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Viral Extracellular Vesicles known as ‘Stealth Spheres’

Pyrrhus

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For those of you wondering what the difference is between an "exosome" and an "extracellular vesicle":
  • A "vesicle" is like a bubble. It is surrounded by a fatty membrane and is filled with water and other things.
  • An "extracellular vesicle" is any vesicle that is found outside a cell.
  • An "exosome" is a special type of extracellular vesicle derived from an "endosome".
I recently found this review paper from the great Karla Kierkegaard:

Escape of non-enveloped virus from intact cells (Bird and Kierkegaard, 2015)
https://pubmed.ncbi.nlm.nih.gov/25890822/

Among other things, it discusses how enteroviruses encase themselves inside intracellular vesicles which then exit the cell as extracellular vesicles.
These extracellular vesicles containing enteroviruses are not exosomes, and appear to have sizes roughly from 40-800nm.

Well, this is interesting.

In addition to enteroviruses using the larger type of extracellular vesicles, here's a publication that found enteroviruses inside smaller-sized exosomes:

Exosomes cloak the virion to transmit Enterovirus 71 non-lytically (Gu et al., 2019)
https://www.tandfonline.com/doi/full/10.1080/21505594.2019.1705022

Screen Shot 2021-06-09 at 7.36.12 PM.png


Excerpt:
Gu et al 2019 said:
Enterovirus 71 (EV71) is a non-enveloped virus and it can be released from host cells through a traditional cytolytic manner. Now, we showed EV71 could be spread non-lytically between cells during early viral infection.

In order to explain this phenomenon, we separated supernatant fluids of rhabdomyosarcoma (RD) cells cultures infected with EV71 by isopycnic gradient centrifugation. Two populations of virus particles were morphology indistinguishable by transmission electron microscope (TEM).

It showed that some EV71 particles were wrapped inside extracellular vesicles which were verified to be exosomes by immunoassay and morphologic analysis. In addition, exosomes containing viral RNA were shed in plasma of EV71-infected encephalitis in children. Our findings indicate that the “non-enveloped” EV71 virions could be wrapped within exosomes which promote their spread in the absence of cell lysis.
[...]
Notably, two populations showed different morphologies. The population at high density (1.18–1.26 g cm−3) contained plentiful 27–30 nm EV71 particles (d in Figure 2(b)). The population at a low density (1.10–1.12 g cm−3) contained some 30–150 nm vesicles (a-c in Figure 2(b)) wrapping between one and four virus-like particles with morphology similar to 27–30 nm EV71 particles in dense fractions (d in Figure 2(b)), and besides empty exosomes, the distribution of the number of virion-like particles inside one vesicle is shown in Figure 2(c).

Furthermore, Nanoparticle Tracking Analysis (NTA) revealed these vesicles with low density (1.10–1.12 g cm-3), with diameter concentrated on the size of 101.8 nm (Figure 2(d)), which was consistent with exosomes in dimension. Besides, exosomes markers CD63 and TSG101 and viral protein VP1 could be detected in these vesicles by immunoblots, while albumin and calnexin(CANX) as a negative control to confirm no cell debris in exosomes preparation (Figure 2(e)). These results suggested that EV71 virions were wrapped within exosomes in the supernatant fluids of EV71-infected RD cells.
 
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246
Hypothesis: me/cfs is a failure of lipid metabolism that manifests in energy deficiencies, and also a deficiency of certain cellular membrane structures that rely on those lipids. The membrane deficiencies lead to problems in the gut, the endothelial system, the blood-brain barrier, connective tissues and problems with production of exosomes.

I don't think that is what is going on in my case, I took all sorts of phospholipids for several weeks and it did help for a bit but then made me worse.
 

Pyrrhus

Senior Member
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I recently found this review paper from the great Karla Kierkegaard:

Escape of non-enveloped virus from intact cells (Bird and Kierkegaard, 2015)
https://pubmed.ncbi.nlm.nih.gov/25890822/

Among other things, it discusses how enteroviruses encase themselves inside intracellular vesicles which then exit the cell as extracellular vesicles.


