As many of you are probably aware, one of my longest-running arguments in regard to SARS-CoV-2 is that this is primarily a bioenergetic disease, basically a mitochondrial dysfunction disease, to what extent only research into specific markers will tell. In regards to many of the other pieces I wrote, you can always search for whatever I wrote + mitochondrial dysfunction and there will be shared pathways at some point. In fact, for most of today I was revisiting shorter analyses I wrote here on complex papers, revisiting after weeks will often bring you clarity and insight (as it just did).
I recently shared this paper on Twitter, where the authors found a new protein mimicry in the virus, this time in the ORF8, I have written about the other proteins of SARS-CoV-2 and here is one about the ORF8 alone, and what they found entails.
From the Twitter post “As I said many times too much focus on the wrong proteins. The ORF8 has a histone (DNA stuff) mimicry, and authors propose ORF has multiple functions that are time dependent. The severity of the disease was related to ORF8 expression. The authors didn't propose this but I will. Cryptic peptides in the ORF8 are what give it multiple functions. With the ARKS possibly being the starting point ? Maybe. ORF8 also affects interferon signaling. Where is the ORFX ?
The paper itself is really good to read, but the point of sharing these is because of the overall “theme” in this one. The other proteins of SARS-CoV-2 and how they related to mitochondrial function. Or better, dysfunction.
SARS-CoV-2 ORF3c impairs mitochondrial respiratory metabolism, oxidative stress and autophagic flow
A first hyper-inflammatory phase, characterized by increased aerobic glycolysis (Warburg effect), increased oxygen consumption, elevated ATP production, and hyperglycemia, occurs as the host tissues react to the virus by increasing energy production and by activating the innate immune response. This is the phase which often culminates with the cytokine storm. A second hypo-inflammatory, immune-tolerant phase is characterized by decreased oxygen consumption, resumption of mitochondrial respiration and ATP production, as well as by increased fatty acid oxidation
For instance, the ORF9b protein, which localizes to mitochondria, antagonizes type I and III interferons by targeting multiple innate antiviral signaling pathways (Han et al, 2021). Another mitochondrial accessory protein, ORF10, inhibits the cell innate immune response inducing mitophagy-mediated MAVS degradation (Li et al, 2022a).
A notable exception among SARS-CoV-2 accessory proteins is accounted for by ORF3c, which has remained uncharacterized and under-investigated. The ORF3c protein has been predicted to be encoded by sarbecoviruses (a subgenus of betacoronaviruses) only (Firth, 2020; Jungreis et al, 2021), including SARS-CoV-2, SARS-CoV, and bat coronavirus RaTG13 (one of the bat betacoronavirus most closely related to SARS-CoV-2 (Zhou et al, 2020)). Analysis of the conservation of ORF3c in sarbecoviruses, together with ribosome-profiling data, strongly suggest that ORF3c is a functional protein (Cagliani et al, 2020; Finkel et al, 2021; Firth, 2020; Jungreis et al, 2021). Herein, we report the first investigation of the effect of ORF3c on cellular innate immune responses, autophagy, and lung cell mitochondrial metabolism.
The first paragraph is of larger interest to understanding the nuances of the viral infection both short and long-term, the reason I highlighted it, there is a shift in mitochondria (bioenergetics) kinetics after your infection is clear, but of course, in many people, this “correction” or back to bioenergetic normalcy just doesn’t happen, and they are kept in a rather dysfunctional metabolic state.
In this paper authors finally characterize the functions of ORF3c, and suggest this is (once again) a functional protein, a recurring trend lately. From the paper “Altogether, these data indicate that ORF3c localizes in the mitochondria and suggest that, at least partially, the protein product of ORF3c localizes on mitochondrial membranes. Further studies are required to clarify whether mitochondrial membrane binding of ORF3c occurs via the predicted transmembrane domain and/or via interaction with host mitochondrial proteins.”
They further find that ORF3c either itself or parts of it localizes itself on mitochondrial membranes, therefore inside the mitochondria which affect how they work.
Mitochondrial Δψ, measured using a DiOC6 (3,3′-dihexyloxacarbocyanine iodide) fluorescent probe, was found to be more negative in both transfected cells compared to the control suggesting oxidative phosphorylation hyperactivation.
The overexpression of each ORF led to an increase of the PER derived from mitochondria and a decrease in glycolytic PER In accordance, the activity of lactate dehydrogenase (LDH) did not significantly increase after transfection, suggesting that pyruvate is predominantly used in the Krebs cycle.
