Molecular mechanisms of stress-induced reactivation in mumps virus condensates, 2023, Xiaojie Zhang et al

[Mij]

Highlights•

• Cellular stress provokes activation of viral replication in an RNA virus model
• The viral replication factories are liquid-like condensates that coarsen under stress
• Stress-triggered IDR phosphorylation stabilizes the viral replication machinery
• In-cell nucleocapsid structures reveal changes in genomic RNA and IDR accessibility

Summary
Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases. Here, we uncover how cellular stress disrupts the metastable host-virus equilibrium in persistent infection and induces viral replication in a culture model of mumps virus.

Using a combination of cell biology, whole-cell proteomics, and cryo-electron tomography, we show that persistent viral replication factories are dynamic condensates and identify the largely disordered viral phosphoprotein as a driver of their assembly. Upon stress, increased phosphorylation of the phosphoprotein at its interaction interface with the viral polymerase coincides with the formation of a stable replication complex.

By obtaining atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events constitute a stress-mediated switch within viral condensates that provide an environment to support upregulation of viral replication.

https://www.cell.com/cell/fulltext/...m/retrieve/pii/S0092867423002763?showall=true

 

[Hutan]

German research
Thanks for posting @Mij. If I had to pick one theory on the cause of ME/CFS, I think it would probably be something to do with reactivations of pathogens that have been sitting latent in cells. I'm only a few paragraphs into the paper, but I think this is looking to be worth reading.

An accidental encounter of persistent viral infection in our cell cultures used for a stress response study al- lowed us to investigate the basis of stress-mediated reactivation in a member of the negative-stranded RNA virus family Paramyx- oviridae, the mumps virus. Mumps is a highly contagious viral illness that was a common childhood disease before the intro- duction of vaccination.4,5 Mumps virus (MuV) is typically self-limiting. Namely, the majority of patients experience a full recovery and acquire lifelong immunity.5 However, chronic com- plications such as myositis,6 chronic arthritis,7 and encephalitis8 arise not due to an immediate effect of the primary infection (such as in parotitis4) but develop months or years thereafter or following vaccination in immunodeficient patients.9

So, it's suggested here that the mumps virus can cause chronic complications in a delayed way, years after the infection. So, yet another disease where 'die or recover' are not the only options. That reference [9] looks like a credible case where a live attenuated mumps vaccine caused a brain infection in an immune-compromised child.

 

[Hutan]

Viral factories
Membrane-less inclusions in cells that can be seen with electron microscopy, containing viral genomes and replication machinery.

MuV transcription and replication occur in subcellular compart- ments termed viral factories (VFs), where the N-encapsidated viral genomes and replication machineries composed of P and L concentrate.11 The formation of VFs is a complex process that has independently evolved in a variety of non-related viruses.12 Biomolecular condensation mediated by liquid-liquid phase sepa- ration of viral proteins or host factors with intrinsically disordered regions (IDRs)13 and viral RNAs has been recently shown to be fundamental for the organization of VFs, regulation of viral replica- tion, and promotion of viral assembly in measles virus (MeV),14 rabies virus,15 vesicular stomatitis virus,16 HIV-1,17–19 and SARS-CoV-2.20,21 MuV replication factories were reported to be membrane-less inclusions by conventional immuno-electron mi- croscopy (EM) on sections of chemically fixed cells11 and typically contain curved tubule-like structures (the nucleocapsids) following viral inoculation into cultures to model acute infection.

Graphical abstract

 

[Hutan]

Viral replication and release are accelerated by stress

We established a culture model of persistent MuV infection in a HeLa cell line (HeLa-MuV) stably expressing the stress granule protein mCherry-G3BP1 (Ras GTPase-activating protein-binding protein 1) as a marker of cellular stress28 (Fig- ure 1A; STAR Methods). Immunofluorescence using an antibody against the native viral N protein showed its localization in micron-sized cytoplasmic compartments that also contain the MuV genomic RNA (detected by RNA fluorescence in situ hybrid- ization [FISH]; Figure 1B; Table S1), which we refer to as MuV replication factories.

I marvel at the techniques. There's a protein (RasGTPase protein-binding protein) that is claimed to be a measure of cellular stress. And fluorescent markers of both viral N protein and viral RNA. They have cells infected with the virus, with a small amount of viral replication going on, such that neighbouring uninfected cells can be infected, but it's at a low level. The cellular stress marker protein isn't detected and the cells are growing and replicating at normal levels.

