Testing for Cellular mRNA translation? (global host mRNA translation shut-off by Enterovirus)

[sometexan84]

Does anyone know how to test for mRNA translation activity?
(or maybe "protein synthesis" testing, or "gene expression" testing...????)

I've just been researching enterovirus a lot lately, and learned how it can completely inhibit host cellular mRNA translation. So, I'm trying to see if there are any tests that can measure (potential) repression of host mRNA translation.

My CFS was brought on by acute enterovirus (exacerbated by vigorous exercise, steroid treatment, and stress), so it's of great interest to me. If nothing else, re-writing all of this here helps me retain the info. So, not a total loss if no one else is interested in this.

More info about Enterovirus shutting down our mRNA...
Enterovirus is a sneaky SOB it turns out. It inhibits our ability to translate our own messenger RNA through multiple means. The mRNA is translated into amino acid and proteins to keep the body working how it's supposed to. But some viruses (like Enterovirus, including Coxsackievirus B and Echovirus) neutralize host mRNA translation, and instead translate viral mRNAs for their own purposes. Leading to host cellular protein synthesis shutoff.

1. eIF4G cleavage (the bad kind of cleavage) - Enteroviral 2A protease "cleaves" eIF4G (eukaryotic initiation factor 4 gamma) proteins. eIF4G cleavage by 2A protease inhibits host cellular mRNA translation. 2Apro is also key in ensuring optimum viral replication.
2. mTORC1 inhibition - This is a signaling pathway that regulates cellular metabolism, growth, proliferation and survival. Enterovirus (CVB3) inhibits mTORC1 activity, leading to repression of host mRNA translation.
   • Study from above link used CVB3. Haven't confirmed regarding the other Enterovirus Bs
3. Host Shut-off - mTORC1 inhibition along with eIF4G cleavage combine to achieve global host mRNA translation shut-off by Enterovirus.
   

Anyway, this is why I want to be able to measure this. This seems like an excellent biomarker for measuring progress in re-gaining control of our body.

 

[Hip]

Interesting, I did not know this, but according to this paper, it's not just enterovirus which inhibits host gene expression:

many viruses inhibit host cell gene expression, which plays a major role in the ability of these viruses to cause disease.

Members of many different virus families inhibit the expression of host genes during the process of virus replication. The typical explanation for this effect given in textbooks is that it provides higher levels of cellular resources such as nucleoside triphosphates to be used for biosynthesis of viral gene products.

However, there are many examples of viral mutants that are defective in their ability to inhibit host gene expression yet replicate as well as wild-type viruses in most cell types.

The cell types that restrict the growth of such mutants compared to wild-type viruses are often those that can mount a vigorous antiviral defense. Such observations support the idea that the role of the virus-induced inhibition of host gene expression is to inhibit the host antiviral response.

Though the paper points out that sometimes in viral infection, the inhibition of host protein synthesis comes from the immune response to viral double-stranded RNA:

The principle that viruses may inhibit host gene expression in order to inhibit the antiviral defense of the host is pretty straightforward. However, sorting out the relationship between viral cytopathogenesis and the host antiviral response can be difficult in practice.

For some viruses, phenomena once considered CPE of the virus are now known to be part of the antiviral response of the host. For example, the inhibition of host protein synthesis in virus-infected cells is often considered a direct effect of viral gene products. Indeed, there are cases, such as most picornaviruses, in which viral gene products act directly to inhibit host protein synthesis without affecting viral protein synthesis.

However, in many cases the inhibition of host protein synthesis results from activation of the host response to viral double-stranded RNA (dsRNA), such as activation of protein kinase R (PKR).

The paper also details what you mentioned about eIF4G-mediated inhibition of host gene expression in picornavirus infections (enterovirus is in the picornavirus family):

The cleavage of translation initiation factor eIF4G in picornavirus-infected cells is a classic example of virus-induced inhibition of host translation. For most picornaviruses this cleavage is mediated by the viral 2A protease.

I have a folder on enterovirus 2A protein. One 2A inhibitor is a French drug in development called 11 FR 33j6 3NL4. And nitric oxide donors inhibit 2A. This patent talks about peptides which might inhibit 2A.