In conclusion, the mitochondria of transfected cells were not only unable to bypass the blockage of the fatty acid pathway through the use of the other two fuels, but they also required fatty acids to maintain basal OCR.
One personal observation, one which the authors themselves hinted at in the abstract, is the timing of measuring (and type of cells) will affect results, therefore the use of fatty acids as fuel rather than glycolysis (sugar) would be more of a timing, stage of infection, in line with papers where they found different metabolic states soon after infection to months later.
Also in line with other papers, they found changes in the Mitochondrial Δψ (Membrane Potential), a recent marker for different states of mitochondrial metabolism. They went to test the levels and ratio of NAD+/NADH (cell fuel) and if there were changes in substrates (things needed for a reaction), and the changes pointed towards the use of fatty acids as fuel.
These changes in NAD+ were further tested to measure if there was more oxidative stress in the mitochondria by measuring different stages of the activity of the Glutathione “cycle” which is one of your body's main mechanisms to clean ROS (cell rust) in which they found a significantly lower level in the presence of ORF3c.
I could explain the meaning of this closing paragraph but the authors themselves do a marvelous job at it.
The changes describe here in the closing section of the paper will definitely play a larger role in the long-term consequences of infection, the long-term changes are metabolic states derived from both severity and viral load, and uncontrolled replication will definitely lead to more expression of these proteins, which in turn is basically a biological roll of the dice if not properly taken care of.
This paper gives another mechanistic perspective on the importance of the other proteins of the virus, especially in the long-term prospectives, changes in mitochondrial function are exponential.
This is complex already and somewhat extensive, but originally I meant to cover three papers, and I will try to make it brief.
SARS-CoV-2 Nsp6 damages Drosophila (fly) heart and mouse cardiomyocytes (heart cells) through MGA/MAX complex-mediated increased glycolysis
2DG should not be new if you have been subscribed to me for a while now, I have covered the effects of this drug, in fact, my whole perspective on this virus was always from a mitochondrial/metabolic perspective. The following piece focused on the brain and sugar has many hints to the overall “hypothesis”, in which the exacerbated use of glucose as fuel early on in the infection (consequentially the exacerbation of insulin release and loss of control of glucose after the mRNA vaccine) leads to the start of a whole cascade.
From draining your body of glutathione to upregulating HIF-1a, and cascading into mitochondrial dysfunction and the Kynurenine Pathway.
In this paper, authors found that the NSP (non-structural protein 6) from SARS-CoV-2 induces glycolysis and (if you read the piece above) causes mitochondria dysfunction, increases ROS, and may explain the cardiac damage seen post-infection.
These results suggest that these SARS-CoV-2 encoded proteins (Nsp6, Orf6, Orf7a and Nsp3) may be associated with cardiac pathology, with Nsp6 having the most damaging effect in our model.
We found both Nsp6 and Pgi overexpression caused disorganization of the cardiac actin filaments, and significant loss of mitochondria activity as visualized by ATP5a (Fig. 3d, e). These results indicate that increased glycolysis pathway activity can lead to mitochondrial defects causing heart damage.
These results indicate that increased glycolysis pathway activity can lead to heart damage, both structurally and functionally.
The NSP6 affects the gene expression of genes directly linked to glycolysis, therefore inducing the proposed damage by the authors, not only affecting the function of cells/heart but damaging the structure (heart damage).
The good news is highlighted in yellow. As a wise man once said “Kudos to the designer”. And as a closing remark from these brilliant researchers. And my argument since March 2020.
Since the scope of this one is already rather extensive, I recommend you to read the following paper in its entirety. Especially because each of these proteins binds to a different protein and has a different effect. For the molecular nerds out there. Amazed to this day by how many things parts of this virus can bind.
