SARS-CoV-2 suppresses mRNA expression of selenoproteins associated with ferroptosis, ER stress and DNA synthesis

Abstract

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.

Full text

SARS-CoV-2 suppresses mRNA expression of selenoproteins associated with ferroptosis,
endoplasmic reticulum stress and DNA synthesis
Yijun Wang1,5, Jinbao Huang1,5, Yong Sun2, Jun He2, Weiwei Li2, Zhirong Liu2, Ethan Will Taylor3,
Margaret P Rayman4, Xiaochun Wan1*, Jinsong Zhang1*
1 The State Key Laboratory of Tea Plant Biology and Utilization, School of Tea & Food Science,
Anhui Agricultural University, Hefei, China.
2 Public Health Research Institute of Anhui Province, Anhui Provincial Center for Disease Control
and Prevention, Hefei, China.
3 Department of Chemistry and Biochemistry, University of North Carolina at Greensboro,
Greensboro, NC, USA.
4 Faculty of Health and Medical Sciences, Department of Nutritional Sciences, University of
Surrey, Guildford, United Kingdom.
5 These authors contributed equally.
*email: xcwan@ahau.edu.cn; zjs@ahau.edu.cn
Abstract
A significant, positive association between selenium status and prognosis of SARS-CoV-2
infection has been identified among COVID-19 patients in China. Moreover, a German study
revealed a pronounced deficit of serum selenium and SELENOP concentrations in COVID-19
patients, and selenium deficiency was associated with mortality risk from COVID-19. The
present study investigated the influence of SARS-CoV-2 on gene expression of host
selenoproteins which mediate many beneficial actions of selenium. We found that SARS-CoV-2
suppressed mRNA expression of selenoproteins associated with ferroptosis (GPX4),
endoplasmic reticulum stress (SELENOF, SELENOK, SELENOM and SELENOS) and DNA synthesis
(TXNRD3), while SARS-CoV-2 increased gene expression of IL-6 (an inflammatory cytokine
positively correlated with severity of COVID-19), in Vero cells. These results provide a deeper
insight into the connection between selenium and SARS-CoV-2 pathogenesis.
Keywords: Selenium; Selenoprotein; SARS-CoV-2; mRNA expression.
Introduction
The world is in the midst of a pandemic of Coronavirus Disease 2019 (COVID-19) caused by
infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Selenium (Se), an
essential micronutrient, is the only trace element to be specified in the genetic code; 25 genes
encode selenoproteins that normally have a selenocysteine residue at their active centre.1
Many selenoproteins participate in anti-oxidant, anti-inflammatory and anti-viral actions of
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Se.2,3 Se has been found to be a significant factor affecting incidence, severity or mortality of
various viral diseases in animals and humans. RNA viruses, such as coxsackievirus B3 and
influenza A/Bangkok/1/79 (H3N2), mutated into more virulent strains in a Se-deficient host. Se
supplementation or higher Se status improves clinical outcomes of infections caused by
evolutionally diverse viruses.2,3
A link between Se status and the prognosis of COVID-19 patients in China has been identified.
When the cure rate (%) in cities outside Hubei province was plotted against population hair Se
concentration (a surrogate for Se intake), a significant, positive linear association was shown,
with a Pearson r of 0.85 (p<0.0001).4 Consistently, a German study found that serum Se and
SELENOP concentrations in COVID-19 patients were significantly lower than healthy controls,
and Se status was significantly higher in samples from surviving COVID patients than in those of
non-survivors.5 Relevant to these observations, ebselen, an organoselenium compound, was
found to have the strongest inhibitory activity out of 10,000 compounds examined (the current
drug arsenal) against the SARS-CoV-2 main protease, which mediates the life cycle of the virus
and is a well-recognized target for inhibition.6 These new findings, together with published
evidence on other viruses,2,3 suggest that Se compounds can achieve both prophylaxis and
therapy in COVID-19. However, whether SARS-CoV-2 in turn affects host selenoproteins is
currently unknown. Hence, in the study described below, we investigated the potential impact
of SARS-CoV-2 infection on the expression of all 25 known host selenoproteins at the mRNA
level.
Results and Discussion
Vero E6 cells were infected with SARS-CoV-2 at 20-fold TCID50 (50% tissue culture infective
dose). Pilot experiments showed that significant cytopathy occurred after 72-h incubation.
Following a 48-h incubation, which did not cause morphological alteration of the cells (verified
by microscopy), viral copy numbers and abundance of mRNAs encoding IL-6 and 25
selenoproteins were measured.
