Original Paper Intervirology Effect of Human Coronavirus OC43 Structural and Accessory Proteins on the Transcriptional Activation of Antiviral Response Elements Meshal Beidas Wassim Chehadeh Department of Microbiology Faculty of Medicine Kuwait University Jabriya Kuwait Received January 29 2018 Accepted June 3 2018 Published online July 24 2018 Dr Wassim Chehadeh Department of Microbiology Faculty of Medicine Kuwait University PO Box 24923 Safat 13310 Kuwait E Mail wchehadeh hsc edu kw 2018 S Karger AG Basel E Mail karger DOI 10 1159 000490566 Keywords Coronavirus OC43 Antiviral response elements Luciferase reporter gene assay Interferon antagonism Abstract Objectives The molecular mechanisms underlying the pathogenesis of human coronavirus OC43 HCoV OC43 in fection are poorly understood In this study we investigated the ability of HCoV OC43 to antagonize the transcriptional activation of antiviral response elements Methods HCoV OC43 structural membrane M and nucleocapsid N and ac cessory proteins ns2a and ns5a were expressed individu ally in human embryonic kidney 293 HEK 293 cells The transcriptional activation of antiviral response elements was assessed by measuring the levels of firefly luciferase ex pressed under the control of interferon IFN stimulated re sponse element ISRE IFN promoter or nuclear factor kappa B response element NF B RE The antiviral gene ex pression profile in HEK 293 cells was determined by PCR ar ray Results The transcriptional activity of ISRE IFN pro moter and NF B RE was significantly reduced in the pres ence of HCoV OC43 ns2a ns5a M or N protein following the challenge of cells with Sendai virus IFN or tumor necrosis factor The expression of antiviral genes involved in the type I IFN and NF B signaling pathways was also downregu lated in the presence of HCoV OC43 structural or accessory proteins Conclusion Both structural and accessory HCoV OC43 proteins are able to inhibit antiviral response elements in HEK 293 cells and to block the activation of different an tiviral signaling pathways 2018 S Karger AG Basel Introduction Human coronavirus OC43 HCoV OC43 is an envel oped positive sense RNA virus classified as a Betacoro navirus the same genus as severe acute respiratory syn drome SARS and Middle East respiratory syndrome MERS coronaviruses HCoV OC43 infection has been associated mainly with upper respiratory tract symptoms and exacerbation of asthma and pneumonia in some groups and institutional settings 1 4 The virus has also been associated with severe neurological disorders like acute disseminated encephalomyelitis in children 5 6 While most studies have focused their attention on the immunopathology of SARS CoV and MERS CoV there has not been the same interest for HCoV OC43 Indeed there are few studies concerned with the molecular mech Downloaded by G teborgs Universitet 130 241 16 16 7 26 2018 4 07 27 AM Beidas Chehadeh Intervirology 2 DOI 10 1159 000490566 anisms governing HCoV OC43 infection and its effect on the intracellular host defenses HCoV OC43 is over 30 kb in length and similarly to the other coronaviruses it is composed of the structural proteins spike S envelope E membrane M and nucleocapsid N 7 Uniquely there are two accessory proteins interspaced between these structural proteins called ns2a and ns5a 8 These proteins are not essential for replication however they might play a role in the pathogenesis of coronavirus infec tion 9 The interferon IFN induction and signaling path way is the major branch in the innate immune response against viruses The induction of type I IFN is initiated by molecular sensing of viral RNA by pattern recogni tion receptors such as TLR RIG I and MDA5 10 The adaptor protein MAVS mediates the signals from RIG I and MDA5 to activate the kinases TBK1 and IKK which in turn phosphorylate IRF3 and IRF7 transcrip tion factors 11 IRF3 and IRF7 dimerize to undergo nuclear translocation and initiate the transcription of type I IFN The adaptor proteins MYD88 and