It's also interesting that in the heart disease called dilated cardiomyopathy, which has long been linked to coxsackievirus B, the viral 2A protease is sufficient just on its own to cause all the damage found in cardiomyopathy. Ref: here.



In terms of measuring host mRNA expression, I am not sure if such tests are commercially available; but even if they were, it's not clear if they could measure the effect of host gene expression suppression by viruses, since most of the cells in the blood would not be infected, and thus would be functioning normally as cells.

 

[sometexan84]

One 2A inhibitor is a French drug in development called 11 FR 33j6 3NL4

Catchy name!

it's not just enterovirus which inhibits host gene expression:

Yea, a lot of them do, maybe all of them. This is epigenetic deregulation. DNA methylation plays a huge role in this. This is where Rich Van Konynenburg's Methylation cycle comes into play.

There are multiple ways a virus can effect gene expression though. Histone modification is another way. And then there's cellular transcription vs translation.

Transcription is where DNA is converted to complementary RNA code. Translation happens afterward, and is where the mRNA is converted into amino acid and protein, at which point it can perform some sort of function in a cell.

EBV for instance causes epigenetic deregulation of the immune system by messing w/ cellular transcription, and by messing w/ DNA methylation. EBV also messes w/ transcription via miRNAs (micro RNAs, not to be confused w/ mRNA or messenger RNAs).

But I think enterovirus is unique in that it pretty much directly, and completely can shut off our core cellular functions (mRNA translation).

It doesn't have the massively widespread disruption potential of EBV, but w/ the ability to completely turn off our cell functioning, it's understandable to me that enterovirus has been implicated as one of the top causes of CFS.

I have a folder on enterovirus 2A protein

gimme

Inhibitors are cool. Actually, I am more concerned w/ what happens after viral clearance. Like, what if changes made by enterovirus stick around, even after the virus is gone? This is why I want to test for cellular function.

since most of the cells in the blood would not be infected, and thus would be functioning normally as cells.

ooooo, that sounds like an assumption Mr. Hip. A very dangerous assumption.

 

[sometexan84]

@Hip
Did you know that Enterovirus can completely control apoptosis in infected cells by disrupting calcium homeostasis?

The 2B protein of Coxsackie modulates the calcium regulation in the cell, where low Ca2+ levels provide conditions for viral replication, and high Ca2+ levels lead to viral release. Complete control of apoptosis and virus release (viral replication).

And supposedly, this is accomplished by modulation of RYR1 (ryanodine receptor) via the 2B protein.

Only reason I went down this road was because of @Pyrrhus post on the new epigenetics study. And it showed that RYR1 was shown as an abnormally hypo-methylated gene in ME/CFS.

Anything you see involving Calcium, I'd think "enterovirus"

 

[Hip]

gimme

Inhibitors are cool. Actually, I am more concerned w/ what happens after viral clearance. Like, what if changes made by enterovirus stick around, even after the virus is gone? This is why I want to test for cellular function.

I posted most of what my 2A folder contains just above, the antiviral substances which block 2A.

Other items it contains are:

2A blocks interferon receptor 1. Ref: 1
2A targets MDA5 and MAVS. Ref: 1
Enterovirus cannot replicate unless 2A binds to SETD3. Ref: 1

ooooo, that sounds like an assumption Mr. Hip. A very dangerous assumption.

It's well-known that levels of virus are very low or not present in the blood of ME/CFS patients. This is why ME/CFS patients typically test negative when viral PCR tests are performed on the blood.

Sometimes patients are positive for a virus by blood PCR, but the general rule is that they are negative.


In fact, these negative blood PCR results in ME/CFS are the main reason why controversy exists as to whether chronic active viral infections are even present in ME/CFS. If blood PCR mostly came out positive in ME/CFS patients, there would be no doubt that ongoing infection is present.

Yes ME/CFS patients often have chronically elevated antibodies to one or more viruses, but this is not direct evidence of viral infection (by detecting antibodies you only measure the immune response to infection, you do not directly detect the infection; whereas PCR directly detects an infection). You will not be able to convince the research community that an infection is present in ME/CFS unless you provide direct evidence, like PCR.

