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Understanding selenium and glutathione as antiviral factors in COVID-19: Does the viral Mpro protease target host selenoproteins and glutathione synthesis?

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Abstract and Figures

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.
Comparison of known and predicted M pro cleavage sites. The most relevant positions P4-P1′ of the 11 known SARS coronavirus Mpro cleavage sites in nonstructural proteins are shown (for nsp4 through nsp16), aligned with predicted sites in several human selenoproteins (GPX1, SelenoF, SelenoP, and TXNRD1), as well as glutaredoxin-1 (GLRX-1) and the rate-limiting enzyme for glutathione synthesis, glutamate-cysteine ligase catalytic subunit (GCLC, with 2 predicted cleavage sites a and b). The known nsp cleavage sites shown are all from SARS-CoV-2, with the addition of the nsp5/6 site from SARS-CoV (marked *). The sites identified in human proteins are displayed next to the known Mpro cleavage site to which they are most similar, highlighted in matching color. The experimentally determined catalytic efficiencies of SARS-CoV Mpro at each of the known nsp cleavage sites are shown as relative kcat/Km ([18]). Three different methods for prediction of cysteine protease cleavage sites were used: PROSPER, with numbers in parenthesis showing the score for the predicted site, any score > 0.8 being significant; NetCorona, where scores > 0.5 are considered positive, and Procleave, where every possible P4-P4′ octamer in a protein sequence is evaluated, scored and ranked. Some aligned non-identical residues highlighted in italics are nonetheless chemically and structurally similar to residues found in the same position in the known cleavage sites, e.g., serine (S) vs. threonine (T), Leucine (L) or isoleucine (I) vs. valine (V), and glutamine (Q) vs. asparagine (N); all of these pairs differ only by a single carbon atom (CH3 or CH2 unit). The predicted Mpro cleavage sites in human proteins labeled A to G are discussed in the text. The computational results cited are collated in Fig. S2.
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PERSPECTIVE
Understanding selenium and glutathione as antiviral factors in COVID-19:
Does the viral Mpro protease target host selenoproteins and glutathione synthesis?
Ethan Will Taylor* & Wilson Radding
Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro
Patricia A. Sullivan Science Building, PO Box 26170, Greensboro, NC 27402-6170, USA
*For correspondence: ewtaylor@uncg.edu
Abstract
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.
_________________________________________________________________________________
Keywords: Coronavirus, COVID-19, selenium, glutathione, glutathione peroxidase 1, protease,
selenoprotein, thioredoxin reductase
Taylor and Radding Selenoproteins as SARS-CoV-2 protease substrates
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Introduction
Several independent studies have now established a significant association between the outcome of
COVID-19 and previously documented regional selenium (Se) status in Chinese cities [1], and a similar
relationship between serum Se and mortality in a European cohort of COVID-19 patients [2]. These
observations invite questions about the mechanisms involved, particularly because they fit into a
consistent pattern of a role for Se that has been reported over several decades for a variety of RNA
viruses (enteroviruses, hantaviruses, and influenza A) and viruses with an RNA stage (HIV-1 and
Hepatitis B virus), as reviewed by various authors [3-5].
Most (but not all) of the biological roles of Se, both as selenocysteine in selenoproteins, and as redox-
active Se-containing metabolites, involve interactions with cysteine thiols and disulfides in proteins and
peptides, and their various oxidized forms. Like Se, the essential antioxidant and free radical scavenger
glutathione (GSH), a tripeptide thiol, has also proven to be an important factor in various viral infections,
particularly in HIV/AIDS, as reviewed in section 2.1.2. of [6], and most recently, in COVID-19 [7,8].
Given their intertwined biochemical roles, there are likely to be common factors and mechanisms
underlying the therapeutic importance of Se and GSH in viral infections.
A confirmed molecular interaction between SARS-CoV-2 protease and a human selenoprotein
Correlations between COVID-19 clinical outcomes and both host Se and GSH status provide important
context to a related observation emerging from a proteomics-based study of possible cellular targets of
SARS-CoV-2 (SCoV2) proteins. Using affinity-purification mass spectrometry, Gordon et al. identified
high-confidence protein-protein interactions between 26 of the SCoV2 proteins and human proteins [9].