A cytokine storm has been identified as hallmark of critical illness in COVID-19 patients; thus it
is relevant that IL-6 levels were found to be positively correlated with disease severity.7 When
SARS-CoV-2 viral copy number in the cultured cells reached 4.4×109 (Figure 1A), IL-6 was
significantly up-regulated by 4.3-fold (Figure 1B). It has been demonstrated that IL-6 can affect
the selenoenzyme, glutathione peroxidase (GPX), in an isozyme-specific manner. Of note,
however, GPX1 mRNA expression remained unaffected while that of GPX4 decreased.8
Consistent with those results, we found that SARS-CoV-2 did not alter GPX1 expression
(Supplemental Table 1) but significantly down-regulated that of GPX4 by 69.4% (Figure 1C). In
contrast to GPX1, which catalyzes intracellular detoxification of hydrogen peroxide to water,
GPX4 is unique in the GPX family in that it protects phospholipids from iron-dependent
ferroptotic cell death, by reversing peroxidation of polyunsaturated fatty acids via their
reduction to non-toxic lipid alcohols in the membrane.9,10 GPX4 is a so-called “housekeeping”
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gene, ranking high in the hierarchy of selenoprotein expression, whereas GPX1 is ranked much
lower and is much less likely to be expressed at low Se status.11 While moderate Se deficiency
causes a significant reduction in GPX1 concentration, that of GPX4 is not much reduced. The
present study reveals that infection with SARS-CoV-2 together with Se deficiency could
synergistically destroy GPX defenses, resulting in severe oxidative stress similar to that which
previous studies have found to cause virus mutation to more virulent strains.12
Seven selenoproteins including SELENOF, SELENOM, SELENOK and SELENOS are residents of the
endoplasmic reticulum (ER).13 SARS-CoV-2 infection significantly down-regulated SELENOF,
SELENOM, SELENOK and SELENOS, by 75.9%, 56.2%, 71.3% and 61.1%, respectively (Figure 1D).
SELENOF and its distant homologue, SELENOM, possess redox-active motifs; thus they regulate
redox homeostasis, catalyze the reduction or rearrangement of disulfide bonds in ER-localized
proteins and facilitate ER protein-folding. Accordingly, impaired SELENOF and SELENOM
increase misfolded proteins, causing ER stress. On the other hand, SELENOK, along with
SELENOS, promotes ER-associated degradation (ERAD) of errant proteins by recruiting cytosolic
valosin-containing protein to increase translocation of misfolded proteins from the ER lumen to
the cytosol. Thus, impaired SELENOK and SELENOS attenuate ERAD of misfolded proteins.14
Concomitant down-regulation of SELENOF, SELENOM, SELENOK and SELENOS provoked by
SARS-CoV-2 is likely to result in increased concentration of misfolded proteins in the ER and
catastrophic ER stress. A direct mechanistic link between the reduced expression of SELENOS
and the production of inflammatory cytokines has been well documented;15 this may well
constitute an underlying mechanism by which SARS-CoV-2 induces marked elevation of IL-6
concentration. Of all of the ER-resident selenoproteins, we find SELENOF to be the most
affected by SARS-CoV-2 infection (75.9% decrease). This is interesting in light of a recent report
that SELENOF may be targeted for proteolysis by the SARS-CoV-2 main protease Mpro, because
the SELENOF protein contains a sequence (TVLQ/AVSA) that is almost identical to a known viral
Mpro cleavage site (TVLQ/AVGA).16 Taken together, these observations suggest that disruption
of SELENOF function may be particularly important for SARS-CoV-2 replication.
Thioredoxin serves as an electron donor for ribonucleotide reductase which catalyzes the
conversion of ribonucleotides to deoxyribonucleotides for DNA synthesis.17 Inhibition of the
selenoenzyme, thioredoxin reductase (TXNRD), decreases DNA synthesis and increases the
ribonucleotide pool for RNA synthesis. Some large DNA viruses are equipped with their own
ribonucleotide reductase to facilitate DNA synthesis for virus production.18 Likewise, RNA
viruses may attempt to suppress the diversion of ribonucleotides for DNA synthesis in order to
enhance RNA synthesis. According to computational analysis, SARS-CoV-2 targets TXNRD3 by
antisense at several sites, with computed interaction energies equivalent to the strongest
microRNA interactions.18 Consistent with the computational prediction, our study found that
SARS-CoV-2 significantly down-regulated TXNRD3, by 36.9% (Figure 1E). TXNRD3 is mainly
expressed in the testis.19 SARS-CoV-2 has been detected in the semen of patients with COVID-
19.20 Orchitis was a complication of SARS-CoV infection.21 GPX4 is essential for sperm
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maturation and is correlated with fertility-related parameters.22,23 The present study suggests
that TXNRD3 knockdown by SARS-CoV-2 and GPX4 down-regulation owing to SARS-CoV-2-
triggered elevation of IL6 together probably deteriorate male fertility. Apart from the testis,
pulmonary TXNRD3 protein levels are high according to the Human Protein Atlas.24 Thus, by
knockdown of TXNRD3, SARS-CoV-2 would boost virus production in the lung, the major site of
SARS-CoV-2 replication.
