The molecular basis for the role of selenium in the pathology of the SARS Coronaviruses SARS-CoV-1 & 2, the causative agents of the 2003 SARS and current COVID-19 epidemics.
Higher selenium status has been shown to improve the clinical outcome of infections caused by a range of evolutionally diverse viruses, including SARS-CoV-2. However, the impact of SARS-CoV-2 on host-cell selenoproteins remains elusive. The present study investigated the influence of SARS-CoV-2 on expression of selenoprotein mRNAs in Vero cells. SARS-CoV-2 triggered an inflammatory response as evidenced by increased IL-6 expression. Of the 25 selenoproteins, SARS-CoV-2 significantly suppressed mRNA expression of ferroptosis-associated GPX4, DNA synthesis-related TXNRD3 and endoplasmic reticulum-resident SELENOF, SELENOK, SELENOM and SELENOS. Computational analysis has predicted an antisense interaction between SARS-CoV-2 and TXNRD3 mRNA, which is translated with high efficiency in the lung. Here, we confirmed the predicted SARS-CoV-2/TXNRD3 antisense interaction in vitro using DNA oligonucleotides, providing a plausible mechanism for the observed mRNA knockdown. Inhibition of TXNRD decreases DNA synthesis which is thereby likely to increase the ribonucleotide pool for RNA synthesis and, accordingly, RNA virus production. The present findings provide evidence for a direct inhibitory effect of SARS-CoV-2 replication on the expression of a specific set of selenoprotein mRNAs, which merits further investigation in light of established evidence for correlations between dietary selenium status and the outcome of SARS-CoV-2 infection.
Selenium is a trace element essential to human health largely because of its incorporation into selenoproteins that have a wide range of protective functions. Selenium has an ongoing history of reducing the incidence and severity of various viral infections; for example, a German study found selenium status to be significantly higher in serum samples from surviving than non-surviving COVID-19 patients. Furthermore, a significant, positive, linear association was found between the cure rate of Chinese patients with COVID-19 and regional selenium status. Moreover, the cure rate continued to rise beyond the selenium intake required to optimise selenoproteins, suggesting that selenoproteins are probably not the whole story. Nonetheless, the significantly reduced expression of a number of selenoproteins, including those involved in controlling ER stress, along with increased expression of IL-6 in SARS-CoV-2 infected cells in culture suggests a potential link between reduced selenoprotein expression and COVID-19-associated inflammation. In this comprehensive review, we describe the history of selenium in viral infections and then go on to assess the potential benefits of adequate and even supra-nutritional selenium status. We discuss the indispensable function of the selenoproteins in coordinating a successful immune response and follow by reviewing cytokine excess, a key mediator of morbidity and mortality in COVID-19, and its relationship to selenium status. We comment on the fact that the synthetic redox-active selenium compound, ebselen, has been found experimentally to be a strong inhibitor of the main SARS-CoV-2 protease that enables viral maturation within the host. That finding suggests that redox-active selenium species formed at high sele-nium intake might hypothetically inhibit SARS-CoV-2 proteases. We consider the tactics that SARS-CoV-2 could employ to evade an adequate host response by interfering with the human selenoprotein system. Recognition of the myriad mechanisms by which selenium might potentially benefit COVID-19 patients provides a rationale for randomised, controlled trials of selenium supplementation in SARS-CoV-2 infection.
