Does SARS-nCoV-2 spike protein expression induce endoplasmic reticulum stress and upregulation of intracellular chemokine mRNA concentrations?

SARS-nCoV spike does... so I suspect the answer is 'YES'.

Update: Thanks to a reader for bringing the pre-print entitled: “Modulation of SARS-CoV-2 Spike-induced Unfolded Protein Response (UPR) in HEK293T cells by selected small chemical molecules” to my attention. It is quite relevant to this article.

A paper was brought to my attention this morning by Walter Chestnut that was published in 2007 in the Journal of Virology entitled: “The coronavirus spike protein induces endoplasmic reticulum stress and upregulation of intracellular chemokine mRNA concentrations”.¹ In this paper, the authors show induction of endoplasmic reticulum (ER) stress, and Cxcl2 mRNA transcription during infection in vitro, specifically, via expression of the spike protein. They used both a mouse coronavirus (Murine Hepatitis Virus (MHV)), and a human SARS-CoV virus in their experiments because they were the quintessential models for coronavirus infection at the time.

Cxcl2 is the mouse version of a human interleukin called Interleukin-8 (IL-8) that is really important to (early) immunological responses. IL-8 is a chemokine (chemotactic cytokine) for neutrophils, which are the very prevalent white blood cells that play vital roles as part of the innate immune system. IL-8 can also induce phagocytosis in macrophages, is involved in histamine release and has many other essential roles as part of immune defenses.²³⁴⁵

So what they showed is induction of transcription of this chemokine in the absence of actual protein synthesis in combination with a spike-induced stressed out ER. The effect of these two things was a build up of intracellular mRNA from the chemokine, and the engagement of ER stress responses to signal relief that apparently, SARS-CoV might be able to modulate to its benefit! The Hepatitis C Virus can do this. The reason this is relevant to all of us now is two-fold:

1. because SARS-CoV and SARS-CoV-2 have very similar spike proteins and thus the effects might be the same for SARS-CoV-2

and

1. because the mRNA template in the COVID-19 injections was mimicked after the template that encodes the spike protein for SARS-CoV-2.

Here is Walter’s article if you want a great overview of the potential problems.

First we need a crash course in some biology stuff.

On the Endoplasmic Reticulum (ER)

The ER is akin to the protein-producing transportation system in eukaryotic cells. Eukaryotic organisms are organisms with nucleated cells, so eukaryotic cells have nuclei - for the most part. RBCs are eukaryotic cells but no, they do not have nuclei or ERs. Nature, wha?

The ER comprises rough (studded with ribosomes) and smooth parts. The rough ER is adjacent to the nucleus of the cell and plays the role of protein maker/folder/transporter (platform for ribosomes). The smooth ER, which is a little more distal from the nucleus, plays the vital role of detoxifier, metabolizer, storage (calcium), lipid, steroid and glycogen synthesizer, depending on the cell type. Phew, that’s a lot of roles. Seems important; this ER. Let’s have a look at the rough and smooth ER in the context of the nucleus of a eukaryotic cell in Figure 1.

Figure 1: The nucleus and ER of a eukaryotic cell. https://rsscience.com/endoplasmic-reticulum/

Here you can see the adjacent position of the ER to the nucleus of this ‘cell’. The little dark blue guys dotting the outside of the rough ER (on the cytosolic side) are ribosomes. By now, you all know that ribosomes are the protein-synthesizers of cells. (They convert messenger RNA (mRNA) into proteins via translation with the help of transfer RNAs (tRNAs).) Proteins whose destinies lie outside of the cell (like chemokines) and proteins whose destinies lie in the cell membrane (like membrane-bound molecular transporter bits) are trafficked by ‘their’ ribosome to the rough ER where the ribosome docks and inserts the protein in the lumen of the rough ER.⁶ That’s where the protein folding magic really happens.

Figure 2: Screenshot of the transfer of secretory protein and ribosome to the membrane of the rough ER from Elizabeth Wright’s video “Synthesis of a secretory protein”. https://www.youtube.com/watch?v=4SHs6-rGrOU

I really encourage everyone to watch Elizabeth Wright’s video description of the synthesis of a secretory protein - a protein whose destiny is outside of the cell.