Taken together, Se deficiency, which compromises the production of Se-sensitive GPX1, and
SARS-CoV-2, which reduces GPX4 expression, could synergistically destroy GPX antioxidant
defenses, resulting in concurrent increase of both intracellular ROS and membrane lipid
peroxidation. Of seven ER-resident selenoproteins, SARS-CoV-2 simultaneously suppressed the
expression of SELENOF, SELENOM, SELENOK and SELENOS, indicating that the ER is an organelle
that is severely adversely affected by SARS-CoV-2. Impaired function of SELENOF and SELENOM
together cause the accumulation of misfolded proteins; if SELENOK and SELENOS are also
compromised, the effect of the accumulation will be aggravated. It is known that carriers of the
A-allele of the SELENOS −105G/A promoter polymorphism (rs28665122) experience increased
ER stress and production of inflammatory cytokines,15 hence concomitant down-regulation of
SELENOF, SELENOM, SELENOK and SELENOS induced by SARS-CoV-2 may have the capacity to
induce a cytokine storm, at least in some susceptible individuals. Furthermore, the current work
confirms the computational prediction that SARS-CoV-2 may boost virus production by down-
regulating TXNRD3.18 These findings, summarized in Figure 2, provide a deeper insight into the
connection between Se and SARS-CoV-2 and reinforce the potential importance of modulation
of COVID-19 by Se.
Experimental Section
Cells, virus and viral inoculation
African green monkey kidney (Vero) cells were obtained from American Type Culture Collection
(ATCC) and maintained in Dulbecco’s Modified Eagle’s media (DMEM, Corning, USA)
supplemented with 10% fetal bovine serum (FBS, Gibco, Invitrogen), 2% L-glutamine and 1%
penicillin/streptomycin at 37 ºC in a humidified atmosphere of 5% CO2. Patient-derived SAS-
CoV2 (SZ005) was isolated by the Anhui Provincial Center for Disease Control and Prevention
(Anhui, China). The viral titer was determined by 50% tissue culture infective dose (TCID50)
according to the cytopathic effect by use of the Karber method. All the infection experiments
were performed in a biosafety level-3 (BSL-3) laboratory. Vero cells were seeded on 6-well
plates with a density of 1×106 cells/well and infected with 20 TCID50 virus at 37 °C.
RNA extraction and quantitative real-time RT-PCR (qRT-PCR)
After viral infection for 48 h, the cell culture was subjected to virus inactivation treatment and
then divided into two replicates. One of them (100 μL) was used for viral RNA isolation on an
automatic nucleic acid extraction workstation (TANBead, Taiwan) according to the
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manufacturer’s instructions. Reverse transcription was performed with TaqMan Fast Virus 1-
Step Master Mix (ThermoFisher, Catalog Numbers 4444432). Briefly, 2 μL viral RNA was used as
template for one-step quantitative PCR. The full-length S gene of SARS-CoV-2 was synthesized
and cloned into pcDNA3.1 as positive control plasmid. Serial dilutions of the positive control
(1,000-1,000,000,000 copies per μL) were used to establish a standard curve for determining
the initial starting amount of the target template in experimental samples. The primes and
probe used for quantitative PCR were: SB-F: GGCTGTTTAATAGGGGCTGAAC, SB-R:
ACCAAGTGACATAGTGTAGGCA, SB probe: 5’ FAM-AGACTAATTCTCCTCGGCGGGCACG-BHQ.
