PCR array profiling of antiviral genes in human embryonic kidney cells expressing human coronavirus OC43 structural and accessory proteins
Identifieur interne : 000256 ( Pmc/Corpus ); précédent : 000255; suivant : 000257PCR array profiling of antiviral genes in human embryonic kidney cells expressing human coronavirus OC43 structural and accessory proteins
Auteurs : Meshal Beidas ; Wassim ChehadehSource :
- Archives of Virology [ 0304-8608 ] ; 2018.
Abstract
Human coronavirus OC43 (HCoV-OC43) is a respiratory virus that usually causes a common cold. However, it has the potential to cause severe infection in young children and immunocompromised adults. Both SARS-CoV and MERS-CoV were shown to express proteins with the potential to evade early innate immune responses. However, the ability of HCoV-OC43 to antagonise the intracellular antiviral defences has not yet been investigated. The potential role of the HCoV-OC43 structural (M and N) and accessory proteins (ns2a and ns5a) in the alteration of antiviral gene expression was investigated in this study. HCoV-OC43M, N, ns2a and ns5a proteins were expressed in human embryonic kidney 293 (HEK-293) cells before challenge with Sendai virus. The Human Antiviral Response PCR array was used to profile the antiviral gene expression in HEK-293 cells. Over 30 genes were downregulated in the presence of one of the HCoV-OC43 proteins, e.g. genes representing mitogen-activated protein kinases, toll-like receptors, interferons, interleukins, and signaling transduction proteins. Our findings suggest that similarly to SARS-CoV and MERS-CoV, HCoV-OC43 has the ability to downregulate the transcription of genes critical for the activation of different antiviral signaling pathways. Further studies are needed to confirm the role of HCoV-OC43 structural and accessory proteins in antagonising antiviral gene expression.
Url:
DOI: 10.1007/s00705-018-3832-8
PubMed: 29619598
PubMed Central: 7086905
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PMC:7086905Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">PCR array profiling of antiviral genes in human embryonic kidney cells expressing human coronavirus OC43 structural and accessory proteins</title><author><name sortKey="Beidas, Meshal" sort="Beidas, Meshal" uniqKey="Beidas M" first="Meshal" last="Beidas">Meshal Beidas</name><affiliation><nlm:aff id="Aff1"></nlm:aff></affiliation></author><author><name sortKey="Chehadeh, Wassim" sort="Chehadeh, Wassim" uniqKey="Chehadeh W" first="Wassim" last="Chehadeh">Wassim Chehadeh</name><affiliation><nlm:aff id="Aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">29619598</idno><idno type="pmc">7086905</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7086905</idno><idno type="RBID">PMC:7086905</idno><idno type="doi">10.1007/s00705-018-3832-8</idno><date when="2018">2018</date><idno type="wicri:Area/Pmc/Corpus">000256</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000256</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">PCR array profiling of antiviral genes in human embryonic kidney cells expressing human coronavirus OC43 structural and accessory proteins</title><author><name sortKey="Beidas, Meshal" sort="Beidas, Meshal" uniqKey="Beidas M" first="Meshal" last="Beidas">Meshal Beidas</name><affiliation><nlm:aff id="Aff1"></nlm:aff></affiliation></author><author><name sortKey="Chehadeh, Wassim" sort="Chehadeh, Wassim" uniqKey="Chehadeh W" first="Wassim" last="Chehadeh">Wassim Chehadeh</name><affiliation><nlm:aff id="Aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">Archives of Virology</title><idno type="ISSN">0304-8608</idno><idno type="eISSN">1432-8798</idno><imprint><date when="2018">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="Par1">Human coronavirus OC43 (HCoV-OC43) is a respiratory virus that usually causes a common cold. However, it has the potential to cause severe infection in young children and immunocompromised adults. Both SARS-CoV and MERS-CoV were shown to express proteins with the potential to evade early innate immune responses. However, the ability of HCoV-OC43 to antagonise the intracellular antiviral defences has not yet been investigated. The potential role of the HCoV-OC43 structural (M and N) and accessory proteins (ns2a and ns5a) in the alteration of antiviral gene expression was investigated in this study. HCoV-OC43M, N, ns2a and ns5a proteins were expressed in human embryonic kidney 293 (HEK-293) cells before challenge with Sendai virus. The Human Antiviral Response PCR array was used to profile the antiviral gene expression in HEK-293 cells. Over 30 genes were downregulated in the presence of one of the HCoV-OC43 proteins, e.g. genes representing mitogen-activated protein kinases, toll-like receptors, interferons, interleukins, and signaling transduction proteins. Our findings suggest that similarly to SARS-CoV and MERS-CoV, HCoV-OC43 has the ability to downregulate the transcription of genes critical for the activation of different antiviral signaling pathways. Further studies are needed to confirm the role of HCoV-OC43 structural and accessory proteins in antagonising antiviral gene expression.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Cavanagh, D" uniqKey="Cavanagh D">D Cavanagh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vijgen, L" uniqKey="Vijgen L">L Vijgen</name></author><author><name sortKey="Keyaerts, E" uniqKey="Keyaerts E">E Keyaerts</name></author><author><name sortKey="Lemey, P" uniqKey="Lemey P">P Lemey</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Larson, He" uniqKey="Larson H">HE Larson</name></author><author><name sortKey="Reed, Se" uniqKey="Reed S">SE Reed</name></author><author><name sortKey="Tyrrell, Da" uniqKey="Tyrrell D">DA Tyrrell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lepiller, Q" uniqKey="Lepiller Q">Q 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Arch Virol</journal-id><journal-id journal-id-type="iso-abbrev">Arch. Virol</journal-id><journal-title-group><journal-title>Archives of Virology</journal-title></journal-title-group><issn pub-type="ppub">0304-8608</issn><issn pub-type="epub">1432-8798</issn><publisher><publisher-name>Springer Vienna</publisher-name><publisher-loc>Vienna</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">29619598</article-id><article-id pub-id-type="pmc">7086905</article-id><article-id pub-id-type="publisher-id">3832</article-id><article-id pub-id-type="doi">10.1007/s00705-018-3832-8</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>PCR array profiling of antiviral genes in human embryonic kidney cells expressing human coronavirus OC43 structural and accessory proteins</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Beidas</surname><given-names>Meshal</given-names></name><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0003-0761-4892</contrib-id><name><surname>Chehadeh</surname><given-names>Wassim</given-names></name><address><phone>+965 24 63 65 19</phone><email>wchehadeh@hsc.edu.kw</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0001 1240 3921</institution-id><institution-id institution-id-type="GRID">grid.411196.a</institution-id><institution>Department of Microbiology, Faculty of Medicine,</institution><institution>Kuwait University,</institution></institution-wrap>PO Box 24923, 13310 Safat, Kuwait</aff></contrib-group><author-notes><fn fn-type="com"><p>Handling Editor: Chan-Shing Lin.