Dr John Chia has really push the boundaries on ME/CFS blood PCR testing for enterovirus: in his research, he used one of the most sensitive enterovirus PCR tests available: reverse transcription PCR (abbreviated to RT-PCR), which can detect enterovirus at levels as low as 80 copies of RNA per ml of blood, which is extremely sensitive.

He found that even with this extreme sensitivity, enterovirus ME/CFS patients would sometimes test positive when the peripheral blood lymphocyte cells were tested, but usually would test negative. Patients who tested positive would often be negative on the next PCR. Chia tested thousands of patients with his PCR assay.

Chia concluded that it is clear the enteroviral RNA present in the blood is at very low levels.



Dr Chia is always trying to get the message across to other ME/CFS researchers that it is pointless testing the blood by PCR or other molecular methods, because the infection is not in the blood in ME/CFS, but resides in the tissues.

There is good evidence for the infection being in the tissues: when British ME/CFS researchers in the 1990s started testing skeletal muscle tissue samples from patients, they found a lot were positive for enterovirus. Molecular methods (= detecting a pathogen via its genes) like PCR only became available in the late 1980s.

So muscle biopsy testing is a reliable way to detect enterovirus in ME/CFS patients. But because muscle biopsies are time consuming, painful and leave a scar, Dr Chia later pioneered stomach tissue testing as a replacement for muscle biopsy testing. This was a great advancement, but sadly not much interest was shown by other researchers.

Again with stomach tissue, you can detect enterovirus directly by PCR, or via enterovirus VP1 protein staining.



When you think of it, it is rather peculiar to expect to be able to detect low-level tissue infections in the blood, if those infections are localize to specific organs. For example, we know that in type 1 diabetes, the insulin-producing beta cells of the pancreas are infected with coxsackievirus B, and one theory is that T1D is caused by this pancreatic CVB infection.

However, if you test T1D patients for enterovirus by PCR blood test, you will not find anything. But that negative result does not rule out that a pancreatic infection may be present (because we know it is), and may be the cause of T1D. It's CVB4, and also CVB1, which are linked to T1D.

So this indicates that we need to be testing tissues. But even great virus hunters like Prof Ian Lipkin test the blood of ME/CFS patients, and not the tissues. Of course, it is much harder and more expensive to conduct a study on tissues samples; whereas ME/CFS blood samples are easy to obtain from ME/CFS blood banks.

Did you know that Enterovirus can completely control apoptosis in infected cells by disrupting calcium homeostasis?

The 2B protein of Coxsackie modulates the calcium regulation in the cell, where low Ca2+ levels provide conditions for viral replication, and high Ca2+ levels lead to viral release. Complete control of apoptosis and virus release (viral replication).

And supposedly, this is accomplished by modulation of RYR1 (ryanodine receptor) via the 2B protein.

Interesting, I had not seen this. It's good to examine the various pathways involved in enteroviral infection, just to see if there might be something that can be done to alter these pathways, which might then result in viral clearance, or at least a reduction in viral load.


EDIT: actually in my folder on coxsackievirus B immune evasion, I just found one of my notes:

CVB 2B protein suppresses apoptotic responses by manipulating intracellular calcium. 2B protein decreases the calcium in endoplasmic reticulum and the Golgi, resulting in down-regulation of calcium signaling between these stores and the mitochondria, and increases the influx of extracellular calcium. Ref: 1

(I always have to use my computer hard drive as a memory prosthetic, as most of the things I learn I forget after a few weeks).

 

[sometexan84]

It's well-known that levels of virus are very low or not present in the blood of ME/CFS patients. This is why ME/CFS patients typically test negative when viral PCR tests are performed on the blood.

Actually, what I meant was that I believe it's an "assumption" that non-infected cells function normally (in blood or tissue).

For example, to quote from the Prusty/Naviaux study:

Could a very lightly smoldering HHV-6 or HHV-7 infection be whacking the ability of the uninfected cells in ME/CFS to produce energy?

