Severe acute respiratory syndrome coronavirus papain like protease suppressed alpha interferon induced responses through downregulation of extracellular signal regulated kinase 1 mediated signalling pathways Shih Wein Li 1 2 Chien Chen Lai 2 3 Jia Fong Ping 1 Fuu Jen Tsai 3 Lei Wan 3 Ying Ju Lin 3 Szu Hao Kung 4 and Cheng Wen Lin 1 5 6 Correspondence Cheng Wen Lin cwlin mail cmu edu tw Chien Chen Lai lailai dragon nchu edu tw Received 15 November 2010 Accepted 21 January 2011 1 Department of Medical Laboratory Science and Biotechnology China Medical University Taichung Taiwan ROC 2 Institute of Molecular Biology National Chung Hsing University Taichung Taiwan ROC 3 Department of Medical Genetics and Medical Research China Medical University Hospital Taichung Taiwan ROC 4 Department of Biotechnology and Laboratory Science in Medicine National Yang Ming University Taipei Taiwan ROC 5 Clinical Virology Laboratory Department of Laboratory Medicine China Medical University Hospital Taichung Taiwan ROC 6 Department of Biotechnology Asia University Wufeng Taichung Taiwan ROC Severe acute respiratory syndrome coronavirus SARS CoV papain like protease PLpro a deubiquitinating enzyme reportedly blocks poly I C induced activation of interferon regulatory factor 3 and nuclear factor kappa B reducing interferon IFN induction This study investigated type I IFN antagonist mechanism of PLpro in human promonocytes PLpro antagonized IFN a induced responses such as interferon stimulated response element and AP 1 driven promoter activation protein kinase R 29 59 oligoadenylate synthetase OAS interleukin IL 6 and IL 8 expression and signal transducers and activators of transcription STAT 1 Tyr701 STAT1 Ser727 and c Jun phosphorylation A proteomics approach demonstrated downregulation of extracellular signal regulated kinase ERK 1 and upregulation of ubiquitin conjugating enzyme UBC E2 25k as inhibitory mechanism of PLpro on IFN a induced responses IFN a treatment significantly induced mRNA expression of UBC E2 25k but not ERK1 causing time dependent decrease of ERK1 but not ERK2 in PLpro expressing cells Poly ubiquitination of ERK1 showed a relationship between ERK1 and ubiquitin proteasome signalling pathways associated with IFN antagonism by PLpro Combination treatment of IFN a and the proteasome inhibitor MG 132 showed a time dependent restoration of ERK1 protein levels and significant increase of ERK1 STAT1 and c Jun phosphorylation in PLpro expressing cells Importantly PD098059 an ERK1 2 inhibitor treatment significantly reduced IFN a induced ERK1 and STAT1 phosphorylation inhibiting IFN a induced expression of 29 59 OAS in vector control cells and PLpro expressing cells Overall results proved downregulation of ERK1 by ubiquitin proteasomes and suppression of interaction between ERK1 and STAT1 as type I IFN antagonist function of SARS CoV PLpro INTRODUCTION Severe acute respiratory syndrome SARS associated coronavirus SARS CoV is a novel pandemic virus causing highly contagious respiratory disease with approximately a 10 mortality rate Hsueh et al 2004 Lee et al 2003 Tsang et al 2003 Pathology entails bronchial epithelial denudation loss of cilia multinucleated syncytial cells squamous metaplasia and transendothelial migration of monocytes macrophages and neutrophils into lung tissue Hsueh et al 2004 Nicholls et al 2003 Haematological examination reveals lymphopenia thrombocytopenia andSupplementary figures are available with the online version of this paper Journal of General Virology 2011 92 1127 1140 DOI 10 1099 vir 0 028936 0 028936 G 2011 SGM Printed in Great Britain 1127 leukopenia Wang et al 2004b Yan et al 2004 accompanied by rapid elevation in serum of inflammatory cytokines like gamma interferon IFN c interleukin IL 18 transforming growth factor beta IL 6 IFN gamma inducible protein 10 monocyte chemoattractant protein 1 MCP 1 monokine induced by IFN gamma and IL 8 which stimulate recruitment of neutrophils monocytes and immune responder cells like natural killer NK T and B cells into the lungs and other organs He et al 2006 Huang et al 2005 Wong et al 2004 SARS CoV genome is a 30 kb positive stranded RNA with a 59 cap and a 39 poly A tract that contains 14 ORFs Marra et al 2003 Rota et al 2003 Ziebuhr 2004 The 59 proximal and largest of these ORFs encodes two large overlapping replicase polyproteins 1a and 1ab 450 and 750 kDa respectively processed to produce non structural proteins nsps primarily involved in RNA replication Two specific embedded proteases papain like PLpro and 3C like 3CLpro mediate processing of 1a and 1ab precursors into 16 nsps termed nsp1 16 PLpro located within nsp3 cleaves at nsp1 2 nsp2 3 and nsp3 4 boundaries using consensus motif LXGG Barretto et al 2005 Lindner et al 2005 Thiel et al 2003 along with consensus cleavage sequence of cellular deubiquitinat ing enzymes Modelling and crystal structures reveal correlation between SARS CoV PLpro and the herpes virus associated ubiquitin specific protease indicating potential deubiquitinating activity Ratia et al 2006 Sulea et al 2005 observed in in vitro cleavage assays Barretto et al 2005 Lindner et al 2005 Interestingly one such in vitro deubiquitination assay measured the cleavage of ubiquitin like protein interferon IFN induced 15 kDa protein ISG15 from an ISG15 fusion protein suggesting de ISGylation by PLpro as a mech anism by which SARS CoV inactivates IFN a b induced innate immune response SARS CoV infection