By Doris Loh
The official documentation of an ASYMPTOMATIC carrier of COVID-19 (SARS-CoV-2) released by China on February 28, 2020 brings into serious question if the global concerted reaction and efforts to contain the coronavirus outbreak are justified.
According to the paper by Weng, Jianping et al. the subject is a 50-year old woman with no significant past medical history, and did not have any direct connections to Wuhan and adjacent areas. Apart from mild elevation of liver enzymes after a 10-day treatment course of antivirals after being confirmed positive for COVID-19 on February 6, she was asymptomatic, with all functions being normal, and free of signs of viral pneumonia. After the treatment course was completed, the patient was re-tested by throat and anal swabs and results still returned as POSITIVE for the COVID-19 coronavirus. The authors of the paper concluded that the discovery of a healthy carrier of the virus increases the difficulty in prevention of transmission [1]
Since the outbreak of the coronavirus in late 2019, the total death rate reported as of February 28, 2020 is 7% of total closed cases, or 3.42% of total confirmed cases reported globally. 43.9% of the confirmed infected recovered. Only 18% of the remaining patients still infected are in serious condition.
How is COVID-19 different from a normal garden-variety flu virus, and why are countries closing borders, placing cities and towns on quarantines; canceling public events; closing schools; suspending air travels; locking down resorts and refusing docking of cruise ships just to prevent the spread of a seemingly non-lethal flu virus? Why are investors around the globe selling off equities in such a panic? The Dow Jones Industrial Average registered its worst one-day point drop in history on February 27, 2020 [2]
Discovery of Furin Cleavage Site in COVID-19
The answer lies in a paper that was first released on Jan 27, 2020 in China by Jishou Ruan et al. from the prestigious Nankai University, Tianjin China. This study has since been revised twice, last edit was Feb 14, 2020 [3]. The authors of this study reported for the first time, that unlike all other betacoronavirus, an important mutation in the Spike (S) protein of COVID-19 allowed it to acquire a cleavage site for the furin enzyme. This furin cleavage site is NOT PRESENT in S proteins of ALL OTHER betacoronavirus, including the HIGHLY INFECTIOUS SARS coronavirus [4].
The furin cleavage allows efficient virus entry into cells, making the COVID-19 easily transmissible at rates up to 1,000 times greater than the virulent SARS coronavirus [3]. The authors compared the packing mechanism of the virus to other furin cleavage viruses like HIV, Ebola, as well as other highly transmissible avian influenza strains. The findings of Ruan and Gao et al. have been independently confirmed by a French team led by Decroly around the same time.
On February 10th, Decroly et al. reported the S-protein sequence of COVID-19 has a specific furin-like cleavage site that is absent in betacoronavirus including SARS-CoV. Importantly, the authors pointed out that this furin cleavage site could have significant implications for the viral life cycle and pathogenicity of COVID-19 [5].
Furins and Coronaviruses – What’s the Big Deal?
In order to infect host cells after they enter the body, envelope viruses, like bacteria and viral exotoxins, remain dormant until they are activated. Common influenza viruses can be activated by proteases typically found in cells in the airway epithelium of the respiratory tract [6].
Coronaviruses are highly adaptive to the environments of the different cells they infect. Each coronavirus may be activated by a specific set of proteases. Proteases catalyzes the breakdown of proteins into smaller polypeptides or amino acids by cleaving the peptide bonds of those proteins.
The fact that COVID-19 has cleavage sites for furin enzymes means that this virus can be extremely pathogenic, with the capacity to replicate in MULTIPLE tissues and organs due to how furins are utilized and distributed in the human body [7].
Cleavage specificity can dictate the tropism and virulence of the virus. Furin-like cleavage in human coronaviruses have been associated with the development of neurological diseases where the invasiveness and efficient establishment of lower pathogenicity can result in persistent infection of the central nervous system [8].
HIV, human immunodeficiency virus, initiates infection by fusing the viral envelope with host cell membranes. Furin is used by HIV to cleave the glycoproteins in the viral envelope to complete the fusion process, gaining entry into various host cells and infecting them [9]. The ability of viruses such as Ebola to exploit furin may heavily influence their pathogenicity.
In addition, bioweapons such as Anthrax require furin to initiate toxicity. Furin has been identified as the cellular protease that cleaves the protective antigen protein of anthrax toxins that unleashes toxic activity [10].
If furins are essential to viral entry and replication, would it not be simple just to inhibit furin expression? That would indeed be an easy solution if only furins are not involved in a multitude of vital cellular processes. Basically all cell types have been observed to contain at least a small amount of furin exposed on their cell surface [10]. What are furins exactly?
Furins in Health & Disease
Two percent of the human genome encodes for more than 550 proteases [11]. Proteases are important because they play important roles in basically ALL physiological processes, including the digestion of proteins in our food and degradation of misfolded or discarded proteins [12].