Regarding the PCR testing, I question the accuracy and reliability. There are a bunch of articles out there that go over the issues w/ real-time PCR... things like inconsistent sample sizes used, and institutions using different positive/negative thresholds.

Actually, here's a new article on this - PCR the Unreliable Test Locking Down the World

 

[Hip]

Actually, what I meant was that I believe it's an "assumption" that non-infected cells function normally (in blood or tissue).

Ah, OK, that's a different issue. Yes, certainly if we look at the work Prusty has done on HHV-6, this suggests infected cells can transmit messages to adjacent non-infected cells, which then alters mitochondrial functioning in these non-infected cells.

Although mitochondrial functioning is not the same as gene expression.

Regarding the PCR testing, I question the accuracy and reliability.

Medical testing is a complex and imperfect issue. No test ever has 100% sensitivity and 100% specificity, so every test will create a certain amount of false positives and false negatives. I was just looking at SIBO glucose breath tests recently, and these have a poor sensitivity and specificity, of 55% and 83% respectively.

This breath test thus leads to many false negatives, and some false positives, as only 55% of people who actually have SIBO will receive a correct positive result on this glucose breath test (the other 45% will receive an incorrect negative result); and only 83% of people who do not actually have SIBO will get a correct negative result (17% will get an incorrect positive result).


Then there are issues of test-retest reliability: will the same blood sample get the same test result when tested a second time? In the case of antibody tests, I read that typically you can move up or down 1 titer level on your retest. So for example, if you tested as 1:160 for your ARUP Lab coxsackievirus B test, on retest with the same blood sample, it might go down to 1:80, or go up to 1:320.


I don't know much about PCR, as PCR blood tests are not generally useful in ME/CFS.

However, I know that for basic PCR tests, the problem is they are not a quantitative tests: they can tell you whether a pathogen is present or not (up to the limit of the PCR test detectability), but cannot tell you how much of the pathogen is present. These tests only provide YES/NO answers.

However, quantitative PCR (also called real-time PCR) is a technique which allows you to quantify how much of the pathogen is present.

Unfortunately real-time PCR and reverse transcription PCR are both abbreviated to RT-PCR, which can be confusing.

 

[Pyrrhus]

The 2B protein of Coxsackie modulates the calcium regulation in the cell, where low Ca2+ levels provide conditions for viral replicatio

actually in my folder on coxsackievirus B immune evasion, I just found one of my notes:
CVB 2B protein suppresses apoptotic responses by manipulating intracellular calcium. 2B protein decreases the calcium in endoplasmic reticulum and the Golgi, resulting in down-regulation of calcium signaling between these stores and the mitochondria, and increases the influx of extracellular calcium. Ref: 1

You're both right. The enterovirus 2B protein is a viroporin, which is a viral protein that creates pores in membranes. It can create pores in the membranes of the endoplasmic reticulum, Golgi bodies, or the cell membrane itself. The effect in each case is the same: Ca2+ ions flow through these pores into the cytoplasm, which normally maintains a low concentration of Ca2+ ions. (the cytoplasm is also called the cytosol)

There are many effects of the increased cytoplasmic Ca2+ concentration, but two effects are notable:

1. The increased cytoplasmic Ca2+ concentration starts the process of apoptosis, which then stalls, resulting in blocked apoptosis. Details are complex and currently escape me.
2. The increased cytoplasmic Ca2+ concentration stabilizes the otherwise unstable double-stranded RNA (dsRNA) intermediate state of the enterovirus. When the dsRNA intermediate state is stabilized, viral replication slows down. When the Ca2+ cytoplasmic concentration is high enough, viral replication halts completely and the virus enters a quasi-latent dsRNA state.

Here are some articles on viral protein 2B and Ca2+ ions:
https://pubmed.ncbi.nlm.nih.gov/15680759/
https://pubmed.ncbi.nlm.nih.gov/9218794/

Hope this helps.

 

[Pyrrhus]

And here is a great diagram showing how the enteroviral protein 2B creates pores in membranes in order to raise the Ca2+ concentration in the cytoplasm:

Reference:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6630369/