The rest of the cell suspension was used for the RNA extraction of host cells. Total RNA was
extracted using an RNeasy mini kit (Qiagen Inc., Valencia, CA) and the reverse transcription
reaction was conducted using a GoscriptTM Reverse Transcription System kit (Promega, Madison,
WI). The RNA quality was confirmed by spectrophotometry and electrophoresis. A Power SYBR®
Green PCR Master Mix kit (Life Technologies, Warrington, UK) was employed to conduct the
qRT-PCR on an ABI QuantStudio 7Pro system. The expression level of a target gene mRNA was
normalized to the mRNA level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The
amount of the target gene expression was calculated by the 2−ΔΔCT method. The sequences of
the genes involved in the present study were obtained from Genbank
(www.ncbi.nlm.nih.gov/Genbank), and the sequences of primers used are listed in
Supplementary Table 2.
Statistical analysis
Data are expressed as the mean ± standard error of the mean (n=6), and analysed using the
IBM SPSS Statistics 22.0 (IBM, Armonk, NY). The Mann Whitney test was used to assess the
difference between two groups. Significant levels of p < 0.05 and p < 0.01 were set for all tests.
Acknowledgements
We thank Zhuhui Zhang, Meng Wang, Yinglu Ge from BSL-3 Laboratory of Anhui Provincial
Center for Disease Control and Prevention for their essential assistance with this study. This
study was supported by the Emergency Research Project of Novel Coronavirus Infection of
Anhui Province (202004a07020002 and 202004a07020004), National Natural Science
Foundation of China (31972459), Key Research and Development Program of Anhui Province
(1804b06020367, 201904b11020038)
Author Contributions
J.Z., X.W., Y.W., J.H., R.M., and T.E. conceived and designed the experiments. Y.W., J.H., Y.S.,
J.H., W.L. participated in multiple experiments; J.Z., X.W., Z.L., Y.W., and J.H. analyzed the data.
J.Z., X.W., Y.W., J.H., R.M., and T.E. wrote the manuscript.
Competing interests: The authors declare no competing interests.
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Figure 1 SARS-CoV-2 suppresses gene expression of selenoproteins in Vero cells. Vero E6 cells
were infected with SARS-CoV-2 at 20-fold TCID50. Following a 48-h incubation, viral copy
numbers and abundance of mRNAs encoding IL-6 and selenoproteins were quantified by
Quantitative real time polymerase chain reaction. A, viral copy number; B-E, gene expression.
Data are expressed as mean ± SEM (n=6), statistical differences were examined by the Mann
Whitney test (*p < 0.05 and **p < 0.01).
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Figure 2 Possible consequences of SARS-CoV-2 induced down-regulation of selenoprotein
genes. LPO, lipid peroxidation; NTP, nucleoside triphosphate; dNTP, deoxyribonucleoside
triphosphate; RNR, ribonucleotide reductase.
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Supplementary Table 1. The relative gene expression of selenoproteins in control and SARS-CoV-2 infected Vero cells
Selenoproteins
GPX1
GPX2
GPX3
GPX4
GPX6
TXNRD1
TXNRD2
TXNRD3
DIO1
DIO2
DIO3
SEPHS2
MSRB1
Control
1.04±0.14
1.00±0.04
1.02±0.10
1.27±0.41
ND
1.01±0.07
1.01±0.07
1.03±0.12
1.01±0.05
1.01±0.04
1.01±0.07
1.00±0.03
1.72±0.67
Virus
1.00±0.03
1.10±0.04
1.14±0.16
0.39±0.07*
ND
0.96±0.10
0.86±0.17
0.65±0.04**
1.06±0.06
1.07±0.04
1.12±0.15
1.24±0.05
0.42±0.05
Selenoproteins
SELENOF
SELENOH
SELENOI
SELENOK
SELENOM
SELENON
SELENOO
SELENOP
SELENOS
SELENOT
SELENOV
SELENOW
Control
1.12±0.23
1.02±0.10
ND
1.08±0.19
1.05±0.15
1.01±0.05
1.13±0.23
1.00±0.04
1.13±0.23
1.01±0.06
1.00±0.02
1.01±0.06
Virus
0.27±0.03**
0.83±0.04
ND
0.31±0.02**
0.46±0.04**
1.12±0.08
1.16±0.20
1.06±0.05
0.44±0.04*
1.10±0.06
1.22±0.10
0.83±0.03
Note: ND, undetected. Data are expressed as mean ± SEM (n=6), *p < 0.05 and **p < 0.01, compared to the control (Mann Whitney test).