NSP4 and ORF9b of SARS-CoV-2 Induce Pro-Inflammatory Mitochondrial DNA Release in Inner Membrane-Derived Vesicles
SARS-CoV-2-induced mitochondrial damage is an emerging pathological determinant in COVID-19 [50,53,54,55,56,57]. Extensive mitochondrial ultrastructural changes and functional impairments are primarily observed in infected airway epithelial cells [31,58] in addition to endothelial cells [50], monocytes [54,57], and T cells [55,59]. Under some conditions, mitochondrial damage is accompanied by pathogenic mtDNA release. Several studies have demonstrated that the circulating mtDNA levels in patients with COVID-19 are positively correlated with disease severity [12,13]. The findings of this study further demonstrated the value of circulating mtDNA as a potential biomarker to predict COVID-19 severity and the pro-inflammatory response. In this study, mtDNA extracted from patients with COVID-19 elicited a pro-inflammatory response and promoted cell death in primary human airway epithelial cells. These novel findings encouraged us to elucidate the molecular mechanism of SARS-CoV-2-induced mtDNA release and develop a rational stem cell-based approach with potential clinical applications for the treatment of other diseases in addition to COVID-19.
The release of mtDNA into the cytosol or the extracellular space is intricately associated with the activation of different PRRs [63,64]. Upon release into the cytosol, mtDNA activates the cGAS-STING1 pathway, which activates the type I interferon response [29,30]. The activation of the cGAS-STING pathway may be beneficial during viral infections and potentiate the anti-viral activity of immune cells [29,30] or result in pathological consequences in conditions, such as autoimmune diseases [65]. In SARS-CoV-2 infection, mtDNA released from endothelial cells activates the pro-inflammatory response through the cGAS-STING pathway [50]. However, the findings of this study indicate that in the airway epithelial cells, mtDNA release is not coupled with intrinsic cGAS-STING activation or NLRP3 inflammasome formation. This study demonstrated that a large pool of released mtDNA is secreted extracellularly via inner membrane vesicles. Thus, mtDNA extruded from the mitochondria is protected from cytosolic DNA sensors, leading to the suppression of an immediate anti-viral interferon response. This may explain the impaired interferon response reported in patients with COVID-19 [32,66]. The presence of a minor percentage of mtDNA released without encapsulation in IMM vesicles cannot be ruled out, although this minor population may not be adequate to activate cytosolic DNA sensors. These effects of mtDNA release and the underlying mechanisms may be cell-type-specific. SARS-CoV-2-infected endothelial cells are reported to activate the cGAS/STING pathway [50]. The immediate activation of the NLRP3 pathway in the infected cells can also be ruled out. This supports the notion that the SARS-CoV-2-infected cells do not undergo immediate cell death, which may otherwise limit the propagation of the virus in the target cells.
To understand the cGAS-STING significance you could read this, here the authors argue for another mechanism, rather independent of what you may read in that piece. Here they found a mechanism by which these two proteins modulate your cells and release mitochondrial DNA, which is correlated with the severity of disease, leading to inflammation, damage, and also worse long-term outcomes.
Here is a good visualization of the inflammatory potential of mtDNA release from your cells and “floating” around, therefore modulating your immune response, metabolic state, and your overall physiology, leading to disease acceleration.
Thus our understanding of the mitochondrial aspect of the virus grew quite large in the last few days. As a closing remark, there is a whole hormonal side to all of this, that is also often overlooked, especially from the Thyroid angle (a master regulator of bioenergy of own body). Thyroid, a reduction of FT3 and mitochondrial dysfunction in regards to SARS-CoV-2 infection.
Big thank you to all supporters and all subscribers who share my Substack.
SARS-2 proteins and mitochondria dysfunction
As part of my work towards my next review the mitochondrial protective properties of Baicalin are worth noting here, not just cardiac but for treating fractionation caused by AD too.
Baicalein protects cardiomyocytes against mitochondrial oxidant injury associated with JNK inhibition and mitochondrial Akt activation
https://pubmed.ncbi.nlm.nih.gov/24467536/
What say you John Paul about XBB?
https://twitter.com/my2pups2/status/1578935400277544960
https://twitter.com/prima__facie/status/1579182779350515712?s=19
https://www.biorxiv.org/content/10.1101/2022.09.15.507787v3.full
"Despite their rapidly divergent evolutionary courses, mutations on their receptor-binding domain (RBD) converge on several hotspots, including R346, K356, K444, L452, N460K and F486. The driving force and destination of such convergent evolution and its impact on humoral immunity established by vaccination and infection remain unclear. Here, we demonstrate that these convergent mutations can cause striking evasion of convalescent plasma, including those from BA.5 breakthrough infection, and existing antibody drugs, including Evusheld and Bebtelovimab. BR.2, CA.1, BQ.1.1, BM.1.1.1, and especially XBB, are the most antibody-evasive strain tested, far exceeding BA.5 and approaching SARS-CoV-1 level."