Glutathione peroxidases (GPX), a family of antioxidant selenoenzymes, functionally link selenium and glutathione, which both show correlations with clinical outcomes in COVID-19. Thus, it is highly significant that cytosolic GPX1 has been shown to interact with an inactive C145A mutant of Mpro , the main cysteine protease of SARS-CoV-2, but not with catalytically active wild-type Mpro. This seemingly anomalous result is what might be expected if GPX1 is a substrate for the active protease, leading to its fragmentation. We show that the GPX1 active site sequence is substantially similar to a known Mpro cleavage site, and is identified as a potential cysteine protease site by the Procleave algorithm. Proteolytic knockdown of GPX1 is highly consistent with previously documented effects of recombinant SARS-CoV Mpro in transfected cells, including increased reactive oxygen species and NF-κB activation. Because NF-κB in turn activates many pro-inflammatory cytokines, this mechanism could contribute to increased inflammation and cytokine storms observed in COVID-19. Using web-based protease cleavage site prediction tools, we show that Mpro may be targeting not only GPX1, but several other selenoproteins including SELENOF and thioredoxin reductase 1, as well as glutamate-cysteine ligase, the rate-limiting enzyme for glutathione synthesis. This hypothesized proteolytic knockdown of components of both the thioredoxin and glutaredoxin systems is consistent with a viral strategy to inhibit DNA synthesis, to increase the pool of ribonucleotides for RNA synthesis, thereby enhancing virion production. The resulting "collateral damage" of increased oxidative stress and inflammation would be exacerbated by dietary deficiencies of selenium and glutathione precursors.
A significant, positive association between selenium status and COVID-19 prognosis has recently been identified. The present study investigated the influence of SARS-CoV-2 on host selenoproteins which mediate many beneficial actions of selenium. We found that SARS-CoV-2 suppressed mRNA expression of selenoproteins associated with ferroptosis (GPX4), ER stress (SELENOF, SELENOK, SELENOM and SELENOS) and DNA synthesis (TXNRD3) in Vero cells, providing a deeper insight into the connection between selenium and SARS-CoV-2.
This paper presents a major new and in fact unprecedented mechanism that may contribute to the highly significant correlation between selenium status and COVID-19 mortality that my collaborators and I recently reported in Am J Clin Nutr (Zhang et al. 2020, full text available on this site). The antioxidant selenoenzyme glutathione peroxidase (GPX) links selenium and glutathione, two important dietary factors that have both been found to have a considerable influence on survival in various viral infections, e.g. HIV-1, and now in COVID-19 patients. However, the mechanisms involved are potentially multifactorial, and not well understood. In the light of these clinical correlations, it is highly significant that the intracellular enzyme GPX1 has been shown to interact with Mpro, the main cysteine protease of SARS-CoV-2. Using well-established computational methods, we show that there are potential cysteine protease cleavage sites in GPX1, in several other selenoproteins, and in two glutathione-linked proteins, that in some cases very closely match known Mpro cleavage sites. These sites also map to the surface of the 3D structures of the targeted proteins, suggesting they could be accessible to proteolytic attack. Selenoproteins and glutathione are critical components of cellular antioxidant defenses; thus, virus-mediated proteolysis of one or more of the proposed target proteins, even at a low efficiency, could increase oxidative stress and activate pro-inflammatory cytokines. Particularly under high viral loads, this could contribute to increased morbidity and mortality in COVID-19, which would be even more severe in patients with dietary deficiencies of selenium or glutathione.
The biosynthesis of DNA inherently competes with RNA synthesis because it depends on the reduction of ribonucleotides (RNA precursors) to 2’-deoxyribonucleotides by ribonucleotide reductase (RNR). Hence, RNA viruses can increase viral RNA production in cells by partially blocking the synthesis of DNA, e.g. by downregulating the mammalian selenoprotein thioredoxin reductase (TR), which normally acts to sustain DNA synthesis by regenerating reduced thioredoxin, a hydrogen donor for RNR. Computational and preliminary experimental evidence supports the hypothesis that a number of pathogenic RNA viruses, including HIV-1, Ebola, Zika, some flu viruses, and SARS-CoV-2, target TR isoforms by antisense. TR knockdown would create a host antioxidant defect that could be partially rectified by increased selenium intake, or be exacerbated by selenium deficiency, contributing to viral pathogenesis. There are several non-selenium-dependent means that viruses might also exploit to slow DNA synthesis, such as targeting RNR itself, or components of the glutaredoxin system, which serves as a backup redox system for RNR. HIV-1 substantially downregulates glutathione synthesis, so it interferes with both the thioredoxin and glutaredoxin systems. Computational results suggest that, like Ebola, SARS-CoV-2 targets TR3 by antisense. TR3 is the only TR isoform that includes an N-terminal glutaredoxin domain, so antisense knockdown of TR3 may also affect both redox systems, favoring RNA synthesis. In contrast, some DNA viruses encode their own glutaredoxins, thioredoxin-like proteins and even RNR homologues – so they are doing just the opposite, favoring DNA synthesis. This is clear evidence that viruses can benefit from shifting the RNA:DNA balance to their advantage.