</p></fn></author-notes><pub-date pub-type="epub"><day>4</day><month>4</month><year>2018</year></pub-date><pub-date pub-type="ppub"><year>2018</year></pub-date><volume>163</volume><issue>8</issue><fpage>2065</fpage><lpage>2072</lpage><history><date date-type="received"><day>9</day><month>11</month><year>2017</year></date><date date-type="accepted"><day>22</day><month>3</month><year>2018</year></date></history><permissions><copyright-statement>© Springer-Verlag GmbH Austria, part of Springer Nature 2018</copyright-statement><license><license-p>This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p></license></permissions><abstract id="Abs1"><p id="Par1">Human coronavirus OC43 (HCoV-OC43) is a respiratory virus that usually causes a common cold. However, it has the potential to cause severe infection in young children and immunocompromised adults. Both SARS-CoV and MERS-CoV were shown to express proteins with the potential to evade early innate immune responses. However, the ability of HCoV-OC43 to antagonise the intracellular antiviral defences has not yet been investigated. The potential role of the HCoV-OC43 structural (M and N) and accessory proteins (ns2a and ns5a) in the alteration of antiviral gene expression was investigated in this study. HCoV-OC43M, N, ns2a and ns5a proteins were expressed in human embryonic kidney 293 (HEK-293) cells before challenge with Sendai virus. The Human Antiviral Response PCR array was used to profile the antiviral gene expression in HEK-293 cells. Over 30 genes were downregulated in the presence of one of the HCoV-OC43 proteins, e.g. genes representing mitogen-activated protein kinases, toll-like receptors, interferons, interleukins, and signaling transduction proteins. Our findings suggest that similarly to SARS-CoV and MERS-CoV, HCoV-OC43 has the ability to downregulate the transcription of genes critical for the activation of different antiviral signaling pathways. Further studies are needed to confirm the role of HCoV-OC43 structural and accessory proteins in antagonising antiviral gene expression.</p></abstract><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100004482</institution-id><institution>Kuwait University</institution></institution-wrap></funding-source><award-id>YM 04/15</award-id><principal-award-recipient><name><surname>Chehadeh</surname><given-names>Wassim</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© Springer-Verlag GmbH Austria, part of Springer Nature 2018</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1"><title>Introduction</title><p id="Par2">Human coronavirus OC43 (HCoV-OC43) is taxonomically classified within the <italic>Coronaviridae</italic> family which also includes severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses. HCoV-OC43 belongs to subgroup A of the betacoronaviruses. These are positive-sense single-stranded RNA viruses with a large genome size at 30,780 bases [<xref ref-type="bibr" rid="CR1">1</xref>]. The viral proteins that contribute to the structure of coronaviruses include the Membrane (M), Nucleocapsid (N), Envelope (E) and Spike (S) glycoprotein. Betacoronaviruses possess a unique protein on the surface alongside the S glycoprotein called Hemagglutinin esterase (HE). Each coronavirus possesses its own set of unique accessory proteins. The accessory proteins of HCoV-OC43 are called ns2a and ns5a [<xref ref-type="bibr" rid="CR2">2</xref>].</p><p id="Par3">HCoV-OC43 and other common circulating coronaviruses cause 10 to 30% of all common colds, and 5 to 10% of all upper and lower respiratory tract infections [<xref ref-type="bibr" rid="CR3">3</xref>–<xref ref-type="bibr" rid="CR6">6</xref>]. Subclinical or very mild infections are common, and can occur throughout the year. However, human coronaviruses other than SARS-CoV and MERS-CoV are responsible for 10 to 20% of hospitalisations of young children and immunocompromised adults with respiratory tract illness [<xref ref-type="bibr" rid="CR7">7</xref>]. HCoV-OC43 seems to have also the potential for both neurotropism and neuroinvasion that are associated with multiple sclerosis [<xref ref-type="bibr" rid="CR8">8</xref>, <xref ref-type="bibr" rid="CR9">9</xref>]. Moreover, HCoV-OC43 was detected in the cerebrospinal fluid of a child with acute disseminated encephalomyelitis [<xref ref-type="bibr" rid="CR10">10</xref>]. A case of fatal encephalitis associated with HCoV-OC43 infection was recently reported in an immunodeficient 11-month-old boy [<xref ref-type="bibr" rid="CR11">11</xref>].</p><p id="Par4">Several studies on human coronavirus interaction with host cells and the innate immune response have been conducted on SARS-CoV and MERS-CoV, because of the high case-fatality rate associated with these infections. In Vero E6 cells expressing SARS-CoV N protein, interleukin 6 (IL6) was activated through nuclear factor kappa B (NF-κB) [<xref ref-type="bibr" rid="CR12">12</xref>]. On the contrary, the M protein of SARS-CoV suppresses NF-κB activity and contributes to the pathogenesis of the virus [<xref ref-type="bibr" rid="CR13">13</xref>]. In addition, many proteins in SARS-CoV were shown to have the ability to antagonise interferon (IFN) and counteract the innate immune response. The ORF3b and ORF6 accessory proteins have the potential to antagonise IFN by different mechanisms [<xref ref-type="bibr" rid="CR14">14</xref>]. SARS-CoV M protein was recently shown to suppress IFN production [<xref ref-type="bibr" rid="CR15">15</xref>]. The structural and accessory proteins M, ORF4a, ORF4b, and ORF5 of MERS-CoV were shown to be potent IFN antagonists [<xref ref-type="bibr" rid="CR16">16</xref>, <xref ref-type="bibr" rid="CR17">17</xref>]. It is still unknown whether the evasion mechanisms described above have a certain role in the severity of SARS and MERS. It is noteworthy that several people infected with SARS-CoV or MERS-CoV do not develop severe symptoms [<xref ref-type="bibr" rid="CR18">18</xref>–<xref ref-type="bibr" rid="CR21">21</xref>].