Viral Extracellular Vesicles known as ‘Stealth Spheres’

[Nuno]

Protease inhibitors generally only work on the specific virus they were designed for.

Thanks for clarifying, I was a bit confused on that matter. So, for example, let's take this new Pfizer antiviral
PF-07321332/Poxlovid, it is an orally active 3CL active protease inhibitor, would you say this is just effective against SARSCOV2 protease or other RNA viruses too?

Impossible to answer, I know but just theorically speaking, has a protease inhibitor for a virus never worked for other viruses?

Lastly, on another note, what about GC376? Both GC373 and GC376 are broad spectrum antivirals, which means should work for other viral proteases? What are your thoughts?

Thx for taking the time!

 

[Pyrrhus]

just theorically speaking, has a protease inhibitor for a virus never worked for other viruses?

It's possible if the viruses happen to have similar proteases.

But proteases are more subject to mutation than the RNA-dependent RNA polymerase (RdRp). This means that different viruses generally have evolved different proteases. This also means that it can be easier for a virus to mutate its protease to develop resistance to a protease inhibitor drug.

 

[Wishful]

Hypothesis: me/cfs is a failure of lipid metabolism

FWIW, I had significant but temporary benefits from Evening Primrose Oil and also from conjugated linoleic acids, so I'm willing to accept that lipid membranes might be involved. I'm not rating it high for being the root cause of ME. If it were, I'd expect more variation in severity from changes in diet.

 

[Pyrrhus]

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/

SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum (Knoops et al., 2008)
https://pubmed.ncbi.nlm.nih.gov/18798692/

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?

Now we have some evidence that the SARS-CoV-2 coronavirus might be able to spread directly from cell to cell using exosomes, bypassing both antibodies and the ACE2 cell entry receptor:

SARS-CoV-2 spreads through cell-to-cell transmission (Zeng et al., 2022)
https://doi.org/10.1073/pnas.2111400119

First they describe the known fact that SARS-CoV-2 can spread by fusing cells together to form large clumps called syncytia:

In this work, we show, by using lentiviral pseudotyped virus, that the spike protein of SARS-CoV-2 mediates the viral cell-to-cell transmission. [...] We also find that cell–cell fusion contributes to cell-to-cell transmission, yet ACE2 is not absolutely required.

Then they describe that endosomal pathway inhibitors can prevent cell-to-cell spread, implicating transmission by exosomes:

Herein, we provide evidence that SARS-CoV-2 spreads through cell–cell contact in cultures, mediated by the spike glycoprotein. [...] Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients.
[...]
Enveloped viruses spread in cultured cells and tissues via two routes: by cell-free particles and through cell–cell contact. The latter mode of viral transmission normally involves tight cell–cell contacts, sometimes forming virological synapses, where local viral particle density increases, resulting in efficient transfer of virus to neighboring cells. Additionally, cell-to-cell transmission has the ability to evade antibody neutralization, accounting for efficient virus spread and pathogenesis, as has been shown for HIV and hepatitis C virus (HCV).
[...]
Indeed, cell–cell fusion has been recognized as an important mechanism of cell-to-cell infection for a number of enveloped viruses, including herpesviruses, paramyxoviruses, and retroviruses. However, it must be emphasized that extensive cell–cell fusion by SARS-CoV-2 spike can lead to giant syncytia formation and cell death, which in turn reduces cell-to-cell transmission. While we were able to confirm the cell-to-cell infection of SARS-CoV-2 using the authentic WA-1 strain, syncytia formation was not evident in most cases.
[...]
A surprising result to emerge from our studies was that, despite the critical role of cell–cell contact and plasma membrane–mediated fusion, endosomal entry pathways were also involved in cell-to-cell transmission of SARS-CoV-2 and SARS-CoV (Fig. 5). This is evidenced by the inhibitory effect of drugs that specifically target the endosomal entry pathway of these viruses.

 

[Wishful]

Researchers are studying exosomes in terms of what they might be carrying into cells, but I wonder whether any are looking at what they do to cell membranes. Exosomes are small spheres of membrane (with or without anything inside). When they bind to a cell membrane, do they not for a period of time, create a 'patch' in the cell wall with different characteristics in terms of what they pass through or block? A cell membrane might be tailored to pass specific molecules through at a specific rate. There's no reason why an exosome should have exactly those same characteristics. So, exosomes could alter cell functions regardless of what might be inside the exosome.

If exosomes were the 'something in the blood' that affects cell function, it's possible that it's due to this change in cell membrane characteristics. Hopefully some researchers will read this and add it to their list of things to think about when trying to figure out why cells are misbehaving.

 

[Pyrrhus]

A cell membrane might be tailored to pass specific molecules through at a specific rate. There's no reason why an exosome should have exactly those same characteristics. So, exosomes could alter cell functions regardless of what might be inside the exosome.