As bait for interacting human proteins, one of the proteins they used was the SCoV2 main viral protease
Mpro, a cysteine protease also known as nonstructural protein 5 (nsp5). The study also included a
catalytically inactive C145A Mpro mutant (lacking the active site cysteine), which was also used as a bait
protein, in order to discriminate false positives that might bind nonspecifically to the Mpro active site
cysteine via disulfide bond formation. One of the interactions they identified involved the cytosolic form
of the selenoprotein glutathione peroxidase, GPX1, which bound strongly to the inactive C145A Mpro
mutant. However, this interaction with GPX1 was not observed with the wild type Mpro. This is precisely
what one might expect to see if GPX1 is a protease substrate, because its cleavage would produce two
fragments that would necessarily have reduced affinity to the enzyme relative to GPX1.
Significantly, as shown in their Extended Data Fig. 6 [9], Gordon et al identified one other host protein
(TRMT1) that bound only to the inactive Mpro C145A mutant, and concluded it was a likely Mpro
substrate, because they were able to identify a putative Mpro cleavage site in the TRMT1 protein sequence
(PRLQ/ANFT), where the slash (/) represents the cleavage site. Thus, the only piece of evidence lacking
to draw a similar conclusion for GPX1 is a candidate Mpro cleavage site, which most algorithms will
fail to find, e.g., if they are highly stringent about requiring a Q in the P1 position (Fig. 1).
As shown in Fig. 1A for the 2003 SARS coronavirus (SARS-CoV, SCoV) and in Fig. 1B for the 2019
SCoV2, the consensus logo patterns of 11 Mpro cleavage sites in the viral 1ab polyprotein are essentially
identical for the 2 viruses. Specifically, 8 of the 11 sites are fully identical over the 10 residues spanning
the P5 to P5′ positions, and only two of the 11 cleavage sites have more than a single amino acid
difference within the 10 residue span (Supplementary Material Fig. S1). This means that (as discussed
below) functional studies of the 2003 SCoV protease are highly relevant to that of the 2019 virus. From
Fig. 1A-B, the most important residue positions for Mpro recognition are, in descending order, P1, P2,
Selenoproteins as SARS-CoV-2 protease substrates
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This is a provisional file, not the final typeset article
P1′, P4 and P3, with the first 3 being the major determinants, with the minimal P2-P1′ consensus being
LQ/(S,A,N,G). The downstream sequence past P1′ is highly variable.
Figure 1. Coronavirus Mpro cleavage site consensus sequence logo plots and comparisons.
Logo plots from multiple alignments of the known Mpro cleavage sites are shown for the 2003
SCoV (A) and 2019 SCoV2 (B). The height of a letter at each position reflects its probability
in the alignment; each of the logos shown represents the consensus of 11 Mpro cleavage sites
from a single virus. Plots were generated using WebLogo (weblogo.berkeley.edu/logo.cgi),
from the alignments in Figure S1. C. Comparison of the GPX1 active site sequence containing
selenocysteine (U) to the known SCoV2 Mpro cleavage site at the nsp13/14 junction; this site
is identical in the 2003 and 2019 coronaviruses. D. Comparison of a predicted Mpro cleavage
site in human selenoprotein F to a known SCoV2 Mpro cleavage site at the nsp12/13 junction.
The GPx1 active site has the sequence LUG, where U is the catalytic selenocysteine; this matches the
observed Mpro consensus core target sequence combination LQ/G in 2 of 3 positions. Furthermore, as
shown in Fig. 1C, the important upstream side of the known Mpro cleavage site at the nsp13/14 junction,
NVATLQ/A, is remarkably similar to that of the active site sequence of GPx1, NVASLU/G, where the
selenocysteine (U) lines up with a glutamine (Q) in the viral sequence. These two amino acids (U and
Q) are not highly similar, but are both midrange in size, and polar in nature, because the selenol residue
is predominantly ionized at physiological pH. The other two “mismatches” in the important positions P3
and P1′, i.e. S vs T and G vs A, both differ only slightly, by the size of a methyl group, and in any case,
having a glycine (G) in the P1′ position is a permitted residue (Fig. 1A), as observed in the SCoV Mpro
cleavage site at the nsp5/6 junction (indicated by * in Fig. 2). Significantly, the Procleave protease
cleavage prediction server (procleave.erc.monash.edu.au) [10] identified the GPX1 active site octameric
sequence ASLU/GTTV as a highly ranked possible cleavage site for a cathepsin S-like cysteine protease
(Fig. 2G).
For several reaction intermediates in the GPX1 mechanism, Se is attached to either oxygen, or to
nitrogen, as a selenenylamide. The latter is quite stable, and may accumulate during GSH depletion,
because GSH is needed to regenerate the selenolate [11]. Selenenylamide is more similar than
selenocysteine to glutamine, so that form might be preferentially targeted by Mpro.