</p><p id="Par5">Most research on SARS and MERS has been done on one specific virus and how it activates or inhibits the innate immune response. None of the studies conducted so far have compared results with those of HCoV-OC43 that is associated with low mortality rate. HCoV-OC43 may have the same ability as SARS-CoV and MERS-CoV to evade innate immunity. Indeed, there are few studies investigating HCoV-OC43 interaction with host cells and the innate immune response. The N protein was shown to cause prolonged activation of NF-κB in N-expressing HEK-293T cells [<xref ref-type="bibr" rid="CR22">22</xref>]. Furthermore, HCoV-OC43 infection of cell lines derived from human respiratory tract epithelia and hepatocytes, was enhanced by all three types of IFN, due to the interaction with the IFN-inducible transmembrane (IFITM) proteins [<xref ref-type="bibr" rid="CR23">23</xref>]. Moreover, the acetyl-esterase activity of HE protein strongly enhances the production of infectious virus [<xref ref-type="bibr" rid="CR24">24</xref>].</p><p id="Par6">While SARS-CoV and MERS-CoV have received much attention from the scientific community, very little research has addressed the effect of other human coronavirus infections on antiviral gene expression. The potential role of the HCoV-OC43 structural and accessory proteins in the alteration of antiviral gene expression in HEK-293 cells was investigated in this study.</p></sec><sec id="Sec2"><title>Materials and methods</title><sec id="Sec3"><title>Cell culture</title><p id="Par7">Human embryonic kidney 293 (HEK-293) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and were grown in Dulbecco’s minimal essential medium (DMEM) containing GlutaMAX™ (Life Technologies Corporation<sup>TM</sup>, Grand Island, NY, USA) using 25 cm<sup>2</sup> tissue culture flasks. The medium was supplemented with 10% fetal bovine serum (FBS) (Life Technologies<sup>TM</sup>), Fungizone® (250 µg/ml) (Life Technologies<sup>TM</sup>), penicillin G (10,000 U/ml) (Life Technologies) and streptomycin sulfate (10,000 pg/ml) (Life Technologies<sup>TM</sup>). Monolayers of HEK-293 cell culture flasks were incubated at 37 °C in the presence of 5% carbon dioxide (CO<sub>2</sub>) and 90% humidity.</p></sec><sec id="Sec4"><title>Viruses</title><p id="Par8">Human coronavirus OC43 (HCoV-OC43; VR-1558), influenza A virus (H3N2, A/Hong Kong/8/68 strain; VR-544), and Sendai virus (VR-105) were obtained from ATCC.</p></sec><sec id="Sec5"><title>Amplification of HCoV-OC43 ns2a, ns5a, M, and N genes by RT-PCR</title><p id="Par9">HCoV-OC43 RNA was extracted using the QIAamp® Viral RNA Minikit (Qiagen<sup>TM</sup> GmbH, Hilden, Germany) according to the manufacturer’s instructions. The HCoV-OC43 <italic>ns2a</italic>, <italic>ns5a</italic>, <italic>M</italic>, and <italic>N</italic> genes were amplified by a two-step reverse transcription-polymerase chain reaction (RT-PCR) using the GeneAmp® RNA Core Kit (Applied Biosystems<sup>TM</sup>, Foster City, CA, USA) and 10 pmol of previously described primers [<xref ref-type="bibr" rid="CR2">2</xref>] on a GeneAmp® PCR System 9700 (Applied Biosystems<sup>TM</sup>). The RT reaction conditions were: annealing at 37 °C for 60 minutes, denaturation at 90 °C for 5 minutes, and cooling at 4 °C. Thereafter, the cDNA was amplified by PCR using the following conditions: denaturation at 94 °C for 10 minutes, then 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 30 seconds, followed by a final extension step at 72 °C for 7 minutes, with cooling at 4 °C. PCR products were run on agarose gels and the bands that were size-specific for the amplified gene of interest were cut and purified by Wizard® SV Gel and PCR Clean Up System (Promega<sup>TM</sup>, Madison, WI, USA) according to the manufacturer’s instructions. Influenza A virus RNA was also isolated and was used to amplify the <italic>NS1</italic> gene by RT-PCR using <italic>NS1</italic>-specific primers [<xref ref-type="bibr" rid="CR25">25</xref>]. Influenza A NS1 protein was used as positive control for IFN antagonism [<xref ref-type="bibr" rid="CR14">14</xref>].</p></sec><sec id="Sec6"><title>Expression of HCoV-OC43 ns5a, ns2a, M and N proteins in HEK-293 cells</title><p id="Par10">The pAcGFP1-N In-Fusion® Ready Vector (Clontech<sup>TM</sup>, Takara Bio Company, Mountain View, CA, USA) was used for cloning RT-PCR products. The In-Fusion® HD Cloning Kit (Clontech<sup>TM</sup>) was used to set up the ligation reaction. Competent TOP10 <italic>E. coli</italic> cells (Invitrogen<sup>TM</sup>, Carlsbad, CA, USA) were used for transformation. The isolation of vector was achieved by using the PureYield™ Plasmid Miniprep Kit (Promega) according to the manufacturer’s instructions. Confirmation of successful cloning was achieved using restriction digestion and direct sequencing. HEK-293 cells were seeded at 5 x 10<sup>5</sup> per well of 24-well plates, and transfected with ns2a-pAcGFP1, ns5a-pAcGFP1, M-pAcGFP1, N-pAcGFP1, or NS1-pAcGFP1 using Lipofectamine® 2000 according to the manufacturer’s instructions. Expression of ns2a, ns5a, M, N and NS1 proteins was confirmed by indirect immunofluorescence assay (IFA) using anti-GFP monoclonal antibody (10 μg/ml) (Clone JL-8, Clontech<sup>TM</sup>). Expression of N and NS1 proteins was further confirmed by IFA using monoclonal antibody against HCoV-OC43N (EMD Millipore<sup>TM</sup>, Billerica, MA, USA) and polyclonal antibody against influenza A NS1 protein (EMD Millipore<sup>TM</sup>), respectively.</p></sec><sec id="Sec7"><title>Challenge of HEK-293 cells with Sendai virus</title><p id="Par11">Transfected and mock-transfected HEK-293 cells were grown to confluency in a 96-well plate at 37 °C in the presence of 5% CO<sub>2</sub>. The media was then removed, and Sendai virus suspension (ATCC) was added to each well at 1 multiplicity of infection (m.o.i). The plates were further incubated at 37°C with 5% CO<sub>2</sub> and at 90% humidity.