisting of β-amyloid peptide (Aβ), a 39-43 amino acid peptide produced by proteolytic cleavage of the amyloid precursor protein. APP is cleaved from Aβ, left intact as a membrane associated fragment (β-CTF). These fragments accumulate intracellularly in multivesicular bodies and are incorporated into exosomes and released into the extracellular environment.

The identification of Aβ in association with exosomes is an important finding, especially since other exosomal proteins such as Alix and Flotillin-1 have been shown to accumulate in the brain plaques of Alzheimer’s patients.

Exosomes could also provide an explanation for the transport of equally toxic Aβ and APP-CTF around the body to the brain, where they contribute to amyloid deposition.

Underlying causes of neurodegenerative diseases are the folding and aggregation of specific proteins , such as amyloid β peptide (Aβ) in AD, scrapie associated prion protein (PrPSc) in prion disease, α-synuclein in PD, and superoxide dismutase 1 (oxidoreductase catalyzing the dismutation of superoxide anions O2–, involved in the neutralization of oxidative stress) in amyotrophic lateral sclerosis.

And all of these enzymes seem to be moving in the same direction: cleaning up, removing metabolic waste. Even if the theory still lacks precision, it seems clear that prion and prion-like diseases are neither infections or diseases, but essential vital clean processes of eliminating denatured molecules, AGEs in particular.

Alongside or at the same time as prions, the main proteins associated with these neurodegenerative disorders are found to colocalize on/in exosomes, containing binding sites for copper, zinc, iron and manganese. All of these have an obvious role in what would normally be detoxification: a dozen zinc transporters, ferroportin, transferrins, serotransferrin, several metalloreductases, ferritins, aconitate hydratase (sequester iron), ferroxidase, for iron.

We have already seen the role of AGEs and ALEs in disorders as varied as cancer, diabetes, aging. They are found colocalizing with AGEs receptors (RAGEs) in exosomes with Alzheimer’s disease ¹⁵.

RAGEs also bind the amyloid substance responsible for plaques, stimulating its production in the brain and regulating its influx across the blood-brain barrier. RAGE also promotes senile plaque formation via tau hyper-phosphorylation, synaptic dysfunction and neuronal death.

It is clear that prions were an extensive set of proteins with several functions unique to the brain (insofar as it is largely cut off from the normal blood and immune circulation), one being the sequestration of denatured molecules. However, the formation of plaque, linked to age and therefore to the level of intoxication, is not their property: rather, it is the consequence of a rate of intoxication completely unforeseen by nature and reaching such a peak in old people that the body is overwhelmed.

So we have seen infectious exosomes that are not viruses, and viruses that are not infectious, not quite infectious. Since the purpose of the phenomenon is not negative for the organism (beyond the individual cells), is it logical to always consider these viruses as foreign when we observe a whole continuum between self and non-self?

As far as the DNA content in eukaryotic extracellular vesicles is concerned, less is known than the RNA content. Single-stranded, mitochondrial, plasmid, and double-stranded DNA have been observed. The DNA seems to be present in the form of small fragments (about 10 to 20 kb) covering the whole genome including mitochondria without apparent order for the moment. However, it is important to note that some EV-associated DNA fragments contain entire genes with promoter and terminator regions. This DNA can be transported from cell to cell by endocytosis or fusion and this transfer can affect the transcriptional pattern of recipient cells, inducing both up- and down-regulation of many genes¹⁶.
Finally, not only RNA from enveloped retroviruses but also non-enveloped DNA viruses, such as herpesviruses, export their products and genetic material in the same way.
Many aspects of this mechanism remain to be clarified.

Just as the majority of viral infections are said to be frustrated, the detection of the pathological PrPSc isoform is not a sign of a disease, there would be thresholds, and an excess of normal forms would be able to trigger the conversion.

Conclusion

The consequent increase in resistance to apoptosis is said to allow the cell to produce a virus for a longer period of time, thus facilitating the spread of HIV. This is true, but the process is not automatic or a priori undergone by the cells and one can decently ask the question: why in the same package, incorporate virions at the same time as deaminases decreasing the efficiency of the viral load?

If we exclude this disturbing fact, the official theory of the pathogen already holds little when we consider that HIV for example would contain only 10 genes…

What an incredible compaction of information, if viruses can with so little orchestrate a whole set of cellular and intercellular reactions, and program exosomes to target specific cells or organs!

Well, essentially the same characteristics must be attributed to a simple deformed protein (the prion), having evolved according to the official theory to take control to the point of pushing distant organs to send their own quota of proteins to the brain.

We urgently need to change our perspective and see it as simply an information exchange protocol, just like sex hairs, more classical viruses, tunnels, circulating RNAs and maybe something else yet to be discovered. From my search of the literature, no one has thought of tracking the movement of a single AGE molecule, for example cells in the gut wall, from a nutrient solution that mimics the natural nutrient concentrations from a meal.
However, this would be technically feasible ¹⁷.

It is important to be able to show directly a correlation between reciprocal behavior in case of infection by different viral agents, and the treatment of this xenobiotic load and/or the packaging in the exosomes themselves. Similarly, tracking a prion or Aβ protein as it travels through the body would provide crucial information about how cells process the signal and pass it on, as on potentially several meters of transit there is a chance that the exosome carrying them will be contacted or even captured by cells for which they are not intended.