Taylor and Radding Selenoproteins as SARS-CoV-2 protease substrates
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Because the mutation rate in cellular genes is thousands of times slower than that for an RNA virus, if
there is a physical interaction between these two proteins as Gordon et al observed using the inactive
mutant Mpro, it is almost certainly because the virus has evolved to target the host for some reason, rather
than the converse. The most parsimonious hypothesis is simply that this is a case of a viral protease
targeting a cellular gene for cleavage, for which there are abundant precedents, including the proposed
Mpro targeting of TRMT1 and HDAC2 [9], and HIV-1 protease, which can be toxic to cells, via its action
at various cellular targets [12].
Functional effects of Mpro in transfected cells are consistent with GPX1 knockdown
As detailed above, the original 2003 SCoV Mpro has essentially identical substrate sequence specificity
as that of SCoV2. More specifically, the 10 residue sequence of the cleavage site at the nsp13/14 junction
shown in Fig. 2A, with the greatest overall similarity to the GPX1 active site sequence, is 100% identical
in the 2003 and 2019 SARS coronaviruses (Fig. S1). Thus the results of a 2006 study on the effects of
transfecting cells with a SCoV Mpro expression vector, in order to study the effects of Mpro alone in the
absence of intact virus [13], are highly relevant for COVID-19. In transfected human cells, the results
were exactly what one would expect from knockdown of GPX1: increased oxidative stress via production
of reactive oxygen species and activation of transcription factor NF-κB [13]. Both of these responses can
be produced by exposing cells to hydrogen peroxide, and inhibited by increasing GPX1 activity, either
by overexpression or via the addition of sodium selenite to cell culture media [14,15]. Cells expressing
the SCoV Mpro also became apoptotic, which is a known consequence of NF-κB activation, especially
in combination with a high burden of ROS [16], which is even more likely to occur if other antioxidant
proteins are also degraded, as discussed below.
Potential Mpro sites in other selenoproteins and glutathione-related proteins
The question then arises, could targeting of host selenoproteins for proteolysis be part of a more
extensive viral strategy contributing to the correlations between Se and GSH status and COVID-19
outcome? To investigate this possibility, we undertook a systematic search for potential Mpro cleavage
sites in the human selenoproteome and in several GSH-related proteins including glutaredoxin, a small
thioredoxin-like CXXC protein that is recycled by GSH, rather than directly by a reductase enzyme. For
this analysis, we used three online resources: 1) NetCorona (cbs.dtu.dk/services/NetCorona) [17], the
only resource specific for predictions of Mpro cleavage sites in coronaviruses (although not limited to
just the SARS type coronaviruses), 2) PROSPER, the Protease Specificity Prediction Server
(prosper.erc.monash.edu.au) [18], which was rated in a recent study as one of the most accurate resources
for prediction of HIV-1 protease sites [19], and 3) Procleave (cited above), which was only used if either
of the first 2 methods failed to identify a site. Both Procleave and PROSPER do not specifically predict
coronavirus cysteine protease sites, but make predictions for more generic cysteine proteases (e.g.
cathepsins B, K, L or S). Thus, for both PROSPER and Procleave, the results had to be screened manually
for matches to the Mpro cleavage site consensus as shown in Fig. 1A-B. This search is also predicated on
the assumption that the approach of Gordon et al. [9] might fail to identify some protease targets, because
their high affinity interactions are mostly limited to an 8-10 residue sequence, and in some cases may be
unable to withstand the conditions needed to be isolable and observable by mass spectrometry.
Fig. 2 presents the most notable and highly ranked potential Mpro sites identified computationally (the
order A-G is not significant, as placement in the listing was determined by similarity to the known nsp
sites). The most remarkable instance is in selenoprotein F (SELENOF), an ER protein involved in
glycoprotein folding quality control. Over the 8 residues spanning P4 to P4′, it has 7 of 8 identical to the
Mpro site at the nsp12/nsp13 junction, with the only mismatch at the highly variable P3′ position (Fig.
1D), giving a near-perfect match to the Mpro consensus. This was the highest scored NetCorona hit, also
Selenoproteins as SARS-CoV-2 protease substrates
5
This is a provisional file, not the final typeset article
highly scored by PROSPER, as shown in Fig. 2F, and is significant because coronavirus assembly begins
in the ER with spike protein accumulation on the ER membrane surface.