</p></sec><sec id="Sec8"><title>Antiviral gene expression profile in cells expressing HCoV-OC43 structural and accessory proteins</title><p id="Par12">The Human Antiviral Response PCR array (Qiagen<sup>TM</sup>) was used to profile the antiviral gene expression in HEK-293 cells in two separate experiments. The PCR array monitored the transcriptional activity of different genes involved in the activation of antiviral and pro-inflammatory proteins following amplification of RNA transcripts by real-time RT-PCR. Total RNA from transfected and mock-transfected HEK-293 cells was extracted using an RNeasy® Kit (Qiagen<sup>TM</sup>) with on-column DNase digestion according to the manufacturer’s instructions. The RT<sup>2</sup> First Strand Kit (Qiagen<sup>TM</sup>) was used for cDNA synthesis. The RT reaction was then added to RT<sup>2</sup> SYBR Green ROX qPCR Mastermix (Qiagen<sup>TM</sup>), and the PCR was carried out in a 96-well plate (Qiagen<sup>TM</sup>) on an ABI 7500 Fast Real-Time PCR system (Applied Biosystems<sup>TM</sup>). Cycle threshold (C<sub>T</sub>) values were exported to an Excel file to create a table of C<sub>T</sub> values. This table was then uploaded on to the Qiagen data analysis web portal (<ext-link ext-link-type="uri" xlink:href="http://www.qiagen.com/geneglobe">http://www.qiagen.com/geneglobe</ext-link>). Quality control was performed as the array contains controls to monitor genomic DNA contamination (GDC), first strand synthesis (RTC) and real-time PCR efficiency (PPC). Samples were assigned to controls and test groups. C<sub>T</sub> values were normalised based on a manual selection of reference genes with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) chosen as the housekeeping reference gene. Fold regulation comparisons were used to profile antiviral gene expression. Fold change/regulation was calculated using delta delta C<sub>T</sub> method (∆∆C<sub>T</sub>) where ∆C<sub>T</sub> is calculated between the gene of interest (GOI) and GAPDH, followed by ∆∆C<sub>T</sub> calculations (∆C<sub>T</sub> (Test Group)-∆C<sub>T</sub> (Control Group)). Fold change is then calculated using 2^ (-∆∆C<sub>T</sub>) formula. Thereafter, the data analysis web portal was used to provide the scatter plot, the volcano plot, the clustergram, and the heat map. The fold regulation threshold was ≥ 2 for upregulation and ≤ -2 for downregulation.</p></sec><sec id="Sec9"><title>Statistical analysis</title><p id="Par13">Fold change in gene expression from 2 different experiments was summarised as mean ± standard deviation. The difference in mean fold change between two groups was determined by independent-samples <italic>t</italic>-test. <italic>P</italic>-value < 0.05 was considered significant. All statistical analyses were performed using IBM SPSS statistics software version 25.0 for windows (IBM Corporation, Armonk, NY, USA).</p></sec></sec><sec id="Sec10"><title>Results</title><sec id="Sec11"><title>PCR array profiling of antiviral genes in HEK-293 cells expressing HCoV-OC43 structural and accessory proteins</title><p id="Par14">Figure <xref rid="Fig1" ref-type="fig">1</xref> shows the expression of HCoV-OC43 and influenza proteins in HEK-293 cells. The antiviral gene expression in transfected HEK-293 cells expressing one HCoV-OC43 protein was compared to that in mock- and non-transfected cells. Only the expression of the oncogene FOS was upregulated across all samples (Table <xref rid="Tab1" ref-type="table">1</xref>). Nevertheless, over 30 genes were downregulated in the presence of one of the HCoV-OC43 proteins, e.g. mitogen-activated protein kinases (<italic>MAP3K7</italic>), toll-like receptors (<italic>TLR3</italic> and <italic>TLR8</italic>), caspases (<italic>CASP1</italic>), cathepsins (<italic>CTSS</italic>), interferons (<italic>IFNA1</italic>), interleukins (<italic>IL1B, IL12A, IL15,</italic> and <italic>IL18</italic>), and signaling transduction proteins (<italic>MYD88</italic> and <italic>MAVS</italic>) (Table <xref rid="Tab1" ref-type="table">1</xref>). Similar results were obtained in the presence of the influenza A NS1 protein, a well-known IFN antagonist [<xref ref-type="bibr" rid="CR14">14</xref>] (Table <xref rid="Tab1" ref-type="table">1</xref>). The mean negative fold change for <italic>CARD9</italic> in the presence of the structural protein M or N was larger than that in the presence of the accessory protein ns2a (<italic>p </italic>< 0.01). In contrast, the expression of the <italic>MAP3K7</italic> gene was more downregulated in the presence of ns2a or ns5a protein than in the presence of M or N protein (<italic>p </italic>< 0.01). Downregulation of <italic>CTSS</italic> gene expression was greater in the presence of ns2a, ns5a or N protein than in the presence of M protein (<italic>p </italic>< 0.01). <italic>IL1B</italic>, <italic>CASP1</italic>, and <italic>TLR3</italic> gene expression was more downregulated in the presence of ns2a or ns5a protein than in the presence of M or N protein (<italic>p </italic>< 0.01). On the contrary, <italic>CD86</italic> was more downregulated in the presence of M protein than in the presence of N, ns2a or ns5a protein (<italic>p </italic>≤ 0.01).<fig id="Fig1"><label>Fig. 1</label><caption><p>Expression of HCoV-OC43 and influenza A proteins in HEK-293 cells. HEK-293 cells were transfected with the expression vector pAcGFP1 coding for HCoV-OC43 ns2a, ns5a, M or N proteins, or for influenza A NS1 protein. Three days following transfection, the expression of viral proteins was confirmed by indirect immunofluorescence assay using anti-GFP monoclonal antibody</p></caption><graphic xlink:href="705_2018_3832_Fig1_HTML" id="MO1"></graphic></fig><table-wrap id="Tab1"><label>Table 1</label><caption><p>Mean fold change in antiviral gene expression in HEK-293 cells expressing HCoV-OC43 structural or accessory proteins</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left"></th><th align="left" colspan="2">ns2a</th><th align="left" colspan="2">ns5a</th><th align="left" colspan="2">M</th><th align="left" colspan="2">N</th><th align="left">NS1 (FluA)</th></tr><tr><th align="left"></th><th align="left">Mean</th><th align="left">±SD</th><th align="left">Mean</th><th align="left">±SD</th><th align="left">Mean</th><th align="left">±SD</th><th align="left">Mean</th><th align="left">±SD</th><th align="left"></th></tr></thead><tbody><tr><td align="left"><p><italic>FOS</italic></p><p><italic>MAP3K7</italic></p><p><italic>TLR3</italic></p><p><italic>CTSS</italic></p><p><italic>CASP1</italic></p><p><italic>MEFV</italic></p><p><italic>IL12A</italic></p><p><italic>PYCARD</italic></p><p><italic>IL1B</italic></p><p><italic>MX1</italic></p><p><italic>IFIH1</italic></p><p><italic>RIPK1</italic></p><p><italic>IFNA1</italic></p><p><italic>CD80</italic></p><p><italic>CD86</italic></p><p><italic>CXCL11</italic></p><p><italic>MYD88</italic></p><p><italic>FADD</italic></p><p><italic>MAVS</italic></p><p><italic>TRIM25</italic></p><p><italic>IL15</italic></p><p><italic>CYLD</italic></p><p><italic>TLR8</italic></p><p><italic>TNF</italic></p><p><italic>IL18</italic></p><p><italic>TRAF6</italic></p><p><italic>IRF3</italic></p><p><italic>TBK1</italic></p><p><italic>IFNAR1</italic></p><p><italic>SUGT1</italic></p><p><italic>CARD9</italic></p></td><td align="left"><p>+9.94</p><p>-83.63</p><p>-55.56</p><p>-21.91</p><p>-16.73</p><p>——</p><p>7.18</p><p>-10.06</p><p>-24.83</p><p>-6.97</p><p>-7.89</p><p>-9.61</p><p>-4.99</p><p>-4.41</p><p>-4.15</p><p>-5.84</p><p>-7.55</p><p>-4.61</p><p>-3.81</p><p>-4.59</p><p>-3.77</p><p>-4.98</p><p>-2.39</p><p>-2.22</p><p>-3.16</p><p>-4.71</p><p>—–</p><p>-3.35</p><p>-3.12</p><p>—–</p><p>-4.81</p></td><td