TRIF me diate signals from TLRs and activate IRAK1 TRAF6 and the aforementioned kinases 12 The IRFs along with nuclear factor kappa B NF B can then be phos phorylated to translocate into the nucleus and establish the transcription of type I IFN by binding cognate sites on the IFN promoter 12 NF B specifically binds to the NF B response element NF B RE to regulate the expression of pro inflammatory and cell survival genes 13 IFN signaling via the JAK STAT pathway is estab lished when type I IFN binds to the IFN receptor This leads to activation of the kinases JAK1 and TYK2 These kinases in turn phosphorylate the transcription factors STAT1 and STAT2 14 15 Phosphorylated STAT1 and STAT2 then dimerize and undergo nuclear translocation where they form a complex with IRF9 called the ISGF3 This complex will bind the IFN stimulated response ele ment ISRE and initiate the transcription of ISGs These ISGs assist the cell in counteracting the viral infection by establishing an antiviral state 16 SARS CoV and MERS CoV were shown to inhibit the transcriptional activity of ISRE IFN promoter or NF B RE 17 24 However the effect of different HCoV OC43 proteins on the transcriptional activation of antivi ral response elements has not yet been investigated In this study the ability of HCoV OC43 structural M and N and accessory proteins ns2a and ns5a to antagonize the transcriptional activation of ISRE IFN promoter and NF B RE was investigated Materials and Methods Cell Culture Human embryonic kidney 293 HEK 293 cells American Type Culture Collection ATCC Manassas VA USA were cul tured in 25 cm 2 tissue culture flasks in Dulbecco s minimal essen tial medium containing GlutaMAX TM Life Technologies Corpo ration TM Grand Island NY USA The medium was supplement ed with 10 fetal bovine serum Fungizone null 250 g mL penicillin G 10 000 U mL and streptomycin sulfate 10 000 pg mL Life Technologies TM Monolayers of HEK 293 cell culture flasks were incubated at 37 C in the presence of 5 carbon dioxide CO 2 and 90 humidity Amplification of HCoV OC43 ns2a ns5a M and N Genes by Reverse Transcription Polymerase Chain Reaction The QIAamp null Viral RNA Minikit Qiagen TM GmbH Hilden Germany was used to isolate RNA from HCoV OC43 ATCC VR 1558 according to the manufacturer s instructions The HCoV OC43 ns2a ns5a M and N genes were amplified by a two step reverse transcription polymerase chain reaction RT PCR using the GeneAmp null RNA Core Kit Applied Biosystems TM Fos ter City CA USA and 10 pmol of previously described primers 8 on GeneAmp null PCR System 9700 Applied Biosystems TM The RT reaction conditions were annealing at 37 C for 60 min dena turation at 90 C for 5 min and cooling at 4 C Thereafter the cDNA was amplified by PCR using the following conditions de naturation at 94 C for 10 min then 35 cycles of denaturation at 95 C for 30 s annealing at 60 C for 30 s and extension at 72 C for 30 s followed by a final extension step at 72 C for 7 min and cooling at 4 C PCR products were run on agarose gel and the bands that were size specific for the amplified gene of interest were cut and purified by Wizard null SV Gel and PCR Clean Up Sys tem Promega TM Madison WI USA according to the manufac turer s instructions RNA of Influenza A virus H3N2 A Hong Kong 8 68 strain ATCC VR 544 was also isolated and used to amplify NS1 gene by RT PCR using NS1 specific primers 25 In fluenza A NS1 protein was used in the following experiments as inhibitor of the innate immune host response 17 26 Cloning and Expression of HCoV OC43 and Influenza A Genes in HEK 293 Cells RT PCR products were cloned using the pAcGFP1 N In Fu sion null Ready Vector Clontech TM Takara Bio Company Mountain View CA USA that encodes a green fluorescent protein GFP from Aequorea coerulescens allowing the expression of the fusion protein to the N terminus of AcGFP1 The ligation reaction was set up by utilizing the In Fusion null HD Cloning Kit Clontech TM Competent TOP10 Escherichia coli cells Invitrogen