It appears that coronaviruses also use this type of intracellular double-membraned vesicles:


Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex (Snijder et al., 2006)
https://pubmed.ncbi.nlm.nih.gov/16731931/
Excerpt:
Snijder et al 2006 said:
The RNA replication complexes of mammalian positive-stranded RNA viruses are generally associated with (modified) intracellular membranes, a feature thought to be important for creating an environment suitable for viral RNA synthesis, recruitment of host components, and possibly evasion of host defense mechanisms.
[...]
Electron microscopy revealed the early formation and accumulation of typical double-membrane vesicles, which probably carry the viral replication complex. [...] In immunoelectron microscopy, the virus-induced vesicles could be labeled with replicase-specific antibodies.
[...]
Similar morphological studies and labeling experiments argued against the previously proposed involvement of the autophagic pathway as the source for the vesicles with which the replicase is associated and instead suggested the endoplasmic reticulum to be the most likely donor of the membranes that carry the SARS-CoV replication complex.


SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum (Knoops et al., 2008)
https://pubmed.ncbi.nlm.nih.gov/18798692/
Excerpt:
Knoops et al 2008 said:
Poorly characterized double-membrane vesicles (DMVs) were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution.

Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200-300 nm), and "vesicle packets" apparently arising from DMV merger.

The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior.

Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms.
(spacing added for readability)


These papers raise the question:

Can coronaviruses export these intracellular double-membraned vesicles as single-membraned extracellular vesicles, and if so, does this represent a mode of transmission between cells?
 

Pyrrhus

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I recently found this review paper from the great Karla Kierkegaard:

Escape of non-enveloped virus from intact cells (Bird and Kierkegaard, 2015)
https://pubmed.ncbi.nlm.nih.gov/25890822/

Among other things, it discusses how enteroviruses encase themselves inside intracellular vesicles which then exit the cell as extracellular vesicles.


And here's a paper from Ralph Feuer's group that describes how researchers used an engineered fluorescent viral protein and time-lapse photography to show how the autophagic-pathway intracellular vesicles exit the cell as extracellular vesicles. Very cool.

Coxsackievirus B Exits the Host Cell in Shed Microvesicles Displaying Autophagosomal Markers (Robinson et al., 2014)
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004045

They refer to this process as "autophagosome-mediated exit without lysis (AWOL)":

1628031215881.png




The size of these extracellular vesicles are similar to the size of exosomes, but these are produced through an autophagic pathway, not through an endosomal pathway, so I presume that they are not exosomes. (green arrows below)

1628031750566.png
 
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sometexan84

Senior Member
Messages
1,229
Wanna hear something interesting?

(hopefully you didn't already cover this, I skimmed... didn't see it...so)

I posted elsewhere, no one seemed to care, but at least it's appropriate for this thread. I'm pretty much just copy/pasting fyi

1) Very interesting seeing Enterovirus virions are transported via EVs of Phosphatidylserine.

Like a Phosphatidylserine lipid Trojan horse.

BTW - I had positive Phosphatidylserine antibodies.. coincidence????

2) Basically, Enterovirus inhibits mTORC1.... which activates TFEB... which then "facilitates the release of virions in extracellular vesicles via secretory autophagy"

from...
https://www.nature.com/articles/s41467-020-18168-3
and https://www.cell.com/cell/fulltext/S0092-8674(15)00075-6


3) I found out the exact same thing is happening in SARS-CoV-2. Exact Same, even through the lipid phosphatidylserine! Which they believe is causing the "thromboinflammation" they've seen. I didn't save the article link though...
 

Pyrrhus

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Very interesting seeing Enterovirus virions are transported via EVs of Phosphatidylserine.

Thanks for highlighting the important role of exposed phosphatidyl-serine (PS) on the surface of the extracellular vesicles.

The role of PS is indeed fascinating, as discussed in the (Chen et al., 2015) paper you mentioned. (also mentioned in the original post of this thread)

BTW - I had positive Phosphatidylserine antibodies.. coincidence????