align="left"><p>0.34</p><p>2.17</p><p>0.79</p><p>1.64</p><p>1.56</p><p>—–</p><p>1.34</p><p>1.24</p><p>0.67</p><p>0.57</p><p>0.23</p><p>1.25</p><p>2.78</p><p>1.23</p><p>1.20</p><p>0.07</p><p>2.22</p><p>1.23</p><p>1.49</p><p>1.93</p><p>0.72</p><p>0.04</p><p>0.16</p><p>0.03</p><p>0.06</p><p>0.45</p><p>—–</p><p>0.02</p><p>0.05</p><p>—–</p><p>0.45</p></td><td align="left"><p>+9.49</p><p>-88.75</p><p>-32.59</p><p>-23.85</p><p>-16.52</p><p>-14.36</p><p>-13.62</p><p>-12.64</p><p>-9.94</p><p>-7.24</p><p>-6.99</p><p>-6.90</p><p>-6.59</p><p>-6.38</p><p>-6.01</p><p>-5.05</p><p>-4.79</p><p>-4.30</p><p>-2.80</p><p>-4.06</p><p>-3.96</p><p>-3.76</p><p>-3.46</p><p>—–</p><p>-3.17</p><p>-3.09</p><p>-2.98</p><p>-2.97</p><p>-2.93</p><p>-2.92</p><p>—–</p></td><td align="left"><p>0.23</p><p>2.45</p><p>1.23</p><p>1.85</p><p>1.93</p><p>1.52</p><p>0.95</p><p>0.97</p><p>0.25</p><p>1.22</p><p>1.24</p><p>0.75</p><p>0.53</p><p>0.54</p><p>0.12</p><p>1.26</p><p>1.89</p><p>0.03</p><p>0.74</p><p>0.08</p><p>0.19</p><p>1.35</p><p>1.23</p><p>—–</p><p>0.04</p><p>0.63</p><p>0.35</p><p>0.69</p><p>0.04</p><p>0.09</p><p>—–</p></td><td align="left"><p>+3.45</p><p>—–</p><p>-14.68</p><p>-7.68</p><p>—–</p><p>-5.21</p><p>-6.74</p><p>-18.10</p><p>—–</p><p>—–</p><p>-2.84</p><p>-5.21</p><p>-3.24</p><p>-5.71</p><p>-13.61</p><p>-2.17</p><p>-2.34</p><p>—–</p><p>—–</p><p>—–</p><p>—–</p><p>—–</p><p>-3.06</p><p>-7.27</p><p>-3.14</p><p>-2.47</p><p>-2.19</p><p>-2.85</p><p>—–</p><p>—–</p><p>-16.75</p></td><td align="left"><p>0.45</p><p>—–</p><p>0.63</p><p>0.93</p><p>—–</p><p>2.22</p><p>1.23</p><p>0.63</p><p>—–</p><p>—–</p><p>0.09</p><p>2.55</p><p>1.24</p><p>0.56</p><p>0.83</p><p>0.11</p><p>0.05</p><p>—–</p><p>—–</p><p>—–</p><p>—–</p><p>—–</p><p>0.05</p><p>0.68</p><p>0.91</p><p>0.33</p><p>0.03</p><p>0.45</p><p>—–</p><p>—–</p><p>0.93</p></td><td align="left"><p>+10.06</p><p>-59.54</p><p>-31.73</p><p>-24.57</p><p>-12.03</p><p>-13.59</p><p>-8.61</p><p>-11.48</p><p>-6.63</p><p>-8.14</p><p>-6.05</p><p>-8.85</p><p>-5.39</p><p>-6.04</p><p>-5.69</p><p>—–</p><p>-5.15</p><p>-5.66</p><p>-3.75</p><p>-5.20</p><p>-3.27</p><p>—–</p><p>-3.28</p><p>—–</p><p>-3.25</p><p>-3.76</p><p>—–</p><p>-3.05</p><p>-2.93</p><p>-3.97</p><p>-12.19</p></td><td align="left"><p>0.29</p><p>1.23</p><p>0.83</p><p>0.73</p><p>0.53</p><p>2.22</p><p>1.23</p><p>2.56</p><p>1.59</p><p>0.03</p><p>0.94</p><p>0.35</p><p>0.48</p><p>0.54</p><p>2.34</p><p>—–</p><p>0.53</p><p>0.23</p><p>0.05</p><p>1.29</p><p>0.45</p><p>—–</p><p>1.24</p><p>—–</p><p>0.53</p><p>0.73</p><p>—–</p><p>0.23</p><p>0.04</p><p>0.05</p><p>0.02</p></td><td align="left"><p>+8.23</p><p>-83.27</p><p>-25.79</p><p>-17.94</p><p>-21.32</p><p>-9.89</p><p>-9.59</p><p>-2.60</p><p>-2.15</p><p>-9.12</p><p>-7.10</p><p>-6.66</p><p>-4.42</p><p>—–</p><p>-4.14</p><p>-2.17</p><p>-5.18</p><p>-4.58</p><p>-4.67</p><p>-4.57</p><p>-4.14</p><p>-3.36</p><p>-2.39</p><p>—–</p><p>-3.14</p><p>-3.50</p><p>-3.15</p><p>-3.20</p><p>-2.93</p><p>-2.99</p><p>—–</p></td></tr></tbody></table><table-wrap-foot><p>Results are shown as the mean ± standard deviation (SD) of two independent experiments. Mean ± SD was not available for influenza NS1 because only one experiment was performed. (—–) indicates no significant fold change in one or two experiments</p></table-wrap-foot></table-wrap></p><p id="Par15">Sendai virus, a well-known inducer of antiviral genes, was used to challenge HEK-293 cells. A total of 22 genes were upregulated in the presence of Sendai virus, whereas 9 genes were downregulated (Table <xref rid="Tab2" ref-type="table">2</xref>). There was strong positive fold change for <italic>MAP3K7, FOS, CXCL9, JUN, TLR7, IRF7, IL12B, NOD2</italic> and <italic>IL6</italic> in HEK-293 cells. In the presence of ns2a, ns5a, M, N or NS1 protein, the challenge of cells with Sendai virus resulted in the upregulation of only 7 genes and downregulation of 25 genes (Table <xref rid="Tab3" ref-type="table">3</xref>). The expression of <italic>OAS2</italic> was upregulated in the presence of HCoV-OC43 ns5a or M protein, or influenza A NS1 protein. A positive fold change was observed for three chemokines (<italic>CCL5, CXCL8, CXCL10</italic>), two RIG-I-like receptors (<italic>IFIH1</italic> and <italic>DDX58</italic>), one interferon (<italic>IFNB1</italic>), and one ubiquitin-like protein (<italic>ISG15</italic>), whereas there was a drastic downregulation of <italic>MAP3K7</italic> expression with negative fold change ranging from -484 to -27,496 (Table <xref rid="Tab3" ref-type="table">3</xref>). <italic>FOS, CXCL9, JUN, TLR7, IRF7, IL12B</italic> and <italic>IL6</italic> also showed substantial negative fold changes (Table <xref rid="Tab3" ref-type="table">3</xref>). The expression of <italic>MAP3K7, TLR7, IL12B, IL6</italic> and <italic>NOD2</italic> genes was more downregulated in the presence of ns5a protein than in the presence of other HCoV-OC43 proteins (<italic>p </italic>< 0.05), whereas the expression of the <italic>CXCL9</italic> gene was more downregulated in the presence of ns2a protein than in the presence of other proteins (<italic>p </italic>< 0.001). Moreover, the expression of <italic>JUN</italic> and <italic>IRF7</italic> genes was most downregulated in the presence of N protein (<italic>p </italic>≤ 0.01). Interestingly, the expression of <italic>IRF7</italic> and <italic>IFNA1</italic> genes was more downregulated in the presence of ns2a, ns5a or N protein than in the presence of influenza NS1 protein. The expression of <italic>NOD2</italic> was more downregulated in the presence of ns2a or ns5a protein than in the presence of influenza NS1 protein, while the expression of <italic>PYCARD</italic> was more downregulated in the presence of one of the tested HCoV-OC43 proteins than in the presence of influenza NS1 protein. Moreover, <italic>FOS</italic> gene expression was more downregulated in the presence of ns5a, M or N protein than in the presence of influenza NS1 protein (Table <xref rid="Tab3" ref-type="table">3</xref>).