The cells were then challenged with oxidative stress or heat shock and both the cellular stress marker protein and viral replication increased markedly.

confirming that cellular stress increases viral transcription, replication, and infectious virion release in a persistent MuV infection cul- ture model.

So, cellular stress increases virus production - that's not surprising, but with their advanced microscopes, they can see what is happening inside the cell.
They used other sorts of cells to check that what they were finding wasn't unique to HeLa cells.

 

[rvallee]

I wanted to know what specifically they mean by stress (definitely not psychosocial stress) and unless I missed something it's oxidative stress that promotes viral replication:

Our culture model recapitulated hallmarks of persistent benign infection: cells did not exhibit stress as indicated by the absence of stress granules, and cell division was maintained at normal levels (Figures 1A and 1C), although released virions were capable of infecting naive cells, indicating basal levels of viral replication (Figure 1D). However, when cells were challenged with established conditions of oxidative stress, viral replication increased by 2- to 4-fold

Prolonged mild oxidative stress (30 μM arsenite; As(III)) also increased viral transcription and replication, albeit at a lower rate or magnitude​

And by "stress granules" they mean:

We established a culture model of persistent MuV infection in a HeLa cell line (HeLa-MuV) stably expressing the stress granule protein mCherry-G3BP1 (Ras GTPase-activating protein-binding protein 1) as a marker of cellular stress​

I can't say I know enough about oxidative stress to understand what this means, but oxidative stress comes up a whole lot in ME studies, and if low level sustained oxidative stress is capable enough of promoting viral replication, this starts like sounding like a model, at least as far as I don't know much about biology.

Seems very interesting. Viral reservoirs, stable and, as they write in the highlights "The viral replication factories are liquid-like condensates that coarsen under stress", change morphology in response to oxidative stress. Sounds a lot like a stalemate that takes every opportunity it gets to breach the defenses. And with viruses, it only takes a few virions to mass produce, reservoirs can be small.

That was always the model that made the most sense here. But making sense is not exactly a feature of immunology so who knows?

 

[Hutan]

what specifically they mean by stress (definitely not psychosocial stress) and unless I missed something it's oxidative stress that promotes viral replication

Yes. Two types of chemical stress, and heat stress. The paper has links to detailed descriptions of methods.

For IF microscopy, HeLa-MuV cells (mCherry-G3BP1) were seeded on 35-mm dishes with a glass bottom the day before stress treat- ment. Cells were stressed by replacing the culture medium with that containing either 0.5 mM sodium arsenite (referred to as As(III)) for up to 3 h, 1 mM potassium arsenate (referred to as As(V)) for up to 6 h, 30 mM As(III) for up to 24 h, or transferring the cell culture dishes to an incubator set to 43 C for 1 h for heat shock.

 

[Hutan]

I've made as thread to discuss Ras GTP-ase activating protein-binding protein 1 here:
Ras GTP-ase-activating protein-binding protein 1

 

[Hutan]

An interplay between viral replication and cellular stress has been documented for many viruses in the acute phase of infec- tion following cell entry. MuV and rabies virus infection lead to the formation of stress granule via activation of the protein ki- nase R (PKR) stress pathway by the viral RNAs.60,61 In contrast, MeV can interfere with stress granule formation62 or others, like Zika virus, take advantage of key stress granule proteins or the stress response pathways (e.g., G3BP1) to promote viral gene expression.63

Different viruses have different impacts on stress granule formation, some increasing it, some decreasing it.

Second, we observed that the magnitude of increase in replication activity correlated with the severity of stress: genomic RNA level under mild As(III) stress was slightly suppressed until 6 h before showing a dramatic upregulation at 24 h, reminiscent of acute phase infection where transcription is prioritized to generate more material (e.g., polymerase and nucleocapsid protein) required for replication.67

So there's a delay of about 24 hours between the stress on the cell and the up regulation of the viral transcription. (Perhaps that fits with PEM?) I think the authors are suggesting that the "viral factories" in the cells gear up over this time, getting ready to produce virions.

