The Spike Protein and IRF3 Degradation: Pathway to Persistent Spike Protein Expression and Mitochondrial Disease

Understanding the presence of Spike in those with Long COVID and those with Spike Protein injuries

SARS-CoV-2 Spike interacts with IRF3 and mediates its proteasomal degradation. (A) Co-immunoprecipitation and immunoblot of Flag IRF3 (1 µg) with C9 Spike (1 µg) co-transfected in HEK 293T cells. (B) Immunoprecipitation and immunoblot of transfected C9 Spike (1 µg) with IRF3 in HEK 293T cells treated with MG132 (10 µM) for 4h. (C) Immunoblot analysis of Flag IRF3 (250 ng) co-transfected with C9 Spike (500 ng and 1 μg) in HEK293T cells. (D) Immunoblot analysis of Flag RIG-I, Flag MAVS, Flag TBK1, Flag IKKi, Flag IRF3, and Flag IRF7 (1 µg each) co-transfected with C9 Spike (2 µg) in HEK 293T cells. (E) Immunoblot analysis of IRF3, p65, and STAT1 in HEK 293T cells transfected with C9 Spike (500 ng and 1 µg). (F) Immunoblot analysis of Flag IRF3 (1 µg) co-transfected with C9 Spike (2 µg) in HEK 293T cells and subsequently treated with or without MG132 (10 µM). GAPDH serves as a loading control. (G) IFN-β luciferase reporter assays in HEK 293T cells co-transfected with plasmids encoding IRF3 (500 ng) and C9 Spike (1 µg) followed by MG132 (20 µM) treatment for 4 h. *Indicates p < 0.05 as determined by the student’s t-test. Results are representative of two independent experiments. The relative band intensity (*/#) of co-immunoprecipitated Flag IRF3 in (A), co-immunoprecipitated endogenous IRF3 in (B), Flag IRF3 in (C, F), Flag RIG-I, Flag MAVS, Flag TBK1, Flag IKKi, Flag IRF3 and Flag IRF7 in (D) and endogenous IRF3 in (E) was measured using ImageJ software.

PREFACE: I believe all individuals admitted to hospital should be screened for the presence of Spike Protein. We should also perform mass screening of individuals for the presence of Spike Protein. The data gathered is most likely critical in understanding the current worldwide excess deaths pandemic.

A paper published in January of last year found that the Spike Protein is promoting the degradation of IRF3.

We found SARS-CoV-2 Spike interacted with IRF3 and further instigated its proteasomal mediated degradation to terminate IFN-I activation. SARS-CoV-2 Spike is comprised of an S1 and S2 domain where SARS-CoV-2 entry requires proteolytic cleavage at these sites (Huang et al., 2020). In many of our immunoblot experiments that examine ectopic Spike expression, we noted the detection of both the full length Spike as well as the faster migrating, but lower intensity S2 domain.

SARS-CoV-2 Spike Antagonizes Innate Antiviral Immunity by Targeting Interferon Regulatory Factor 3
https://www.frontiersin.org/articles/10.3389/fcimb.2021.789462/full

What the paper does not discuss, but I believe is extremely important, is that the absence of IRF3 allows for cells to be persistently infected and to continue to express virus, AND VIRAL PARTICLES.

Interferon regulatory transcription factor 3 (IRF3) is a transcription factor that upon activation by virus infection promotes the synthesis of antiviral genes, such as the interferons (Hiscott, 2007). In addition to inducing genes, IRF3 triggers antiviral apoptosis by RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA), which is independent of its transcriptional activity. RIPA protects against lethal virus infection in cells and mice (Chattopadhyay et al., 2016). In the absence of RIPA, caused by genetic ablation, chemical mutagenesis or inhibition of the pattern recognition receptor (PRR) retinoic acid-inducible gene I (RIG-I), Sendai virus (SeV) infection does not trigger cellular apoptosis and become persistently infected (Peters et al., 2008; Chattopadhyay et al., 2013).

Please note this very important finding:

Here, we outline a detailed procedure for the development of a persistently SeV-infected human cell line (Figure 2), which continuously expresses viral protein and produces low levels of infectious viral particles.

Figure 2. Development of SeV-persistent cell lines using P2.1 parental cells.

THE MITOCHONDRIAL IMPLICATION, SPIKE PROTEIN INJURY/DISEASE AND LONG COVID

Another very important observation to consider is that the Spike’s induced degradation of IRF3 is allowing “sick” cells to not only produce Spike, but also not die and dysfunction. I believe this is at the core of Long COVID and Spike Protein injury/disease. If the mitochondria are not telling the infected/spike protein factory cell to die, not only will it continue to express spike, it will also continue to dysfunction. Obviously, when enough cells dysfunction in an organ, that organ dysfunctions.

IRF3 is also critical for triggering apoptosis via a distinct pathway, which does not require its transcriptional activity. In a series of previous studies, we have discovered the pathway, which we named RIPA that triggers apoptosis in virus-infected cells. In RIPA, IRF3 interacts with BCL-2-Associated X protein (BAX), a pro-apoptotic factor (Chattopadhyay et al., 2010). Upon binding to BAX, IRF3 translocates to the mitochondria, and initiates a signaling cascade that ultimately promotes apoptosis (Chattopadhyay et al., 2010). In the absence of IRF3 or other components of RIPA, the cells establish viral persistence when infected with Sendai virus (SeV) (Peters et al., 2008; Chattopadhyay et al., 2013).

Establishment of a Human Cell Line Persistently Infected with Sendai Virus
https://en.bio-protocol.org/en/bpdetail?id=2512&type=0

Perhaps an additional effective therapeutic against the Spike and SARS-CoV-2 would be to induce the upregulation of IRF3. This may clear the virus (and Spike, of course) out of the body.