Furin is a gene that encodes a type 1 membrane bound protease that is expressed in many tissues including neuroendocrine, liver, gut, and brain. Many important biological proteins are inactive when they are first synthesized. In order to become active, certain sections must be removed, or cleaved, by proteases.
Furin enzymes cleave their substrates in order to activate them. More than 100 furin cleavage sites in mammalian proteins have been identified to date. These proteins include growth factors; cytokines such as IGF1, IGF2, TGFβ, PDGFα, PDGFβ, VEGF‐C, NGF, CXCL10; hormones like PTH, TRH, GHRH; adhesion molecules including integrins, vitronectin; collagens, metalloproteinases; coagulation factors; receptors; membrane channels and albumin [13].
Once cleaved by furin enzymes, these proteins and peptides become bioactive, performing key functions in cell proliferation, immunity and inflammation. While most of the target substrates of furins are activated upon cleavage, furins can also inactivate substrates via cleavage [14]. Mutations in proteases like furins or dysregulation in furin activity have been associated with numerous pathological diseases including cardiovascular and neurological disorders; autoimmune diseases and cancer [15, 16, 8, 17, 18].
In a similar way, envelope proteins of viruses including HIV, influenza, dengue fever, ebola, and marburg virus, MUST be cleaved by furin or furin-like proteases to become fully active and functional. COVID-19, a novel coronavirus that originated from Wuhan, China, has now been identified to possess furin-cleavage sites in the S protein.
Thus COVID-19 and a host of important substrates found in the human body, including cytokines, hormones, growth factors and receptor all need to be cleaved by furin enzymes for activation. It is now becoming much clearer why furin-cleavage sites in viruses can increase their virulence and pathogenicity.
What is most interesting about furins, is that because it has pleiotropic properties, variations in furin expression levels and/or its enzymatic activity may result in the progression and pathogenesis of a wide array of disorders, including rheumatoid arthritis, amyloid dementia, but most of all, tumorigenesis [15].
Furins & Cancer
Furins hold the ‘master switch’ that controls tumor growth and development [19]. Furin expression levels are positively correlated with cancer aggressiveness, and are used as a prognostic biomarker for advanced cancers [20]. Aberrant expression or activation of furin promote formation and enhance malignancies of various tumors including colon, head and neck, lung, skin and brain cancers [20].
Since adhesion molecules are substrates of furin, this enzyme can promote migration and extravasation of malignant cells as it processes adhesion molecules that mediate cell to cell interactions. Furin mediated cleavage of integrins can regulate cell growth, division and survival [21]. Cancer metastasis is further increased by the activation of matrix metalloproteinases (MMP14) when they are cleaved by furin enzymes [20].
Furins are now widely accepted to be oncogenic and prometastatic, because they are able to cleave and activate proteins that promote cell proliferation, angiogenesis, migration and tissue invasion. These proteins include growth factors such as IGFs, PDGFs, NGF, or angiogenic factors like VEGF, all of which promote tumorigenesis [20].
The cancer microenvironment is extremely hypoxic. Tumor cells exhibit increased oxygen demand as a result of cell expansion in a background of decreased oxygen supply due to defective tumor vascularization [22]. In response to inadequate oxygen supply, complex mechanisms, like hypoxia-inducible factors (HIFs) and their downstream gene expression networks are activated to allow cells to adapt and survive in a hostile environment [23].
Hypoxia inducible factor 1α (HIF1α) is a transcription factor that can be activated under hypoxia. HIF1a is now regarded as an effective prognostic marker of cancer aggressiveness because it is considered to be the master regulator of neoangiogenesis in cancer development. HIF1a is able to control the expression of a wide array of genes involved in cancer progression.
It is therefore not surprising that furin expression is induced by hypoxia, and ALL THREE promoters for the FUR gene that encodes furin proteins have binding sites for HIF1 [24].
The Love-Hate Relationship Between Furins & HIF1a
During hypoxia, messenger RNA of the FUR gene are dramatically enhanced. Hypoxia activates both HIF-1a AND furin promoters. Expression of furin mRNA was found to be increased rapidly in oxygen-deprived cells by HIF-1, which binds to a sequence in the gene that promotes its rapid expression [25].
Hypoxia triggers the translocation of furins from the trans-Golgi network [26] to the cell surface where furins may further enhance the processing of growth factors and other extracellular cancer precursor proteins [27]. The relationship between HIF-1a and furins is further complicated by the negative feed-back loop between furins and HIF-1a.
As it turns out, although the expression of furins can be significantly increased by HIF-1a [25], furins can also turn around and suppress HIF-1a protein translation in a regulatory feed-back loop that is related to IGF (insulin-like growth factor) signaling [28].
Although furin inhibition is a promising target to treat COVID-19, the use of systemic inhibition will inevitably result in detrimental effects, as furins are involved in a multitude of important physiological activities. Furin knock-out mice die at day 11 during embryonic development due to cardial ventral closure defects and hemodynamic insufficiency [29, 36]. Eliminating furin-expression in endothelial cells of mice resulted in cardiac malformation and death shortly after birth [30].