Supplementary Table 2. Primers used for Real-Time PCR
Gene ID
Gene Name
Primer Sequence-Forward
Primer Sequence-Reverse
103226390
IL-6
5'- TCCTGCGCAGCTTTAAGGAG-3'
5'- CCCAGTGGACAGGTTTCTGA -3'
103227628
GPX1
5'- CAGGAGAACGCCAAGAACGAAGAG -3'
5'- GCACCGTTCACCTCGCACTTC -3'
103229177
GPX2
5'- GGGAGGCGGCTTTGTTCAGTC -3'
5'- GGAGCTAGGAAGGAGGACAGAAGG -3'
103244806
GPX3
5'- TCCGACCAGGTGGAGGCTTTG -3'
5'- CGAGGTGGGAGGACAGGAGTTC -3'
103233593
GPX4
5'- CAGTGAGGCAAGACCGAAGTGAAC -3'
5'- TTACTCCCTGGCTCCTGCTTCC -3'
103221913
GPX6
5'- CCTGCTGTCTTGTCCTGCTGTTC -3'
5'- ATGGTGCCTGTCACTCCTTTGTTG -3'
103238986
TXNRD1
5'- TGACTCGTTTCCGTGCCCAAATC -3'
5'- TGTGATGCTGCCTGCCTTCTATTC -3'
103222987
TXNRD2
5'- CTTTGTTGACGAGCACACGGTTTG -3'
5'- CGCCCTCCAGTAGCAATGATGATG -3'
103228118
TXNRD3
5'- ACGAGGAGACAGGACAGCAGTG -3'
5'- GCCTTGGCTCACCTCACAACAG -3'
103224787
DIO1
5'- CCAGACAGAGTCAAGCGGAACATC -3'
5'- CCAACGGACCTTCAAGACGAACC -3'
103229434
DIO2
5'- GAAGCACCAGAACCAGGAAGATCG -3'
5'- CCATGCGGTCAGCCACAACTC -3'
103229715
DIO3
5'- TCGTGCCTCGTGCTCTTCCC -3'
5'- CACCTCCTCGCCTTCACTGTTG -3'
103230930
SEPHS2
5'- GCCCTCTTCACCCCTCCCTTC -3'
5'- CATTGCCGCCATCGCCTCTC -3'
103226422
MSRB1
5'- GTTCTCCAGCCGCTCGAAGTATG -3'
5'- ATTGCCACACTTGCCACAGGAC -3'
103224494
SELENOF
5'- TACGGTTGTTGTTGGCGACTGTG -3'
5'- AAGCAGGTTGAACTGTCCGAGAAG -3'
103235310
SELENOH
5'- GGAGGAGGCAACCGTTGTTATCG -3'
5'- GTCGGGTTCACCTTTACTGGAAGC -3'
103220638
SELENOI
5'- ATGTGCCTGACTGGGTTTGGATTG -3'
5'- TGGTTCTGCGAGCTTGCTTTCC -3'
103227769
SELENOK
5'- GAGGGAGATACAGAAGCCGAGAGG -3'
5'- TCCAACACTTGTCCGTTCGAGATG -3'
103223180
SELENOM
5'- TGCCTGAGTCCTGGAGACAGAATG -3'
5'- AGTGGAGCTGGAGAGGGAAGAAAG -3'
103225351
SELENON
5'- TGGAGGTGGACATCGGCTACATAC -3'
5'- CGATCACGCTGCCATCCTTATCC -3'
103223548
SELENOO
5'- GACCGACAAGGCAGCCAATTAGAG -3'
5'- CCGCAACCCAATCGCCAGTG -3'
103215202
SELENOP
5'- ATCAGCACCTTGGCAGCAGTAAG -3'
5'- GGTCTGGAGGAGCAGGATGAGTAG -3'
103231289
SELENOS
5'- CCTTCCACTTCATCTGTCGTCGTG -3'
5'- GCCTCTGCGTCCAGGTCTCC -3'
103241480
SELENOT
5'- CCTGAGGTTATAGGCGGGTGTTTG -3'
5'- CTCTCCTTCAATGCGGATGTCTGG -3'
103234677
SELENOV
5'- AGTGACCTACTGTGGCCTCTGAAG -3'
5'- CTGGGCAGCTCTGTCCTCCTC -3'
103225230
SELENOW
5'- GGAAGATGATGGCTACGTGGACAC -3'
5'- CATGAAGCGTCTGCTGAGAGGAG -3'
103218453
GAPDH
5'- CATGACCACAGTCCACGCCATC -3'
5'- GATGACCTTGCCCACAGCCTTG -3'
Note: IL-6, interleukin 6; GPX, glutathione peroxidase; TXNRD, thioredoxin reductase; DIO, iodothyronine deiodinase; MSRB1, methionine sulfoxide reductase B1; SEPHS2,
selenophosphate synthetase 2.