Figure 2. Comparison of known and predicted M
pro
cleavage sites. The most relevant positions
P4-P1′ of the 11 known SARS coronavirus M
pro
cleavage sites in nonstructural proteins are shown
(for nsp4 through nsp16), aligned with predicted sites in several human selenoproteins (GPX1,
SelenoF, SelenoP, and TXNRD1), as well as glutaredoxin-1 (GLRX-1) and the rate-limiting
enzyme for glutathione synthesis, glutamate-cysteine ligase catalytic subunit (GCLC, with 2
predicted cleavage sites a and b). The known nsp cleavage sites shown are all from SARS-CoV-
2, with the addition of the nsp5/6 site from SARS-CoV (marked *). The sites identified in human
proteins are displayed next to the known M
pro
cleavage site to which they are most similar,
highlighted in matching color. The experimentally determined catalytic efficiencies of SARS-CoV
M
pro
at each of the known nsp cleavage sites are shown as relative k
cat
/K
m
(33). Three different
methods for prediction of cysteine protease cleavage sites were used: PROSPER (18), with
numbers in parenthesis showing the score for the predicted site, any score > 0.8 being significant;
NetCorona (17), where scores > 0.5 are considered positive, and Procleave (10), where every
possible P4-P4′ octamer in a protein sequence is evaluated, scored and ranked. Some aligned non-
identical residues highlighted in italics are nonetheless chemically and structurally similar to
residues found in the same position in the known cleavage sites, e.g., serine (S) vs. threonine (T),
Leucine (L) or isoleucine (I) vs. valine (V), and glutamine (Q) vs. asparagine (N); all of these
pairs differ only by a single carbon atom (CH
3
or CH
2
unit). The predicted M
pro
cleavage sites in
human proteins labeled A to G are discussed in the text. The computational results cited are
collated in Fig. S2.
Taylor and Radding Selenoproteins as SARS-CoV-2 protease substrates
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This is a provisional file, not the final typeset article
Equally important is the hit on the selenoprotein thioredoxin reductase 1 (TXNRD1), which also was
highly scored by both NetCorona and PROSPER (Fig. 2E). Note that in a larger set of 77 human
coronaviruses, a serine (S) in P4 is very common [17], and in addition the sequence SI in the TXNRD1
site is isosteric to TV, the aligned residues in the nsp12/13 site, because transfer of a methyl group from
threonine to valine would result in serine and isoleucine. Thus SI should occupy about the same volume
as TV in the Mpro active site. Functionally, this predicted cleavage site is only 5 residues from the C
terminal of TXNRD1, and would result in removal of the C-terminal redox center of TXNRD1
(AGCUG), making the enzyme incapable of regenerating reduced thioredoxin.
A predicted site in selenoprotein P (SELENOP, Fig. 2C) is at position 56, 3 residues prior to the first
selenocysteine, such that cleavage here would likely interfere with the N-terminal redox activity,
possibly without affecting the Se-transport function of the rest of the protein.
Two possible sites were identified in the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate
limiting enzyme for GSH synthesis. The first of these, GCLC-a (Fig. 2B), would cleave after position
217, and is an exact match to P4-P1 of nsp5/6, but with an A in P1′, a highly favorable residue for that
position, although SCoV has a G there, and SCoV2 has an S. Site GCLC-b (Fig. 2A), at position 443,
is an exact match to P4-P1 of nsp4/5, but with a G in P1′. The two coronavirus sites with exact P4-P1
matches to GCLC are those at either end of nsp5, i.e., Mpro itself, which have the highest catalytic
efficiency for cleavage of any of the viral Mpro targets (100% and 41%). This suggests that Mpro might
be particularly efficient at disrupting GSH synthesis, as compared to action at some of the other target
sites predicted here, thereby contributing to the clinical findings in COVID-19 [7].
Finally (Fig. 2D), we include a non-canonical hit that would cleave at position 90 of glutaredoxin-1
(GLRX-1). This site was very highly scored by PROSPER, as well as Procleave, despite the glutamine
(Q) at P1′, which has an additional carbon atom relative to the asparagine (N) found at P1′ in the nsp8/9
site, to which it is otherwise most similar.
Why would SARS-COV-2 target components of the thioredoxin and glutaredoxin systems?
Along with GSH reductase, GSH and glutaredoxin comprise the glutaredoxin system, one of two cellular
redox systems involved in maintaining reduced thiol states, protein folding and repair, and providing
electrons to ribonucleotide reductase (RNR). The other redox system with similar roles is the thioredoxin
system, comprised of thioredoxin and thioredoxin reductase [20].