<table-wrap id="Tab2"><label>Table 2</label><caption><p>Mean fold change in antiviral gene expression in HEK-293 cells challenged with Sendai virus</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Upregulated genes</th><th align="left" colspan="2">Cells (w/ Sendai)</th><th align="left">Downregulated genes</th><th align="left" colspan="2">Cells (w/ Sendai)</th></tr><tr><th align="left"></th><th align="left">Mean</th><th align="left">±SD</th><th align="left"></th><th align="left">Mean</th><th align="left">±SD</th></tr></thead><tbody><tr><td align="left"><italic>MAP3K7</italic></td><td char="." align="char">+276.05</td><td char="." align="char">8.27</td><td align="left"><italic>IFIH1</italic></td><td char="." align="char">-16.21</td><td char="." align="char">3.45</td></tr><tr><td align="left"><italic>FOS</italic></td><td char="." align="char">+177.10</td><td char="." align="char">2.13</td><td align="left"><italic>CCL5</italic></td><td char="." align="char">-14.95</td><td char="." align="char">3.43</td></tr><tr><td align="left"><italic>CXCL9</italic></td><td char="." align="char">+25.64</td><td char="." align="char">2.71</td><td align="left"><italic>CTSS</italic></td><td char="." align="char">-13.06</td><td char="." align="char">1.54</td></tr><tr><td align="left"><italic>JUN</italic></td><td char="." align="char">+24.61</td><td char="." align="char">2.34</td><td align="left"><italic>IL12A</italic></td><td char="." align="char">-8.27</td><td char="." align="char">1.73</td></tr><tr><td align="left"><italic>TLR7</italic></td><td char="." align="char">+21.87</td><td char="." align="char">1.87</td><td align="left"><italic>TLR3</italic></td><td char="." align="char">-6.53</td><td char="." align="char">1.35</td></tr><tr><td align="left"><italic>IRF7</italic></td><td char="." align="char">+15.61</td><td char="." align="char">2.30</td><td align="left"><italic>DDX58</italic></td><td char="." align="char">-5.18</td><td char="." align="char">2.69</td></tr><tr><td align="left"><italic>IL12B</italic></td><td char="." align="char">+15.54</td><td char="." align="char">0.89</td><td align="left"><italic>RIPK1</italic></td><td char="." align="char">-5.03</td><td char="." align="char">2.27</td></tr><tr><td align="left"><italic>NOD2</italic></td><td char="." align="char">+15.26</td><td char="." align="char">1.74</td><td align="left"><italic>CASP1</italic></td><td char="." align="char">-4.01</td><td char="." align="char">0.37</td></tr><tr><td align="left"><italic>IL6</italic></td><td char="." align="char">+15.17</td><td char="." align="char">4.78</td><td align="left"><italic>MEFV</italic></td><td char="." align="char">-2.29</td><td char="." align="char">0.01</td></tr><tr><td align="left"><italic>SPP1</italic></td><td char="." align="char">+4.20</td><td char="." align="char">0.01</td><td align="left"></td><td align="left"></td><td align="left"></td></tr><tr><td align="left"><italic>NLRP3</italic></td><td char="." align="char">+4.16</td><td char="." align="char">0.01</td><td align="left"></td><td align="left"></td><td align="left"></td></tr><tr><td align="left"><italic>IFNA2</italic></td><td char="." align="char">+3.25</td><td char="." align="char">0.62</td><td align="left"></td><td align="left"></td><td align="left"></td></tr><tr><td align="left"><italic>TRADD</italic></td><td char="." align="char">+3.04</td><td char="." align="char">0.45</td><td align="left"></td><td align="left"></td><td align="left"></td></tr><tr><td align="left"><italic>MAVS</italic></td><td char="." align="char">+3.02</td><td char="." align="char">0.75</td><td align="left"></td><td align="left"></td><td align="left"></td></tr><tr><td align="left"><italic>IFNA1</italic></td><td char="." align="char">+2.90</td><td char="." align="char">0.06</td><td align="left"></td><td align="left"></td><td align="left"></td></tr><tr><td align="left"><italic>CCL3</italic></td><td char="." align="char">+2.81</td><td char="." align="char">0.01</td><td align="left"></td><td align="left"></td><td align="left"></td></tr><tr><td align="left"><italic>IRAK1</italic></td><td char="." align="char">+2.78</td><td char="." align="char">0.12</td><td align="left"></td><td align="left"></td><td align="left"></td></tr><tr><td align="left"><italic>CTSB</italic></td><td char="." align="char">+2.72</td><td char="." align="char">0.06</td><td align="left"></td><td align="left"></td><td align="left"></td></tr><tr><td align="left"><italic>PIN1</italic></td><td char="." align="char">+2.50</td><td char="." align="char">0.05</td><td align="left"></td><td align="left"></td><td align="left"></td></tr><tr><td align="left"><italic>TLR8</italic></td><td char="." align="char">+2.48</td><td char="." align="char">0.32</td><td align="left"></td><td align="left"></td><td align="left"></td></tr><tr><td align="left"><italic>IRF5</italic></td><td char="." align="char">+2.36</td><td char="." align="char">0.06</td><td align="left"></td><td align="left"></td><td align="left"></td></tr><tr><td align="left"><italic>MAP2K3</italic></td><td char="." align="char">+2.31</td><td char="." align="char">0.21</td><td align="left"></td><td align="left"></td><td align="left"></td></tr></tbody></table><table-wrap-foot><p>Results are shown as the mean ± standard deviation (SD) of two independent experiments</p></table-wrap-foot></table-wrap><table-wrap id="Tab3"><label>Table 3</label><caption><p>Mean fold change in antiviral gene expression in Sendai-infected HEK-293 cells expressing HCoV-OC43 structural or accessory proteins</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left"></th><th align="left" colspan="2">ns2a</th><th align="left" colspan="2">ns5a</th><th align="left" colspan="2">M</th><th align="left" colspan="2">N</th><th align="left">NS1 (FluA)</th></tr><tr><th align="left"></th><th align="left">Mean</th><th align="left">±SD</th><th align="left">Mean</th><th align="left">±SD</th><th align="left">Mean</th><th align="left">±SD</th><th align="left">Mean</th><th align="left">±SD</th><th align="left"></th></tr></thead><tbody><tr><td align="left"><p><italic>CCL5</italic></p><p><italic>IFIH1</italic></p><p><italic>IFNB1</italic></p><p><italic>ISG15</italic></p><p><italic>DDX58</italic></p><p><italic>CXCL10</italic></p><p><italic>OAS2</italic></p><p><italic>CXCL8</italic></p><p><italic>MAP3K7</italic></p><p><italic>FOS</italic></p><p><italic>CXCL9</italic></p><p><italic>JUN</italic></p><p><italic>TLR7</italic></p><p><italic>IRF7</italic></p><p><italic>IL12B</italic></p><p><italic>NOD2</italic></p><p><italic>IL6</italic></p><p><italic>IL1B</italic></p><p><italic>PYCARD</italic></p><p><italic>MYD88</italic></p><p><italic>CARD9</italic></p><p><italic>IKBKB</italic></p><p><italic>FADD</italic></p><p><italic>TRADD</italic></p><p><italic>MAVS</italic></p><p><italic>IFNA1</italic></p><p><italic>IRAK1</italic></p><p><italic>CTSB</italic></p><p><italic>PIN1</italic></p><p><italic>TLR8</italic></p><p><italic>IRF5</italic></p><p><italic>MAP2K3</italic></p><p><italic>TICAM1</italic></p></td><td align="left"><p>+53.45</p><p>+16.78</p><p>+17.76</p><p>+8.68</p><p>+14.00</p><p>+10.20</p><p>—–</p><p>+9.80</p><p>-12141.84</p><p>-12.68</p><p>-104.94</p><p>-33.49</p><p>-16.23</p><p>-59.17</p><p>-20.00</p><p>-14.32</p><p>-12.75</p><p>-13.65</p><p>-6.51</p><p>-3.72</p><p>-3.71</p><p>-3.12</p><p>-4.87</p><p>-12.70</p><p>-9.74</p><p>-18.51</p><p>-5.31</p><p>—–</p><p>-5.73</p><p>-3.54</p><p>-4.87</p><p>-5.34</p><p>—–</p></td><td