TM Carlsbad CA USA were used for transformation The PureYield TM Plasmid Miniprep Kit Promega TM was used to isolate the vector according to the manufacturer s instructions Confirmation of successful cloning was achieved using restriction digestion and direct se quencing data not shown HEK 293 cells were seeded at 5 10 5 per well of 96 well plates and transfected with ns2a pAcGFP1 ns5a pAcGFP1 M pAcGFP1 N pAcGFP1 or NS1 pAcGFP1 vector using Lipofectamine null 2000 Promega TM according to the manufacturer s instructions Expression of ns2a ns5a M N and NS1 proteins was confirmed by indirect immunofluorescence as say using anti GFP monoclonal antibody 10 g mL Clone JL 8 Downloaded by G teborgs Universitet 130 241 16 16 7 26 2018 4 07 27 AM Coronavirus OC43 Inhibition of Antiviral Response Elements 3 Intervirology DOI 10 1159 000490566 Clontech TM data not shown Expression of N and NS1 proteins was further confirmed by immunofluorescence assay using mono clonal antibody against HCoV OC43 N EMD Millipore TM Bill erica MA USA and polyclonal antibody against influenza A NS1 protein EMD Millipore TM respectively data not shown Transcriptional Activation of Antiviral Response Elements in the Presence of HCoV OC43 and Influenza A Proteins The IFN promoter was amplified using previously described primers 27 and inserted into the reporter pGL4 19 luc2CP Ne vector Promega TM between the restriction sites BglII and HindIII according to the manufacturer s instructions creating the pIFN luc plasmid The reporter vectors coding for firefly luciferase un der the control of ISRE pISRE luc pGL4 45 luc2P ISRE Hygro or NF B RE pNF B RE luc pGL4 32 luc2P NF B RE Hy gro were purchased from Promega TM Following 48 h of transfec tion with one of the expression vectors cited above HEK 293 cells were co transfected with pIFN luc pISRE luc or pNF B RE luc vector at 0 1 g well using Lipofectamine null 2000 Promega TM Re nilla luciferase vector pRL TK Promega TM was used at 10 ng well as an internal control for transfection efficiency Following 24 h of incubation the cells were stimulated for 6 or 24 h with IFN PBL Assay Science TM Piscataway NJ USA at 5 10 4 units well Sendai virus SeV ATCC VR 105 at multiple of infection 1 or tumor necrosis factor TNF Life Technologies TM at 0 1 g mL Thereafter promoter activity was assayed using the Dual Luciferase null Reporter Assay System Promega TM and the GloMax null Multi Jr luminometer Promega TM according to the manufactur er s instructions Firefly luciferase levels were determined in rela tive light units and normalized against Renilla luciferase levels Antiviral Gene Expression Profile in Cells Expressing HCoV OC43 ns2a ns5a M and N Protein Total RNA from transfected and mock transfected HEK 293 cells was extracted using RNeasy null Kit Qiagen TM with on column DNase digestion according to the manufacturer s instructions The RT 2 First Strand Kit Qiagen TM was used for cDNA synthesis The RT reaction was then added to RT 2 SYBR Green ROX qPCR Mastermix Qiagen TM and the Human Antiviral Response PCR array Qiagen TM was used to profile the antiviral gene expression in HEK 293 cells on ABI 7500 Fast Real Time PCR system Ap plied Biosystems TM The PCR array monitored the transcriptional activity of different genes involved in the activation of antiviral and pro inflammatory proteins following amplification of RNA tran scripts by real time RT PCR However we only profiled the ex pression of genes relevant to the antiviral response elements Cycle threshold C T values were exported to an Excel file to create a table of C T values This table was then uploaded on to the Qiagen data analysis web portal C T values were normalized to glyceraldehyde 3 phosphate dehydro genase Fold regulation comparison was used to profile antiviral gene expression Fold change regulation was calculated using the delta delta C T method C T The fold regulation threshold was 2 for upregulation and 2 for downregulation Statistical Analysis Fold