Alright, so the mRNA genetic template that originates from the nucleus gets to the cytoplasmic ribosomes for protein synthesis via the nuclear pores, as shown in Figure 1. If the protein encoded by this mRNA is a secretory protein, it gets shuttled with its ribosome to the rough ER where the protein is dumped into the rough ER lumen for folding and additional finishing touches essential to function. The folding of a protein is a spontaneous process guided by forces and bonds, and depends on many factors such as salt concentration, pH and temperature. The folding environment must be thermodynamically favorable in order for folding to be a spontaneous reaction, so this favorable environment is promoted in the rough ER. The rough ER also prepares the proteins for the eventual entry into the different biochemical environments that they will encounter, by stabilizing them in a biochemical environment that is more oxidizing than other environments like the cytoplasm.

The (hopefully correctly) folded proteins eventually move into the lumen of the smooth ER where they undergo additional modifications for functionality.

Aside: The ER is kind of like the gut of the cell, in my mind: 1. it’s closed to the cytosol - separated by a phospholipid bi-layer to create a special space inside the ER called the cisternal space or lumen, and 2. it’s a highly folded structure to increase surface area. Their functions are very different but the structures are alike. Well, to me anyway.

The Golgi Apparatus is close to the smooth ER. Its role is to accept the proteins from the lumen of the smooth ER, to ensure final modifications to the proteins, and to produce secretory vesicles to transport them to their respective final destinations by using signal peptides and tubule networks. These signal peptides are tags to indicate the final destination for the protein which may be inside or outside of the cell.

Figure 3: The guts of a eukaryotic cell. https://en.wikipedia.org/wiki/Cell_(biology)#/media/File:Endomembrane_system_diagram_en.svg.

That was my crack at describing the normal journey of DNA-derived messenger RNA, through a secretory protein ER-necessitated pathway to produce a properly-folded functional secretory protein.

But spike protein is not an endogenous protein and its coding material is not in our genome. So how does this work?

Is spike protein considered to be a secretory protein? I would say most certainly, yes.

Spike proteins begin their journeys in that cell as messenger RNA (mRNA). The mRNA that encodes the SARS-CoV-2 spike protein is dumped into the cytoplasm of a cell from its lipid nanoparticle (LNP) carrier molecule. This mRNA, for lack of a better way of describing it, eventually ‘finds’ a ribosome and the ribosome does its job to synthesize spike protein. The mRNA was codon-optimized for optimal protein expression in humans, remember, so lots of protein expression should ensue. These proteins are subsequently trafficked to the rough ER for further synthesis and modification. Let’s stop at this point in the secretory pathway even though, by design, the proteins should move to the smooth ER and subsequently onto the Golgi Apparatus for exocytosis. It’s important to stop here and address what the authors found in the paper.

We need to get into something called the unfolded-protein response (UPR).⁷ I recommend watching the following video. It’s a little creepy how the TGA is used as an analogy here, but so be it.

If un- or mis-folded proteins start to accumulate in the ER, then the ER becomes stressed. Think of it like when you get really bad gas from eating too many burritos. The burritos are the mis-folded proteins and the stress response is your fart-fest - the UPR is a way to release the pressure to restore balance. Or kill the cell. The ER stress response involves the engagement of the ER-associated protein degradation (ERAD) system to destroy mis-folded proteins⁸ and the engagement of the UPR to inhibit translation and prevent additional accumulation of unfolded proteins. What's neat about certain viruses is that they can modulate the UPR responses to their benefit. Sneaky little guys. It appears as though SARS-CoV might be one of these viruses.

The UPR is an essential biological process that when malfunctioning, is linked to many diseases such as Parkinson’s and Diabetes. It works by monitoring the proteins in the ER to ensure that they are properly folded and that improperly folded proteins are not accumulating. This involves ER transmembrane sensors such as Ire1.⁹ The UPR ‘kicks in’ when these sensors sense that there are too many improperly-folded proteins being produced as part of the ER stress response. The end-product can be UPR-mediated restoration of homeostasis in the cell or, if things go really south, cell destruction by apoptosis. Again, what's really neat is that certain viruses can help with these life or death decisions. Convenient, eh?