align="left"><p>0.28</p><p>0.03</p><p>0.77</p><p>1.27</p><p>0.25</p><p>0.39</p><p>—–</p><p>1.29</p><p>0.30</p><p>1.25</p><p>0.91</p><p>2.22</p><p>0.01</p><p>0.03</p><p>0.01</p><p>0.01</p><p>0.78</p><p>0.33</p><p>0.56</p><p>0.07</p><p>0.01</p><p>0.19</p><p>1.22</p><p>1.05</p><p>0.07</p><p>0.04</p><p>1.49</p><p>—–</p><p>1.26</p><p>0.01</p><p>0.76</p><p>0.10</p><p>—–</p></td><td char="." align="char"><p>+23.24</p><p>+8.15</p><p>+7.39</p><p>+5.84</p><p>+4.80</p><p>+4.40</p><p>+3.23</p><p>+3.04</p><p>-27496.30</p><p>-22.91</p><p>-54.81</p><p>-51.34</p><p>-33.86</p><p>-27.66</p><p>-41.70</p><p>-18.64</p><p>-37.00</p><p>-30.81</p><p>-9.06</p><p>-4.96</p><p>-3.38</p><p>-5.14</p><p>-4.80</p><p>-7.15</p><p>-10.70</p><p>-24.28</p><p>-3.92</p><p>-7.12</p><p>-4.34</p><p>-7.38</p><p>-4.14</p><p>-10.28</p><p>-3.51</p></td><td char="." align="char"><p>0.27</p><p>0.59</p><p>0.04</p><p>0.51</p><p>0.59</p><p>0.10</p><p>0.32</p><p>0.51</p><p>0.44</p><p>0.60</p><p>0.62</p><p>1.18</p><p>0.01</p><p>0.72</p><p>0.01</p><p>0.42</p><p>0.15</p><p>1.16</p><p>2.71</p><p>0.01</p><p>0.94</p><p>0.53</p><p>1.49</p><p>0.94</p><p>1.07</p><p>0.20</p><p>0.96</p><p>0.30</p><p>0.19</p><p>0.01</p><p>0.11</p><p>0.27</p><p>0.69</p></td><td align="left"><p>+30.00</p><p>+15.23</p><p>+9.16</p><p>+6.87</p><p>+8.20</p><p>+8.03</p><p>+4.67</p><p>+4.68</p><p>-484.06</p><p>-17.63</p><p>-46.57</p><p>-35.35</p><p>-19.42</p><p>-18.05</p><p>-9.86</p><p>-5.00</p><p>-15.16</p><p>-11.86</p><p>-4.77</p><p>-3.33</p><p>-3.85</p><p>-3.57</p><p>—–</p><p>-5.20</p><p>-6.03</p><p>-11.37</p><p>-5.30</p><p>-4.16</p><p>—–</p><p>-6.31</p><p>-5.32</p><p>-8.57</p><p>-7.15</p></td><td align="left"><p>0.22</p><p>0.16</p><p>0.14</p><p>0.70</p><p>0.45</p><p>0.31</p><p>0.63</p><p>0.01</p><p>7.02</p><p>0.91</p><p>3.77</p><p>1.94</p><p>3.91</p><p>1.63</p><p>1.60</p><p>2.60</p><p>2.13</p><p>0.16</p><p>0.34</p><p>0.37</p><p>1.60</p><p>0.07</p><p>—–</p><p>2.32</p><p>1.13</p><p>2.32</p><p>1.13</p><p>0.16</p><p>—–</p><p>0.49</p><p>1.15</p><p>0.10</p><p>1.74</p></td><td align="left"><p>+50.47</p><p>+13.60</p><p>+22.13</p><p>+11.68</p><p>+10.98</p><p>+10.24</p><p>—–</p><p>+8.88</p><p>-16781.69</p><p>-29.61</p><p>-25.86</p><p>-74.12</p><p>-14.61</p><p>-87.48</p><p>-13.24</p><p>-5.40</p><p>-10.19</p><p>-35.01</p><p>-7.36</p><p>—–</p><p>-3.34</p><p>-7.50</p><p>-3.44</p><p>-3.91</p><p>-9.98</p><p>-21.84</p><p>-8.89</p><p>-4.91</p><p>—–</p><p>-3.18</p><p>-5.86</p><p>-4.83</p><p>—–</p></td><td align="left"><p>0.07</p><p>0.36</p><p>0.22</p><p>0.17</p><p>0.42</p><p>0.08</p><p>—–</p><p>0.71</p><p>1.26</p><p>2.67</p><p>0.63</p><p>3.17</p><p>0.01</p><p>3.49</p><p>0.63</p><p>1.78</p><p>0.51</p><p>2.34</p><p>1.43</p><p>—–</p><p>0.01</p><p>2.91</p><p>1.10</p><p>0.83</p><p>1.71</p><p>1.85</p><p>3.91</p><p>0.17</p><p>—–</p><p>0.01</p><p>2.59</p><p>0.42</p><p>—–</p></td><td char="." align="char"><p>+19.74</p><p>+7.47</p><p>+2.96</p><p>+3.85</p><p>+4.07</p><p>+2.91</p><p>+4.52</p><p>+3.61</p><p>-31317.90</p><p>-14.76</p><p>-95.64</p><p>-58.19</p><p>-53.61</p><p>-20.75</p><p>-66.03</p><p>-7.68</p><p>-33.09</p><p>-28.98</p><p>-3.62</p><p>-4.12</p><p>-3.21</p><p>-4.82</p><p>-5.67</p><p>-10.01</p><p>-10.45</p><p>-12.83</p><p>-2.47</p><p>-4.17</p><p>-4.94</p><p>-11.68</p><p>-2.98</p><p>-8.88</p><p>-3.51</p></td></tr></tbody></table><table-wrap-foot><p>Results are shown as mean ± standard deviation (SD) of two independent experiments. Mean ± SD was not available for influenza NS1 as only one experiment was performed. (—–) indicates no significant fold change in one or two experiments</p></table-wrap-foot></table-wrap></p></sec></sec><sec id="Sec12"><title>Discussion</title><p id="Par16">The current study shows for the first time that different genes involved in the activation of antiviral and inflammatory responses, e.g. those representing interleukins, IFNs, MAPKs, PRRs and adaptor proteins, are downregulated in the presence of HCoV-OC43 structural or accessory proteins. Similar antiviral gene expression profiles were observed between influenza NS1 protein, which is known to antagonise type I IFN activity [<xref ref-type="bibr" rid="CR14">14</xref>, <xref ref-type="bibr" rid="CR26">26</xref>–<xref ref-type="bibr" rid="CR28">28</xref>], and HCoV-OC43 proteins. Interestingly, some genes such as <italic>IFNA1</italic> and <italic>IRF7</italic> were more downregulated in the presence of HCoV-OC43 proteins than in the presence of influenza NS1 protein.</p><p id="Par17">Accessory proteins are often not essential for virus replication <italic>in vitro</italic>, but required for optimal replication and virulence in the natural host [<xref ref-type="bibr" rid="CR29">29</xref>]. HCoV-OC43 ns5a accessory protein is known to be a viroporin involved in virion morphogenesis and pathogenesis [<xref ref-type="bibr" rid="CR30">30</xref>]. In its absence, there is a reduction of virulence, inflammation and replication in infected mice [<xref ref-type="bibr" rid="CR30">30</xref>]. Murine coronavirus accessory protein 5a is homologous to ns5a protein, and it has been found to antagonise IFN induction [<xref ref-type="bibr" rid="CR31">31</xref>]. Our results showed that the expression of PRRs, <italic>IFNA1</italic>, <italic>IRF7</italic>, <italic>MYD88</italic>, and <italic>MAVS</italic>, all important for the induction of an antiviral response, was downregulated in the presence of HCoV-OC43 accessory proteins ns2a and ns5a. Moreover, the expression of interleukins, chemokines, <italic>FOS</italic>, <italic>JUN</italic>, IRFs, and <italic>IFNA1</italic> was downregulated in the presence of ns5a protein. FOS and JUN are transcription factors involved in the induction of inflammatory cytokines [<xref ref-type="bibr" rid="CR32">32</xref>, <xref ref-type="bibr" rid="CR33">33</xref>]. Altogether, our results suggest HCoV-OC43 ns2a and ns5a have the potential to induce a shift towards an anti-inflammatory response and blockage of the type I IFN pathway. However, <italic>in vivo</italic> studies are required to confirm the type of immune response HCoV-OC43 elicits during infection. In the absence of adequate immune responses, the virus will be able to replicate and spread freely.