change in firefly luciferase from three different experi ments was summarized as mean standard deviation The differ ence in fold change mean between two groups was determined by independent samples t test p values 0 05 were considered sig nificant All statistical analyses were performed using SPSS statis tics software version 25 0 for windows IBM Corporation Ar monk NY USA GraphPad Prism 7 software GraphPad Soft ware Inc La Jolla CA USA was used to generate all charts Results Effect of HCoV OC43 proteins on the transcriptional activation of antiviral response elements The transcriptional activity of ISRE IFN promoter and NF B RE was assessed by measuring the firefly lu ciferase levels in HEK 293 cells transfected with ns2a pAcGFP1 ns5a pAcGFP1 M pAcGFP1 N pAcGFP1 or NS1 pAcGFP1 vector In control cells nonexpressing viral protein the firefly luciferase levels were highest fol lowing 6 h of stimulation with one of the inducers data Firefly luciferase RL U 1 5 10 7 1 10 7 5 10 6 0 Mock ns2a ns5a Ma ISRE No inducer IFN N NS1 Firefly luciferase RL U 4 10 6 3 10 6 1 10 6 2 10 6 0 Mock ns2a ns5a Mb ISRE No inducer SeV N NS1 Fig 1 Mean fold change in firefly luciferase levels under the control of ISRE in IFN treated a and SeV treated b HEK 293 cells expressing HCoV OC43 structural M or N or accessory ns2a or ns5a protein Results are shown as the mean standard deviation SD of three independent experiments Firefly luciferase activity measurements were normalized against Renilla luciferase levels Downloaded by G teborgs Universitet 130 241 16 16 7 26 2018 4 07 27 AM Beidas Chehadeh Intervirology 4 DOI 10 1159 000490566 not shown In the absence of an inducer the firefly lucif erase levels in HEK 293 cells expressing one HCoV OC43 protein were similar to the background levels Fig 1a b In IFN treated HEK 293 cells the expres sion of firefly luciferase under the control of ISRE or IFN promoter was inhibited in the presence of one of the tested HCoV OC43 proteins or in the presence of in fluenza A NS1 protein Fig 1a 2 The transcriptional ac tivity of ISRE was also inhibited in SeV stimulated HEK 293 cells in the presence of one HCoV OC43 protein Fig 1b In mock transfected HEK 293 cells SeV in duced only low levels of firefly luciferase under the con trol of IFN promoter data not shown and therefore the effect of HCoV OC43 proteins on the transcriptional activity of IFN promoter in SeV stimulated HEK 293 cells could not be assessed Following the challenge with TNF the transcriptional activity of NF B RE was in hibited in the presence of one of the tested HCoV OC43 proteins or in the presence of influenza A NS1 protein Fig 3 PCR Array Profiling of Antiviral Genes in HEK 293 Cells Expressing HCoV OC43 Structural and Accessory Proteins To investigate whether the inhibition of the transcrip tional activity of antiviral response elements in the pres ence of HCoV OC43 proteins can be associated with a downregulation of the expression of antiviral genes PCR array profiling of antiviral genes was carried out in trans fected and mock transfected HEK 293 cells Following SeV challenge of HEK 293 cells the expression of genes involved in the type I IFN and NF B signaling pathways was downregulated in the presence of HCoV OC43 struc tural or accessory proteins Fig 4 Similar results were obtained in the presence of the influenza A NS1 protein The mean negative fold change for TRADD gene in the presence of accessory protein ns2a was greater than in the presence of other HCoV OC43 proteins p 0 05 The expression of IRF7 gene was more downregulated in the presence of ns2a or N protein than in the presence of oth er HCoV OC43 proteins p 0 001 whereas the expres sion of TLR7 gene was more downregulated in the pres ence of ns5a protein than in the presence of other HCoV OC43 proteins p 0 01 Remarkably IRF7 and IFNA1 gene expression was more downregulated in the presence of ns2a ns5a or N protein than in the presence of influ enza A NS1 protein Discussion Similar to