Because DNA is an “add-on” to RNA biosynthesis, deoxyribonucleotides can only be synthesized via
the reduction of ribonucleotides by RNR, a process which is unsustainable without the participation of
one or both of the thioredoxin and glutaredoxin systems. As a consequence of this basic fact of
biochemistry, one should expect that RNA viruses might exploit various mechanisms to interfere with
components of the thioredoxin and glutaredoxin systems, in order to minimize the diversion of
ribonucleotides into DNA synthesis. This is simply the inverse of a strategy used by some large DNA
viruses to maximize DNA production, by encoding their own thioredoxin-like proteins, glutaredoxins
and even entire RNR genes [21]. Consistent with this hypothesis, the results presented here suggest
coronaviral targeting of TXNRD1, glutaredoxin-1, and GCLC, a key enzyme for GSH synthesis, for
proteolytic cleavage. This would lead to knockdown of both of the essential redox systems required to
sustain DNA synthesis, and thereby conserve ribonucleotides to enhance RNA synthesis for virus
production.
Selenoproteins as SARS-CoV-2 protease substrates
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The potential Mpro targets TXNRD1 and GCLC are strongly upregulated by vitamin D3
In light of accruing evidence that vitamin D3 status is inversely correlated with severity of COVID-19
[22-25], it is significant that, via its hormone-like actions on gene expression, D3 has been shown to be a
potent activator of both TXNRD1 and GCLC. In a microarray study, 6 hours after exposure to 1,25-
dihydroxyvitamin D3, TXNRD1 mRNA was upregulated 7.1X, and GCLC was upregulated 2.5X [26].
These effects of vitamin D3, as well as a resulting increase in GSH levels, have been functionally
documented in cell culture studies [27,28] and in human subjects [29]. Vitamin D3-mediated activation of
both TXNRD1 and GSH biosynthesis could substantially counteract the proteolytic knockdown of
TXNRD1 and GCLC predicted by our hypothesis, which, if validated, would thus represent an important
mechanism contributing to a role for vitamin D3 in moderating the severity of COVID-19. However, the
therapeutic potential of D3 in COVID-19 is the subject of ongoing debate [30]. Perhaps inconsistencies
in the findings of various studies of this question may be explained in part by a 20-year old observation,
that the effective upregulation of TXNRD1 by vitamin D3 requires the presence of an adequate level of
Se [27]. So the full potential of vitamin D3 vs. COVID-19 may only be seen in combination with optimal
Se intake, and possibly, vice-versa.
Discussion and Conclusions
Altogether, given the protein interaction and functional data [9,13], it is a very strong hypothesis that
GPX1 is an Mpro substrate. Of the other sites we propose, some are more likely than others, but those in
TXNRD1 and SELENOF are particularly convincing, and all of the predicted sites map to surface
accessible regions of the targeted proteins (Fig. S3). If validated, these results offer new insights into
COVID-19 pathogenesis. If SCoV2 is targeting GSH biosynthesis as well as TXNRD1 and GPx1 for
proteolytic knockdown, in infected cells, the resulting decreases in these critical antioxidant molecules
would contribute to increased oxidative stress, NF-κB activation and pro-apoptotic signaling [14,15].
Because NF-κB is an activator of many pro-inflammatory cytokines, including IL-6 [31], this could
contribute to increased inflammation and the cytokine storms observed in COVID-19, and be a
significant basis of pathogenic effects associated with SCoV2 infection of various tissues, including the
lung, gastrointestinal tract and cardiovascular system. Importantly, these consequences of virus-mediated
proteolysis would be taking place in everyone who is actively infected, regardless of their Se status, and
would thus be consistent with results suggesting that Se intakes up to several times the minimal dietary
requirement are associated with an increase in cure rate from COVID-19 [1]. But people with suboptimal
nutritional status could be particularly at risk, because their GSH and selenoprotein levels might be low
to begin with, making them more vulnerable to the detrimental effects of virus-induced proteolysis. Still,
an infection resulting from low dose exposure to virus and limited by a strong immune response in a
person with excellent dietary status still might have minimal impact on cellular and patient health,
because proteolytic knockdown of host proteins is likely to be incomplete, due to low catalytic
efficiencies at some target sites (Fig. 2) and the stochastic nature of such molecular interactions.
These results are not unprecedented our lab has shown that some RNA viruses target host mRNAs
encoding isoforms of thioredoxin reductase via RNA:RNA antisense interactions, which, like
proteolysis, would likely also result in host selenoprotein knockdown, but by a different mechanism
[21,32]. If our hypothesis is confirmed (i.e. some of these host cleavage sites prove to be functional), that
leaves us with an interesting question – what evolutionary advantages are driving some viruses to go so
far as to degrade or block the synthesis of GSH and specific host selenoproteins?