</p><p id="Par18">The M protein is indispensable in virion assembly between the Golgi apparatus and endoplasmic reticulum, and is the most abundant protein among the coronavirus proteins [<xref ref-type="bibr" rid="CR34">34</xref>]. SARS-CoV M protein suppresses the activation of NF-κB [<xref ref-type="bibr" rid="CR13">13</xref>]. It can also halt IFN production by blocking the formation of the TRAF3 complex [<xref ref-type="bibr" rid="CR15">15</xref>]. MERS-CoV M protein is also a potent IFN antagonist, targeting the TRAF3-TBK1 interaction to inhibit phosphorylation of IRF3 [<xref ref-type="bibr" rid="CR17">17</xref>, <xref ref-type="bibr" rid="CR35">35</xref>]. Moreover, TRAF6, TRADD and FADD are key players in the NF-κB and apoptosis pathways [<xref ref-type="bibr" rid="CR36">36</xref>]. Our results showed that the expression of <italic>TRAF6</italic> was downregulated in the presence of M or other tested HCoV-OC43 proteins, but this downregulation could not be confirmed after stimulation with Sendai virus. In contrast, the downregulation of <italic>TRADD</italic> was only observed after challenge with Sendai virus. However, the expression of <italic>FADD</italic> was not downregulated in the presence of HCoV-OC43M protein before or after challenge with Sendai virus. Furthermore, the expression of <italic>MAVS</italic>, <italic>MYD88</italic>, <italic>IRFs</italic> and <italic>IFNA1</italic> that are essential in the induction of the type I IFN signaling pathway, was downregulated in the presence of M protein. This suggests that HCoV-OC43M protein has similar downregulatory effects on the induction of type I IFN and NF-κB as SARS-CoV and MERS-CoV M protein. Further studies are needed to confirm the effects of HCoV-OC43M protein on the type I IFN and NF-κB signaling pathways.</p><p id="Par19">The function of the N protein is to ensure the replication and transcription of viral RNA while disrupting the cell cycle [<xref ref-type="bibr" rid="CR37">37</xref>, <xref ref-type="bibr" rid="CR38">38</xref>]. Indeed, SARS-CoV N protein inhibits the progression of the cell cycle and activates the proinflammatory factor cyclooxygenase-2 [<xref ref-type="bibr" rid="CR39">39</xref>]. Moreover, it was shown that SARS-CoV N protein inhibits <italic>IFN-β</italic> expression [<xref ref-type="bibr" rid="CR14">14</xref>], while it activates <italic>IL-6</italic> expression [<xref ref-type="bibr" rid="CR13">13</xref>]. HCoV-OC43N protein potentiates NF-κB by binding its inhibitor, microRNA 9 [<xref ref-type="bibr" rid="CR22">22</xref>]. Our study did not show significant fold changes in NF-κB expression in the presence of N protein; however there was a significant downregulation in expression of PRRs (<italic>NOD2</italic> and <italic>TLR7</italic>), adaptor proteins (<italic>MYD88, MAVS, CARD9</italic>) and signal mediators (<italic>IKBKB, IRAK1, TBK1, RIPK1, TRADD, TRAF</italic>), all critical in the induction of NF-κB signaling pathway. Moreover, our results showed that expression of MAPKs (<italic>MAP3K7</italic> and <italic>MAP2K3</italic>) was downregulated in the presence of N protein, which is in line with a previous study showing that murine coronavirus N protein inhibits signaling of the AP-1 complex [<xref ref-type="bibr" rid="CR40">40</xref>], which is composed of FOS and JUN oncogenes, whose expression was also downregulated in our investigation - in the presence of HCoV-OC43 ns2a, ns5a, N or M proteins after challenge with Sendai virus.</p><p id="Par20"><italic>MAP3K7</italic>, also called <italic>Transforming growth factor-b (TGF-b)-activated kinase 1</italic> (<italic>TAK1</italic>), was the most downregulated gene in the presence of any HCoV-OC43 protein tested after challenging cells with Sendai virus. TAK1 is a protein kinase known to be a critical mediator of the inflammatory response as it is activated by lipopolysaccharide (LPS) and other TLR/NLR agonists, TNF-α and IL-1 [<xref ref-type="bibr" rid="CR41">41</xref>]. Binding of IL-1 and LPS to their receptors activates TAK1 through a common pathway whereby the kinases IRAK1 and IRAK4 recruit TRAF6. TNF-α activates TAK1 via a similar mechanism, which is mediated by RIP1 and TRAF2. Activation of TRAF2 and TRAF6 leads to the generation of K63-linked polyubiquitin chains, which bind to the adaptor proteins TAB<xref rid="Tab2" ref-type="table">2</xref> or TAB<xref rid="Tab3" ref-type="table">3</xref>. TAK1 is bound constitutively to the accessory protein TAB<xref rid="Tab1" ref-type="table">1</xref> and to the homologs TAB<xref rid="Tab2" ref-type="table">2</xref> or TAB<xref rid="Tab3" ref-type="table">3</xref>. Once activated, TAK1 transduces the signal to NF-κB, c-Jun N-terminal kinase (JNK), and p38 via phosphorylation of IKK, mitogen-activated protein kinase kinase 4/7 (MKK4/7), and MKK3/6, respectively. Ultimately, NF-κB and other transcription factors downstream of p38 and JNK are activated, resulting in the transcription of genes important for inflammatory and immune responses. Interestingly, Protein kinase R (PKR) interacts with TRAF6, TAK1, and TAB<xref rid="Tab2" ref-type="table">2</xref> as a scaffold protein to mediate TLR3-dependent NF-κB and MAPK activation [<xref ref-type="bibr" rid="CR42">42</xref>, <xref ref-type="bibr" rid="CR43">43</xref>]. The strong downregulation of TAK1 expression observed in our study could be due to: 1) the inhibition of TAK1 expression by direct interaction of HCoV-OC43 protein with TAK1 preventing binding of other mediators like TRAF2/6 or TAB2/3; 2) the upregulation of expression of negative regulators of TAK1 like PP2C or PP6 phosphatases, and deubiquitinases such as ubiquitin-specific peptidase 4 (USP4) [<xref ref-type="bibr" rid="CR41">41</xref>].</p><p id="Par21">In certain cell lines, IFITM proteins were shown to facilitate HCoV-OC43 entry into host cells [<xref ref-type="bibr" rid="CR23">23</xref>]. Our study showed that all tested HCoV-OC43 proteins were able to downregulate the expression of IFNA1, suggesting resistance to IFN-α induction. The interaction between HCoV-OC43 proteins and the host cell requires further investigation to understand the processes that could contribute to overall resistance to the antiviral state. Having all these HCoV-OC43 proteins with potential inhibitory effects expressed in close proximity in infected cells indicates a high replicative capacity of the virus.</p><p id="Par22">In conclusion, our findings suggest that similarly to SARS-CoV and MERS-CoV, HCoV-OC43 has the ability to downregulate the transcription of genes critical for the activation of different antiviral signaling pathways. While SARS-CoV and MERS-CoV were shown to induce proinflammatory cytokines <italic>in vitro</italic> [<xref ref-type="bibr" rid="CR44">44</xref>, <xref ref-type="bibr" rid="CR45">45</xref>], HCoV-OC43 accessory and structural proteins have the potential to inhibit inflammatory response in HEK-293 cells. Further studies are in progress to confirm the role of HCoV-OC43 structural and accessory proteins in antagonising antiviral gene expression, and to elucidate the nature of the immune response and the level of inflammation seen during HCoV-OC43 infection.
</p></sec></body><back><notes notes-type="ethics"><title>Compliance with ethical standards</title><notes notes-type="funding"><title>Funding</title><p id="Par23">This work was supported by Kuwait University Research Administration Grant no. 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