Before I continue, I want to be clear about the distinction between viruses as complete entities and viral proteins like spike protein of coronaviruses. Again, the experiments reported on in this paper were done in the context of viruses - MHV and SARS-CoV. The presence of a virus (infection of a cell) can induce ER stress due to accumulation of viral glycoproteins, and potential incorrect folding (and accumulation) of these proteins in the ER lumen.

The authors discovered in their experiments that the spike protein affected the ER in a very specific way. Besides inducing the ER stress and UPR responses, SARS-CoV was found to modulate the UPR response, that is, it was found to upregulate certain UPR genes.

In this study we show that the MHV spike protein, like that of SARS-CoV, induces ER stress. MHV infection resulted in extensive expression of unfolded-protein response (UPR) markers such as Herpud1 and XBP1s, while SARS-CoV infection upregulated only a limited set of UPR genes.

This discernation may seem subtle, but it is a really important distinguishing feature of SARS-CoV, and may also be a distinguishing feature, therefore, of SARS-CoV-2. They also found that the overload of the viral glycoproteins induced innate immune response mechanisms, as I mentioned earlier. This also has enormous potential implications pathologically.

The data reported indicate for the first time that in addition to the innate immune sensors that have been described so far, the overload of the ER with viral glycoproteins could also mediate induction of innate immune responses such as transcriptional activation of chemokine genes. Moreover, our data demonstrate that in productively infected cells, CoV-induced translational attenuation contributes to viral evasion of potentially harmful host proteins, such as chemokines.

So they showed very distinctive repercussive activities in the context of spike protein for SARS-CoV that implicate pathologies related to protein mis-folding diseases and immune system dysregulation. It also had implications for cancer.

More on this soon when I learn more about the UPR genes that SARS upregulates.

1. Versteeg GA, van de Nes PS, Bredenbeek PJ, Spaan WJ. The coronavirus spike protein induces endoplasmic reticulum stress and upregulation of intracellular chemokine mRNA concentrations. J Virol. 2007 Oct;81(20):10981-90. doi: 10.1128/JVI.01033-07. Epub 2007 Aug 1. PMID: 17670839; PMCID: PMC2045536.

2. Möller A, Lippert U, Lessmann D, Kolde G, Hamann K, Welker P, Schadendorf D, Rosenbach T, Luger T, Czarnetzki BM. Human mast cells produce IL-8. J Immunol. 1993 Sep 15;151(6):3261-6. PMID: 8376778.

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4. Bickel M. The role of interleukin-8 in inflammation and mechanisms of regulation. J Periodontol. 1993 May;64(5 Suppl):456-60. PMID: 8315568.

5. Cesta Maria Candida, Zippoli Mara, Marsiglia Carolina, Gavioli Elizabeth Marie, Mantelli Flavio, Allegretti Marcello, Balk Robert A. The Role of Interleukin-8 in Lung Inflammation and Injury: Implications for the Management of COVID-19 and Hyperinflammatory Acute Respiratory Distress Syndrome. Frontiers in Pharmacology. Volume 12. 2022. doi:10.3389/fphar.2021.808797.

6. Other proteins like ones who have intracellular destinies, like for mitochondria or nuclear use, are made fully in the cytoplasm.

7. Schröder M, Kaufman RJ. The mammalian unfolded protein response. Annu Rev Biochem. 2005;74:739-89. doi: 10.1146/annurev.biochem.73.011303.074134. PMID: 15952902.

8. https://en.wikipedia.org/wiki/HERPUD1

9. Chen Y, Brandizzi F. IRE1: ER stress sensor and cell fate executor. Trends Cell Biol. 2013 Nov;23(11):547-55. doi: 10.1016/j.tcb.2013.06.005. Epub 2013 Jul 21. PMID: 23880584; PMCID: PMC3818365.