influenza A NS1 protein HCoV OC43 structural M and N and accessory ns2a and ns5a pro teins were able to inhibit the transcriptional activity of antiviral response elements ISRE IFN promoter and NF B RE and to downregulate the expression of sev eral genes involved in the activation of an antiviral re sponse Firefly luciferase RL U 3 10 4 2 10 4 1 10 4 0 Mock ns2a ns5a M IFN promoter No inducer IFN N NS1 Firefly luciferase RL U 4 10 8 3 10 8 1 10 8 2 10 8 0 Mock ns2a ns5a M NF B RE No inducer TNF N NS1 Fig 2 Mean fold change in firefly luciferase levels under the con trol of IFN promoter in IFN treated HEK 293 cells expressing HCoV OC43 structural M or N or accessory ns2a or ns5a pro tein Results are shown as the mean standard deviation SD of three independent experiments Firefly luciferase activity mea surements were normalized against Renilla luciferase levels Fig 3 Mean fold change in firefly luciferase under the control of NF B RE in TNF treated HEK 293 cells expressing HCoV OC43 structural M or N or accessory ns2a or ns5a protein Re sults are shown as the mean standard deviation SD of three independent experiments Firefly luciferase activity measurements were normalized against Renilla luciferase levels Downloaded by G teborgs Universitet 130 241 16 16 7 26 2018 4 07 27 AM Coronavirus OC43 Inhibition of Antiviral Response Elements 5 Intervirology DOI 10 1159 000490566 The inhibition of the ISRE IFN promoter and NF B RE activity and the downregulation of the expression of IFNA1 IRF7 TLR7 MYD88 TRADD and MAVS in the presence of each HCoV OC43 accessory protein sug gest that HCoV OC43 ns2a and ns5a have the potential to block the type I IFN and NF B pathways Previous studies have shown that SARS CoV and MERS CoV accessory proteins have a role in innate immune evasion 9 17 20 22 However HCoV OC43 accessory proteins are not ho mologous to SARS CoV and MERS CoV accessory pro teins Also of interest the murine coronavirus accessory protein 5a which is homologous to HCoV OC43 ns5a protein was shown to antagonize IFN induction 28 SARS CoV and MERS CoV M proteins can suppress the activity of IFN promoter and ISRE 18 19 22 NF B activation is also inhibited when SARS CoV M protein is expressed in Vero or HeLa cells 23 Our data showed that the transcriptional activity of ISRE IFN promoter and NF B RE was inhibited in the presence of HCoV OC43 M protein In addition like the accessory proteins the HCoV OC43 M protein was able to downregulate the expression of IFNA1 IRF7 TLR7 MYD88 TRADD and MAVS These findings suggest that in addition to its func tion in the assembly of virions 29 HCoV OC43 M pro tein is an antagonist of the host antiviral defenses The coronavirus N protein binds genomic RNA to form the helical capsid and has a critical role in coronavi rus replication 30 31 The SARS CoV N protein can ac tivate NF B RE in Vero E6 cells 32 However SARS CoV N protein inhibits the promoter activity of ISRE IFN promoter and NF B RE in 293T cells 17 Our study showed that the transcriptional activity of ISRE IFN promoter and NF B RE was inhibited in the pres ence of HCoV OC43 N protein Lai et al 33 showed that after 24 h of TNF treatment HCoV OC43 N protein potentiates NF B expression in 293T cells by binding its inhibitor miRNA 9 miRNA 9 expression is induced by stimuli which activate NF B such as TNF and lipo polysaccharide 34 Unfortunately the authors did not show results for NF B RE activation after 6 h of TNF treatment the optimal time for NF B RE activation in our experiments The influenza A virus that is known to inhibit IRF3 AP 1 and NF B activation 35 36 was shown to upregulate the miRNA 9 expression 37 We speculate that the expression of N protein results in tran sient downregulation of NF B expression through down regulation of its mediators e g TLR7 MYD88 TRADD leading to early inhibition of NF B RE activation and that the accumulation of miRNA9 in cells with time will lead to the formation of N protein miRNA 9 complex and the remov