That is very interesting. As you know, PS is also exposed on proto-apoptotic cells, so I wonder if the development of the antibodies could have been due to either PS-enriched extracellular vesicles or exposed-PS proto-apoptotic cells...


That's a good paper. I'm going to quote an excerpt here for the benefit of others:

Dynamic remodelling of the human host cell proteome and phosphoproteome upon enterovirus infection (Giansanti et al., 2020)
https://www.nature.com/articles/s41467-020-18168-3

Excerpt:
Giansanti et al 2020 said:
The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3.

Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later.

Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy.

Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.
 

sometexan84

Senior Member
Messages
1,229
That is very interesting. As you know, PS is also exposed on proto-apoptotic cells, so I wonder if the development of the antibodies could have been due to either PS-enriched extracellular vesicles or exposed-PS proto-apoptotic cells...
I'm not sure I follow. You're saying PS antibodies might be increased due to an increase in damaged cells?

I know they've found PS antibodies more in long covid and ME/CFS than in healthy people. Though, it's like 5% compared to 1%.

I just see molecular mimicry.. though maybe i'm over simplifying.
 

Pyrrhus

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I'm not sure I follow. You're saying PS antibodies might be increased due to an increase in damaged cells?

Yes, one of the first events in apoptosis is the "flipping" of phosphatidyl-serine (PS) from its normally hidden location on the inside surface of the cell membrane to the outside surface of the cell membrane, where it can then be seen by the immune system.

Therefore, damaged or infected cells may display PS on its outside surface, whereas healthy cells conceal the PS on its inside surface. (This is the fundamental principle behind some antiviral strategies.)

I just see molecular mimicry.. though maybe i'm over simplifying.

I doubt that "molecular mimicry" would be involved here- I think that "epitope spreading" might be a more applicable explanation for the emergence of auto-antibodies.

EDIT:
For more information on molecular mimicry and epitope spreading, see:
How Viruses Cause Autoimmunity
https://forums.phoenixrising.me/threads/how-viruses-cause-autoimmunity.86598/
 
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Pyrrhus

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Therefore, damaged or infected cells may display PS on its outside surface, whereas healthy cells conceal the PS on its inside surface. (This is the fundamental principle behind DRACO.)

My bad. I mixed up DRACO/VTose with a different broad-spectrum antiviral strategy.

DRACO/VTose targets cells with double-stranded RNA (dsRNA).

The other broad-spectrum antiviral strategy targets cells with exposed phosphatidylserine (PS).

:headslap:
 

Learner1

Senior Member
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I did a search for PS and HHV6 and this article popped up, which seems to fit with what you're saying above.

To explore whether an alternative apoptosis-initiated replication program is a common feature of herpesvirus biology, we studied cell lines latently infected with Epstein-Barr virus/HHV-4, HHV-6A, HHV-6B, HHV-7, and KSHV.

We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication. An alternative apoptosis-initiated replication program appears to be a common feature of HHV biology.

We also found that commonly used cytotoxic chemotherapeutic agents activate HHV replication, which suggests that treatments that promote apoptosis may lead to activation of latent herpesviruses, with potential clinical significance.
From:

https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC3807386/



This makes me more than a little annoyed, having had cytotoxic chemotherapeutic agents reactivate HHV6 and EBV it to be gaslit by the cancer community.


On a more hopeful note, one of my doctors is suggesting exosomes to tackle my HHV6 and HSV2 if the Famvir and Valganciclovir I'm currently on don't work. I'm skeptical, but interested in how to apply the above conversation to this problem. At the very least, seems like the right exosomes should be used.
 

Pyrrhus

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The other broad-spectrum antiviral strategy targets cells with exposed phosphatidylserine (PS).

Here is that approach I was talking about:

Targeting Inside-Out Phosphatidylserine as a Therapeutic Strategy For Viral Diseases (Soares et al., 2008)
https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC2597367/pdf/nihms71262.pdf

Commercialized as "Bavituximab":
https://en.wikipedia.org/wiki/Bavituximab


We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication.
From:

https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC3807386/

Wow. Well there you have it.

Herpesviruses certainly know how to make the best of a bad situation.