In line with this, feedback mechanisms must exist to maintain a long-term balance between the MuV and the host. Finally, the increase in virion release may result in a new infection or slow progression of severe chronic diseases.6,9

Reference 6 is Inclusion body myositis: A chronic persistent mumps myositis?, 1986

Among the generalized chronic idiopathic inflammatory myopathies, inclusion body myositis (IBM) has emerged as a clinicopathologic variant during the past two decades. It occurs primarily in elderly persons (in approximately the sixth decade of life), but young adults (in approximately the second decade of life) may also be affected. Slowly progressive weakness of distal as well as proximal muscle groups in IBM is usually not associated with skin rash, malignancy or collagen-vascular disease, and is refractory to treatment with steroids or other immunosuppressants. Exceptions to each of these general rules have been found. Muscle biopsy and electromyography may suggest a neurogenic process mixed with myopathic features.

That's an old reference suggesting a persistent mumps infection causes progressive muscle weakness.

It is tempting to speculate that such molecular switches induced by stress may have broad implications across a diverse family of viruses that establish chronic infection in the human host and for the diseases they elicit.

 

[Mij]

So there's a delay of about 24 hours between the stress on the cell and the up regulation of the viral transcription. (Perhaps that fits with PEM?)

 

[Mij]

Given that G3BP1 is a well-described target for many viruses with a clear functional role during infection could also offer novel therapeutic opportunities.

 

[rvallee]

Maybe it's only part of the answer, but the intensity with which latent infections have been denied and dismissed, despite being common and well-documented, as a potential source of illness, and contrasting with the conditions where 1) viral particles are as small compared to a human body as a human body is to the volume of the entire damn planet Earth and 2) it only takes a handful of particles for a full infection.

The hubris. It's just staggering. I'm pretty convinced that this is the main explanation for most diseases, even most of those wrongly attributes to "lifestyles". Even most aging is probably a consequence of accumulated injuries and stalemates with various infections that found a niche somewhere.

As in this is probably the most important idea in all of medicine, or one of the top 5. And it's dismissed with the same ease as flat earth, even though it's common and well-documented. This feels as bad as having completely missed electricity, having dismissed it as of no interest and made taboo, turned into superstition and described as magical.

 

[Hutan]

Maybe it's only part of the answer, but the intensity with which latent infections have been denied and dismissed

I agree. The more you look, the more there are many reports, not at all flaky, of latent infections that cause problems some time down the track. It may not be the answer to ME/CFS, although it could well be. But certainly medicine should be giving these a lot more attention.

And, I also agree, that aging, which actually looks a lot like ME/CFS, might have a lot to do with carrying an accumulated burden of latent infections.

 

[SNT Gatchaman]

And, I also agree, that aging, which actually looks a lot like ME/CFS, might have a lot to do with carrying an accumulated burden of latent infections.

Or, as our immune systems degrade, losing the ability to control the deleterious effects of those latent infections. I think the partial reactivation of herpesviruses will be key and the mechanisms are starting to come in to focus. Eg if HHV6 partial reactivation prevents the host cell's own microRNA machinery that could have systemic consequences, losing more and more ability to maintain homeostasis through mechanisms that rely on those microRNAs.

 

[rvallee]

I agree. The more you look, the more there are many reports, not at all flaky, of latent infections that cause problems some time down the track. It may not be the answer to ME/CFS, although it could well be. But certainly medicine should be giving these a lot more attention.

And, I also agree, that aging, which actually looks a lot like ME/CFS, might have a lot to do with carrying an accumulated burden of latent infections.

As best as I can tell, this hypothesis is mostly brushed off because there is basically nothing we can do about it. No lifestyle changes can change the fact that we regularly get exposed with stuff that can cause health issues. And it's not just pathogens, so many toxins, metals, now plastics everywhere. It's everywhere, in the air we breathe, the water we drink, pathogens in food from faraway. Medicine massively struggles when they don't know the source of a problem, and this is basically a medical uncertainty principle, they can't ever really know for sure.

It removes control. If regular, even mild, infections can cause lifelong disease like this, barely possible to detect, it just makes everything so much harder. And it really seems like the ignorant bliss is simply preferred, mostly in favor of BS "adjust your lifestyle and nothing can touch you" woo. Even though it's mostly BS. There are centenarians who never exercised a day in their life, drank every day, smoked for decades. And then there are people like my aunt, one of the fittest and healthiest person you could find, died before 40. Did everything right, and still lost the biological lottery.

Everything I see out of medicine confirms this. It makes them powerless against it, at least temporarily, and that's just unacceptable. It's best to have the flawed perception of being in control, even if it's just an illusion. Even though swallowing the hard pill, working at it, would change everything. Very defeatist.