Understanding why RNA viruses may have developed such strategies presents an interesting challenge
for future research.
Taylor and Radding Selenoproteins as SARS-CoV-2 protease substrates
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Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest.
Author Contributions
The study was conceived and designed by EWT, who wrote the initial draft. Both EWT and WR worked
on the data analysis and revisions of the manuscript.
Funding
This work has been supported by a recurring unrestricted gift from the Dr. Arthur and Bonnie Ennis
Foundation, Decatur, IL, USA.
Data Availability Statement
The datasets and listings of source sequence files for this study are all included in the figure legends of the
article and in the online Supplementary Materials.
© 2020 by the authors. Submitted for possible open access publication
under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Taylor and Radding Selenoproteins as SARS-CoV-2 protease substrates
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This is a provisional file, not the final typeset article
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Supplementary Materials
for
Taylor & Radding, 2020
Mpro site 2003 SARS-CoV 2019 SARS-CoV-2 # Non-identical
nsp4/5 SAVLQ/SGFRK SAVLQ/SGFRK 0
nsp5/6 GVTFQ/GKFKK GVTFQ/SAVKR 4
nsp6/7 VATVQ/SKMSD VATVQ/SKMSD 0
nsp7/8 RATLQ/AIASE RATLQ/AIASE 0
nsp8/9 AVKLQ/NNELS AVKLQ/NNELS 0
nsp9/10 TVRLQ/AGNAT TVRLQ/AGNAT 0
nsp10/11 EPLMQ/SADAS EPMLQ/SADAQ 3
nsp12/13 HTVLQ/AVGAC HTVLQ/AVGAC 0
nsp13/14 VATLQ/AENVT VATLQ/AENVT 0
nsp14/15 FTRLQ/SLENV FTRLQ/SLENV 0
nsp15/16 YPKLQ/ASQAW YPKLQ/SSQAW 1
Figure S1. The 11 known Mpro cleavage sites in the 2003 and 2019 SARS coronaviruses with a
comparison of the 10-residue protein sequences spanning the cleavages sites in the two viruses.
The alignments underneath each of the virus names (2003 SARS-CoV and 2019 SARS-CoV-2)
were used to generate the logos shown in Fig. 1A and 1B. The cleavage sites and sequences for
the SARS-CoV (Genbank NC_004718.3) are given in Ref. 33, and the equivalent sequences in
SARS-CoV-2 were taken from its Genbank Reference Sequence, NC_045512.
6:HEVHUYHUSURWHDVHFOHDYDJHVLWHSUHGLFWLRQUHVXOWVFLWHGLQ)LJXUH
*HQEDQNVRXUFHILOHVIRUWKHSURWHLQVHTXHQFHV*&/&13B6(/(12313B
*/5;13B7;15'13B6(/(12)13B*3;13B
61HW&RURQDUHVXOWVIURPFEVGWXGNVHUYLFHV1HW&RURQD
1HW&RURQD6HUYHUSUHGLFWLRQUHVXOWV
7HFKQLFDO8QLYHUVLW\RI'HQPDUN
165 SelenoF
MVAMAAGPSGCLVPAFGLRLLLATVLQAVSAFGAEFSSEACRELGFSSNLLCSSCDLLGQFNLLQLDPDCRGCCQEEAQF 80
ETKKLYAGAILEVCGCKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSDPVLKLLDDNGNIAEELSILKWNTDSVEEFLSE 160
KLERI Selenocysteine (U) at #96 changed to C for search as NetCorona ignores U
..........................C..................................................... 80
................................................................................ 160
.....
Pos Score Cleavage
___________________________
27 0.846 ATVLQ^AVSAF SelenoF
60 0.067 none SelenoF
65 0.073 none SelenoF
75 0.066 none SelenoF
79 0.064 none SelenoF
103 0.064 none SelenoF
105 0.219 none SelenoF
120 0.136 none SelenoF
___________________________
499 TXNRD1
MNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQAL 80
QDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIA 160
TGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHM 240
EEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTD 320
EEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIE 400
VYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFT 480
TLSVTKRSGASILQAGCCG (Penultimate U changed to C as NetCorona ignores U)
................................................................................ 80
................................................................................ 160
................................................................................ 240
...............................C................................................ 320
................................................................................ 400
................................................................................ 480
.............C.....