When a herpesvirus-infected cell is dying, the herpesvirus jumps into action to preserve itself and spread to new cells.
 

Pyrrhus

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Here's another intriguing broad-spectrum anti-viral strategy, more applicable to the current discussion:

FGI-104: a broad-spectrum small molecule inhibitor of viral infection (Kinch et al., 2009)
https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC2776286/


This approach targets cells with exposed TSG101, which is a protein used in the viral export of intracellular vesicles as extracellular vesicles.
 

Pyrrhus

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And here's a paper from the great Raul Andino (of quasispecies fame) which describes how the intracellular vesicles of enteroviruses crowd around lipid droplets in order to use the lipids to rapidly construct new intracellular vesicles.

Since viral replication occurs in these intracellular vesicles, the intracellular vesicles are referred to in the paper as "replication organelles" or "replication compartments (RC)". The 2C viral protein is shown in the paper to connect the replication compartments to the lipid droplets.

Viral Generated Inter-Organelle Contacts Redirect Lipid Flux for Genome Replication (Laufman, Perrino, and Andino, 2019)
https://www.cell.com/cell/fulltext/S0092-8674(19)30558-6

Graphical abstract:
1630526517652.png



And here are some Electron Micrographs where you can see the replication complexes (RC) crowding around the lipid droplets (LD):
1630526747425.png
 

sometexan84

Senior Member
Messages
1,229
And here's a paper from the great Raul Andino (of quasispecies fame) which describes how the intracellular vesicles of enteroviruses crowd around lipid droplets in order to use the lipids to rapidly construct new intracellular vesicles.

Since viral replication occurs in these intracellular vesicles, the intracellular vesicles are referred to in the paper as "replication organelles" or "replication compartments (RC)". The 2C viral protein is shown in the paper to connect the replication compartments to the lipid droplets.

Viral Generated Inter-Organelle Contacts Redirect Lipid Flux for Genome Replication (Laufman, Perrino, and Andino, 2019)
https://www.cell.com/cell/fulltext/S0092-8674(19)30558-6

Graphical abstract:
View attachment 44582


And here are some Electron Micrographs where you can see the replication complexes (RC) crowding around the lipid droplets (LD):
View attachment 44583
Shows that a 3C protease inhibitor would be nice. Or confirms it really.

3C cleaving the 2BC, which turn into 2B, and 2C. And it's these guys here that appear to be involved in this process of replication.

Stop 3C, and it prevents this. Among other things.

There's actually big news w/ Pfizer, and it's new 3C inhibitor of SARS-CoV-2 (PF-07321332). About to start trials - https://www.pfizer.com/news/press-r...-initiates-phase-1-study-novel-oral-antiviral
 

Pyrrhus

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And here's a 2020 paper that clearly shows that the enteroviral extracellular vesicles protect the virus from any anti-enterovirus antibodies:

Extracellular Vesicles Released by Enterovirus-Infected EndoC-βH1 Cells Mediate Non-Lytic Viral Spread (Netanyah et al., 2020)
https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC7695210/pdf/microorganisms-08-01753.pdf

Our results showed that [extracellular vesicles] from echovirus 16-infected EndoC-βH1 cells harbor infectious viruses and promote their spread during the pre-lytic phase of infection. Furthermore, the [extracellular vesicles]-mediated infection was not inhibited by virus-specific neutralizing antibodies. In summary, this study demonstrated that enteroviruses could exit beta cells non-lytically within infectious [extracellular vesicles], thereby thwarting the access of neutralizing antibodies to viral particles.
1636237837757.png



And here's a picture of one of these extracellular vesicles, for size comparison. The virus inside the extracellular vesicle is denoted with a black arrow:
1636237726845.png
 

Nuno

Senior Member
Messages
112
Great work on here @Pyrrhus I've been following this thread all along, can't deny 😅

Anyways, do we know if 3C Protease Inhibitors have ever been tried in MECFS? Either on trials or by some experiments from someone? Would be interesting to know.

As they were quite a game changer when first developed for HIV, and now theyre also on work on the new Pfizer Poxlavid (I think thats the name) antiviral which I am interested aswell.
 
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