That's an interesting point. I assume that once the exosome's membrane is incorporated into the receptor cell's membrane, the membrane is "re-modeled" to conform to the receptor cell's requirements. But how quickly and how well is it remodeled?

 

[Wishful]

But how quickly and how well is it remodeled?

Yes, and it probably depends on the type of cell. Something stable such as bone cells might take a long time to return to normal. Microglia, which constantly send out 'tentacles' might fix themselves faster. If exosome membranes do have effects on cells, the different characteristics could explain why ME varies so much between individuals.

This came about because I was thinking about exosomes, and realized that the 'wrappers' don't simply vanish. That made me wonder whether anyone had researched what effects those wrappers have.

 

[Pyrrhus]

Now we have some evidence that the SARS-CoV-2 coronavirus might be able to spread directly from cell to cell using exosomes, bypassing both antibodies and the ACE2 cell entry receptor:

SARS-CoV-2 spreads through cell-to-cell transmission (Zeng et al., 2022)
https://doi.org/10.1073/pnas.2111400119

And here's a paper that provides evidence that coronaviruses can use a form of direct vesicular cell-to-cell spread across neurological synapses, which helps to explain how coronaviruses can travel along cranial nerves to infect the brain, bypassing both the blood-brain-barrier as well as the cerebrospinal fluid that surrounds the brain:

Neurotropic virus tracing suggests a membranous-coating-mediated mechanism for transsynaptic communication (Li et al., 2012)
https://doi.org/10.1002/cne.23171

Swine hemagglutinating encephalomyelitis virus (HEV) [ is a coronavirus that ] has been shown to have a capability to propagate via neural circuits to the central nervous system after peripheral inoculation, resulting in acute deadly encephalomyelitis in natural host piglets as well as in experimental younger rodents.

This study has systematically examined the assembly and dissemination of HEV 67N in the primary motor cortex of infected rats and provides additional evidence indicating that membranous-coating-mediated endo-/exocytosis can be used by HEV for its transsynaptic transfer.

In addition, our results suggested that this transsynaptic pathway could be adapted for larger granular materials, such as viruses. These findings should help in understanding the mechanisms underlying coronavirus infections as well as the intercellular exchanges occurring at the synaptic junctions.

NB: Lead author is neuroscientist Yan-Chao Li of Jilin University, who has spent the last 15 years studying coronavirus infections of the nervous system. Spacing added for readability.

 

[Pyrrhus]

Now we have some evidence that the SARS-CoV-2 coronavirus might be able to spread directly from cell to cell using exosomes, bypassing both antibodies and the ACE2 cell entry receptor:

And now we have some evidence that the SARS-CoV-2 coronavirus might be able to spread directly from cell to cell using Tunneling Nano-Tubes (TNT):

Tunneling nanotubes provide a route for SARS-CoV-2 spreading (Pepe et al., 2022)
https://doi.org/10.1126/sciadv.abo0171

Excerpt:

Neurological manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection represent a major issue in long coronavirus disease. How SARS-CoV-2 gains access to the brain and how infection leads to neurological symptoms are not clear because the principal means of viral entry by endocytosis, the angiotensin-converting enzyme 2 receptor, are barely detectable in the brain.

We report that human neuronal cells, nonpermissive to infection through the endocytic pathway, can be infected when cocultured with permissive infected epithelial cells.

SARS-CoV-2 induces the formation of tunneling nanotubes (TNTs) and exploits this route to spread to uninfected cells. In cellulo correlative fluorescence and cryo–electron tomography reveal that SARS-CoV-2 is associated with TNTs between permissive cells.

Furthermore, multiple vesicular structures such as double-membrane vesicles, sites of viral replication, are observed inside TNTs between permissive and nonpermissive cells. Our data highlight a previously unknown mechanism of SARS-CoV-2 spreading, likely used as a route to invade nonpermissive cells and potentiate infection in permissive cells.

(spacing added for readability)

 

[Pyrrhus]

And here's a paper that provides evidence that coronaviruses can use a form of direct vesicular cell-to-cell spread across neurological synapses, which helps to explain how coronaviruses can travel along cranial nerves to infect the brain, bypassing both the blood-brain-barrier as well as the cerebrospinal fluid that surrounds the brain:

And here's a paper that found direct cell-to-cell transfer of coronavirus by both extracellular vesicles and by tunneling nanotubes:

An intra-cytoplasmic route for SARS-CoV-2 transmission unveiled by Helium-ion microscopy (Merolli et al., 2022)
https://doi.org/10.1038/s41598-022-07867-0

Excerpt:

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism.

However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2.

Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes.

Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.

(spacing and emphasis added for readability)

 

[Pyrrhus]

Related discussion:

How Enteroviruses May Spread From Cell to Cell
https://forums.phoenixrising.me/thr...roviruses-may-spread-from-cell-to-cell.19597/