Pos Score Cleavage
___________________________
33 0.068 none TXNRD1
72 0.104 none TXNRD1
78 0.071 none TXNRD1
81 0.123 none TXNRD1
106 0.073 none TXNRD1
133 0.071 none TXNRD1
230 0.075 none TXNRD1
250 0.070 none TXNRD1
258 0.065 none TXNRD1
272 0.505 RVVAQ^STNSE TXNRD1
323 0.061 none TXNRD1
348 0.191 none TXNRD1
355 0.064 none TXNRD1
450 0.107 none TXNRD1
464 0.074 none TXNRD1
494 0.640 ASILQ^AGCCG TXNRD1
___________________________
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6%
B. GCLC-a VTFQ/A WZK^WZ;ϭ͘ϬϬͿ
nsp5/6* VTFQ/G
6'
D. GLRX-1 VSLQ/Q WZK^WZ;ϭ͘ϯϬͿ
nsp8/9 VKLQ/N


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6(
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SILQ/A WZK^WZ;ϭ͘ϭϰͿ
nsp12/13 TVLQ/A
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6)
F. SelenoF TVLQ/A WZK^WZ;ϭ͘ϬϮͿ
nsp12/13 TVLQ/A
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6$
A. GCLC-b AVLQ/G WƌŽůĞĂǀĞηϳͬϱϵϮĨŽƌĂƚŚĞƉƐŝŶ^
nsp4/5 AVLQ/S
 
6%
B. GCLC-a VTFQ/A WƌŽůĞĂǀĞηϭϯŽĨϱϵϮĨŽƌĂƚŚĞƉƐŝŶ>
nsp5/6* VTFQ/G
6&
C. SelenoP
ALLQ/A WƌŽůĞĂǀĞηϯͬϯϳϰĨŽƌĂƚŚĞƉƐŝŶ>
nsp7/8 ATLQ/A
6'
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nsp8/9 VKLQ/N
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nsp13/14 ATLQ/A
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Background SARS‐CoV‐2 coronavirus infection ranges from asymptomatic through to fatal COVID‐19 characterised by a “cytokine storm” and lung failure. Vitamin D deficiency has been postulated as a determinant of severity. Objectives To review the evidence relevant to vitamin D and COVID‐19 Methods Narrative review Results Regression modelling shows that more northerly countries in the Northern Hemisphere are currently (May 2020) showing relatively high COVID‐19 mortality, with an estimated 4.4% increase in mortality for each 1 degree latitude north of 28 degrees North (P=0.031) after adjustment for age of population. This supports a role for ultraviolet B acting via vitamin D synthesis. Factors associated with worse COVID‐19 prognosis include old age, ethnicity, male sex, obesity, diabetes and hypertension and these also associate with deficiency of vitamin D or its response. Vitamin D deficiency is also linked to severity of childhood respiratory illness. Experimentally, vitamin D increases the ratio of angiotensin converting enzyme 2 (ACE2) to ACE, thus increasing angiotensin II hydrolysis and reducing subsequent inflammatory cytokine response to pathogens and lung injury. Conclusions Substantial evidence supports a link between vitamin D deficiency and COVID‐19 severity but it is all indirect. Community‐based placebo‐controlled trials of vitamin D supplementation may be difficult. Further evidence could come from study of COVID‐19 outcomes in large cohorts with information on prescribing data for vitamin D supplementation or assay of serum unbound 25(OH) vitamin D levels. Meanwhile vitamin D supplementation should be strongly advised for people likely to be deficient.
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Higher rates of serious illness and death from coronavirus SARS-CoV-2 (COVID-19) infection among older people and those who have comorbidities suggest that age- and disease-related biological processes make such individuals more sensitive to environmental stress factors including infectious agents like coronavirus SARS-CoV-2. Specifically, impaired redox homeostasis and associated oxidative stress appear to be important biological processes that may account for increased individual susceptibility to diverse environmental insults. The aim of this Viewpoint is to justify (1) the crucial roles of glutathione in determining individual responsiveness to COVID-19 infection and disease pathogenesis and (2) the feasibility of using glutathione as a means for the treatment and prevention of COVID-19 illness. The hypothesis that glutathione deficiency is the most plausible explanation for serious manifestation and death in COVID-19 patients was proposed on the basis of an exhaustive literature analysis and observations. The hypothesis unravels the mysteries of epidemiological data on the risk factors determining serious manifestations of COVID-19 infection and the high risk of death and opens real opportunities for effective treatment and prevention of the disease.