Selenium status is an established factor in the incidence, outcome or virulence of various RNA viral infections. Because China has geographic regions that range from extremely high to extremely low selenium intakes, we hypothesized that selenium status might influence the outcome of COVID-19 in these areas. For our analysis, we used reported cumulative case and outcome data for a snapshot of the COVID-19 outbreak, as of 2-18-2020. We found that in the city of Enshi, which is renown for having the highest selenium intakes in China, the cure rate was 3 times as high as that for all the other cities in Hubei Province, where Wuhan is located (p < 0.0001). In contrast, in Heilongjiang Province, where Keshan is located and extreme selenium deficiency is endemic, the death rate was almost 5 times as high as that for all the other Provinces and Municipalities outside of Hubei (p < 0.0001). Finally, for a set of 17 cities outside of Hubei, using published city data on average levels of selenium in human hair (a reliable measure of dietary intake), a significant linear correlation with the cure rate for COVID-19 was observed (R squared = 0.72, P < 0.0001).
This is the first in a set of presentations or research articles that will explore a body of evidence relevant to the question of whether selenium supplementation could be beneficial in COVID-19, and for whom. The primary evidence justifying this investigation is the new paper published 4-28-2020 in Am. J. Clin. Nutr., "Association between regional selenium status and reported outcome of COVID-19 cases in China", https://doi.org/10.1093/ajcn/nqaa095. The short presentation attached here reviews historical precedents, primarily for viral infections in which a similar variation in incidence (case frequency) or virulence of the disease has been associated with selenium distribution in soils and human intake. The main point of this presentation is to show that for every previous case where such correlations have been made, selenium supplementation, either as a long term dietary adjustment, or as a pharmacological application in an outbreak of an acute infection, led to significant decreases in mortality and/or incidence of the disease. Because the correlations we have now shown between regional selenium intakes and COVID-19 outcomes are probably the most significant ever documented for an infectious disease, the inference (to me at least) is that it is therefore highly probable that in cases of suspected SARS-CoV-2 exposure, Se supplementation within the recommended range of safe daily intakes have the potential to be at least somewhat protective, with negligible chance of harm. Additional presentations or preprints that I will post will delve into possible molecular mechanisms that can explain WHY such correlations exist...
This poster provides a brief introduction to the concepts that were later published as: Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry (E.W. Taylor et al. 2016, Current Topics in Medicinal Chemistry, 16:1530-35. DOI : 10.2174/1568026615666150915121633. In Figure 5, the poster includes additional experimental evidence for the proposed mechanism by demonstrating the existence of extended isoforms of the HIV-1 nef protein and the Ebola nucleoprotein, via readthrough of their 3'-terminal UGA stop codons. Later work from our lab (published in L.Premadasa PhD Dissertation, 2016) showed that readthrough of the nef 3'-UGA is dependent on both the selenium concentration and the presence of thioredoxin reductase 1 mRNA, which is the antisense target of the HIV-1 nef coding region demonstrated here .
When the SARS genomic sequence became available early in 2003, I was able to identify 2 programmed ribosomal frameshift sites in the virus, both associated with an in-frame UGA (potential selenocysteine) codon in the overlapping reading frame. Both of these frameshift sites were later shown to be functional via in vitro reporter gene assays. Dr. Jinsong Zhang contacted me with evidence of selenium abnormalities in the blood of SARS patients, leading to the joint abstract. These results were never published at the time because the SARS outbreak was contained and with the threat no longer imminent, it was not possible to get funding to pursue the findings. The 2 frameshift sites, their in-frame UGA codons, and glutathione peroxidase homology region are also found in the 2019 SARS-CoV-2 virus, supporting a role for selenium in the pathology of COVID-19.