Even mutations in the cleavage site of a single furin target protein have been shown to result in genetic disorders such as haemophilia B or X‐linked hypohidrotic ectodermal dysplasia [31, 32] Underexpression or knock-out of furin expression in retinal myeloid inhibit revascularization and angiogenesis [33]. Furin expression in the eyes is not only important for maintaining ocular health, it also allows transmission of viruses that have furin-cleavage sites. COVID-19 has recently been shown to be transmitted through ocular pathways [34].
Clearly the systemic inhibition of furins to control COVID-19 infection is fraught with obstacles. Yet there is one molecule that holds promise for the treatment of COVID-19
Vitamin C & Immunity
Clinical trials on the use of high-dose ascorbic acid (24 g), Vitamin C are currently underway in China [35]. One of the most obvious reasons for using Vitamin C is the effect on lymphocytes.
Infected patients in critical condition who were able to elevate lymphocyte counts during the third week of infection could usually escape death and recover. Whereas patients whose lymphocyte counts continue to plummet could not reverse the complete destruction of their immune systems. These patients inevitably die due to multiple organ failures [37].
During critical illness, lymphocytes (white blood cells) are the slowest to restore, endangering patients’ chances for recovery. The regeneration of a subset of lymphocytes known as T-lymphocytes are especially important in the fight against viral infections [38].
Ascorbic acid (Vitamin C) has been found to enhance human T cell proliferation in vitro [39]. In addition, ascorbic acid promotes the maturation of T cells and has been found to be INDISPENSABLE for T cell development in vitro. Without ascorbic acid, a preT cell-stage arrest would occur, inhibiting development and maturation [40].
Ascorbic acid also speeds the regeneration and proliferation of natural killer (NK) cells [41]. Natural killer (NK) cells are unique lymphocytes without a clonally specific receptor and NK cells play a key role in our defense against viral infections [42]. NK cells are critical for the control of viral infections [43].
Ascorbic acid not only is able to protect mitochondria during viral infections [44], it can strengthen the immune system by protecting and regenerating critical lymphocytes [45, 46]. Yet in the context of furin cleavage sites, the importance of ascorbic acid cannot be understated.
Vitamin C, COVID-19 & Furins – The HIF-1a Connection
Hypoxia inducible factor 1α (HIF1α) is a transcription factor that can be activated under hypoxia. HIF-1a levels are normally kept low in the presence of oxygen. When there is adequate oxygen, HIF-1a is bound by the von Hippel-Lindau (VHL) protein, which then prepares HIF-1a for degradation [47].
If HIF-1a is not degraded properly, it will be able to bind with its heterodimeric component, HIF-1β, and becomes stabilized. Once HIF-1a is stabilized, it can activate the expression of a large number of genes associated with survival, apoptosis, and metabolic reprogramming, and of course, increase the expression of furins.
When a person is critically ill, oxidative stress levels are significantly elevated [48, 49]. Oxidative stress can activate HIF-1a [50, 51]. The activation of HIF-1a inevitably triggers the increased expression of furins, leading to cleavage and activation of COVID-19.
Ascorbic acid, Vitamin C has been convincingly demonstrated to modulate the HIF pathway in a dose dependent manner. Increased intracellular ascorbate dramatically elevated activity levels prolyl hydroxylase, which prevented the stabilization of HIF-1a [52].
Prolyl hydroxylase is an enzyme that can add a hydroxyl group (hydroxylation) to the α-subunits of HIF. This hydroxylation process allows HIF-1a to be bound to the von Hippel-Lindau protein (VHL), which then initiates the degradation process of HIF-1a. Prolyl hydroxylases are therefore, critical in preventing HIF-1a from activation and stabilization.
What is truly fascinating is that prolyl hydroxylases not only depend on ascorbic acid as substrates, but ascorbic acid MUST BE IN THE SPECIFIC MOLECULAR STRUCTURE of L-ASCORBATE in order to suppress HIF-1a [53]!!
These are the reasons why 50 tons of Vitamin C were sent to Wuhan to help citizens combat COVID-19 [54], and doctors from China are reporting excellent patient recovery rates from the use of Vitamin C in the treatment of COVID-19.
As of March 3rd, 2020, 80 countries around the world reported confirmed cases of COVID-19, that is a 100% increase from 40 countries that reported confirmed cases on February 25th [55]. The furin cleavage site of COVID-19 confers high transmissibility. We do not yet understand the true extent of the pathogenicity of this novel coronavirus from Wuhan. Only time will tell. In the meantime,
Have you had your AA today?
References
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[54] https://twitter.com/dsm?fbclid=IwAR2M_5xD7N2ccbVFM1iSvPfEjcOd110DljWWWq0bTgSo3PLTuS3k5DI8LfU