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SARS-CoV-2 is an RNA virus responsible for the COVID-19 pandemic that already claimed more than 340,000 lives worldwide as of May 23, 2020, the majority of which are elderly. Selenium (Se), a natural trace element, has a key and complex role in the immune system. It is well-documented that Se deficiency is associated with higher susceptibility to RNA viral infections and more severe disease outcome. In this article, we firstly present evidence on how Se deficiency promotes mutations, replication and virulence of RNA viruses. Next, we review how Se might be beneficial via restoration of host antioxidant capacity, reduction of apoptosis and endothelial cell damages as well as platelet aggregation. It also appears that low Se status is a common finding in conditions considered at risk of severe COVID-19, especially in the elderly. Finally, we present a rationale for Se use at different stages of COVID-19. Se has been overlooked but may have a significant place in COVID-19 spectrum management, particularly in vulnerable elderly, and might represent a game changer in the global response to COVID-19. https://doi.org/10.3389/fnut.2020.00164
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The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 2.3 million people, killed over 160,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven clinical efficacy, nor are there vaccines for its prevention, and these efforts are hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To address this, we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), identifying 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (29 FDA-approved drugs, 12 drugs in clinical trials, and 28 preclinical compounds). Screening a subset of these in multiple viral assays identified two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the Sigma1 and Sigma2 receptors. Further studies of these host factor targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.
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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).
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SARS-CoV-2 infections underlie the current coronavirus disease (COVID-19) pandemic and are causative for a high death toll particularly among elderly subjects and those with comorbidities. Selenium (Se) is an essential trace element of high importance for human health and particularly for a well-balanced immune response. The mortality risk from a severe disease like sepsis or polytrauma is inversely related to Se status. We hypothesized that this relation also applies to COVID-19. Serum samples (n = 166) from COVID-19 patients (n = 33) were collected consecutively and analyzed for total Se by X-ray fluorescence and selenoprotein P (SELENOP) by a validated ELISA. Both biomarkers showed the expected strong correlation (r = 0.7758, p < 0.001), pointing to an insufficient Se availability for optimal selenoprotein expression. In comparison with reference data from a European cross-sectional analysis (EPIC, n = 1915), the patients showed a pronounced deficit in total serum Se (mean ± SD, 50.8 ± 15.7 vs. 84.4 ± 23.4 µg/L) and SELENOP (3.0 ± 1.4 vs. 4.3 ± 1.0 mg/L) concentrations. A Se status below the 2.5th percentile of the reference population, i.e., [Se] < 45.7 µg/L and [SELENOP] < 2.56 mg/L, was present in 43.4% and 39.2% of COVID samples, respectively. The Se status was significantly higher in samples from surviving COVID patients as compared with non-survivors (Se; 53.3 ± 16.2 vs. 40.8 ± 8.1 µg/L, SELENOP; 3.3 ± 1.3 vs. 2.1 ± 0.9 mg/L), recovering with time in survivors while remaining low or even declining in non-survivors. We conclude that Se status analysis in COVID patients provides diagnostic information. However, causality remains unknown due to the observational nature of this study. Nevertheless, the findings strengthen the notion of a relevant role of Se for COVID convalescence and support the discussion on adjuvant Se supplementation in severely diseased and Se-deficient patients.
Preprint
Background: COVID-19 is a major pandemic that has killed more than 196,000 people. The COVID-19 disease course is strikingly divergent. Approximately 80-85% of patients experience mild or no symptoms, while the remainder develop severe disease. The mechanisms underlying these divergent outcomes are unclear. Emerging health disparities data regarding African American and homeless populations suggest that vitamin D insufficiency (VDI) may be an underlying driver of COVID-19 severity. To better define the VDI-COVID-19 link, we determined the prevalence of VDI among our COVID-19 intensive care unit (ICU) patients. Methods: In an Institutional Review Board approved study performed at a single, tertiary care academic medical center, the medical records of COVID-19 patients were retrospectively reviewed. Subjects were included for whom serum 25-hydroxycholecalcifoerol (25OHD) levels were determined. COVID-19-relevant data were compiled and analyzed. We determined the frequency of VDI among COVID-19 patients to evaluate the likelihood of a VDI-COVID-19 relationship. Results: Twenty COVID-19 patients with serum 25OHD levels were identified; 65.0% required ICU admission.The VDI prevalence in ICU patients was 84.6%, vs. 57.1% in floor patients. Strikingly, 100% of ICU patients less than 75 years old had VDI. Coagulopathy was present in 62.5% of ICU COVID-19 patients, and 92.3% were lymphocytopenic. Conclusions: VDI is highly prevalent in severe COVID-19 patients. VDI and severe COVID-19 share numerous associations including hypertension, obesity, male sex, advanced age, concentration in northern climates, coagulopathy, and immune dysfunction. Thus, we suggest that prospective, randomized controlled studies of VDI in COVID-19 patients are warranted.