does not induce type I IFNs in cell culture Spiegel et al 2005 Recent reports reveal PLpro inhibiting the phosphorylation of interferon regulatory factor 3 IRF 3 and type I IFN synthesis Devaraj et al 2007 and antagonizing both IRF 3 and nuclear factor kappa B NF kB signalling pathways Frieman et al 2009 Still the mechanisms of type I IFN antagonism by which SARS CoV PLpro does this remain unclear Type I interferons IFNs IFN a IFN b and IFN v mediatea wide range of biological activities antiviral activity immune response differentiation cell growth and apoptosis Biron 2001 IFN a b binds to a common heterodimeric receptor composed of IFN a b receptor 1 IFNAR1 and IFN a b receptor 2 IFNAR2 then activates Janus kinase JAK family plus signal transdu cers and activators of transcription STATs family Tang et al 2007 Phosphorylation of STAT1 at tyrosine 701 by JAK1 is required for STAT1 STAT2 heterodimer formation and nuclear translocation Banninger Uddin et al 2002 Currently IFN a is also a widely used cytokine for treating human solid and haematologic malignancies Tagliaferri et al 2005 IFN a mediated anti tumour effect correlates with activation of JAK STAT signalling pathway resulting in upregula tion of Fas FasL and Jnk1 p38 stimulation signalling pathways Escape mechanisms of IFN a mediated anti tumour effect are likewise reported e g EGF mediated Ras Raf ERK1 2 dependent pathway Akt and NF kB dependent pathways and STAT3 PI3K mediated signal ling Tagliaferri et al 2005 Some key regulators of signal transduction e g JAK1 STAT1 ERK1 are de monstrably modified by ubiquitin conjugation Mala khov et al 2003 Lu Zhao et al 2005 e g NF kB inducing kinase critical regulator of non canonical NF kB pathway is ubiquitinated and degraded by RING finger E3 ligases Varfolomeev et al 2007 With SARS CoV PLpro as a deubiquitinat ing enzyme this points to specifically disrupting signal transduction of innate immune system against SARS CoV infection Investigating possible effect of PLpro on the responses to type I IFNs is vital to the understanding of SARS pathogenesis This study first demonstrated stable expression of SARS CoV PLpro significantly inhibited IFN a induced responses like interferon stimulated re sponse element ISRE and AP 1 driven promoter activation gene expression of PKR 29 59 OAS IL 6 and IL 8 and phosphorylation of STAT1 and c Jun Downregulation of ERK1 was identified by comparative proteomic analysis of PLpro expressing cells against control cells with respect to IFN a response correlating with potential antagonistic mechanism of SARS CoV PLpro in response to IFN a RESULTS Expression of the SARS CoV PLpro in human promonocytes To characterize the effect of SARS CoV PLpro on the intracellular innate immune response human promono cyte HL CZ human promonocyte cell line cells were co transfected with the plasmid pSARS CoV PLpro expres sing PLpro with herpes simplex virus HSV epitope tag or empty control vector and GFP reporter plasmid followed by 2 weeks of treatment with G418 to select stably S W Li and others 1128 Journal of General Virology 92 transfected cells Expression of PLpro was detected by immunofluorescent staining Fig 1a and Western blotting Fig 1b with vector derived His tag found in both empty vector and pSARS CoV PLpro transfected cells and HSV tag detected only in pSARS CoV PLpro transfected cells Western blotting of transfected cells lysates with anti HSV tag antibodies revealed a 60 kDa band in pSARS CoV PLpro transfected cells Fig 1b but not in empty vector transfected cells To determine if expressed PLpro was active proteolytic activity in cell lysates was assayed by in vitro trans cleavage with HRP containing the LXGG motif recognized by PLpro as substrate Fig 1 c shows significant reduction in HRP enzyme activity in the reaction containing lysates of PLpro expressing cells but not in the reaction with lysates from vector control cells Lysates of PLpro expressing cells also exhibited time dependent trans cleavage activity SARS CoV PLpro expressed in human promonocyte cells was thus enzymically active Inhibition of PLpro on IFN a induced ISRE and AP 1 mediated activation To test the effect of SARS CoV PLpro on ISRE mediated responses to IFN a activity of ISRE driven reporter and Fig 1 Expression of SARS CoV PLpro in human promonocyte HL CZ cells Cells transfected with pcDNA3 1 control vector plus pEGFP N1 or pSARS CoV PLpro plus pEGFP N1 were selected by a 2 week incubation with G418 The HSV tag fusion protein was detected using immunofluorescence staining of anti HSV tag antibody and rhodamine conjugated anti mouse IgG antibody a Lysates from cells transfected with pcDNA3 1 plus pEGFP N1 lane 1 or pSARS CoV PLpro plus pEGFP N1 lane 2 were analysed by 10 SDS PAGE prior to blotting b The blot s upper half of was probed with anti HSV antibody the lower with anti b actin antibody as internal control Trans cleavage activity of SARS CoV PLpro in transfected cell lysates was further analysed c Following incubation of lysates from 10 6 PLpro expressing cells and control vector cells with substrate HRP residual HRP activity was measured as a mean of three independent experiments error bars show SEM SARS CoV PLpro suppressed ERK1 STAT1 signalling http vir sgmjournals org 1129 mRNA expression of ISRE driven gene PKR in empty vector controls and PLpro expressing cells were exam ined by dual luciferase reporter assay system Fig 2a and quantitative real time RT PCR Fig 2b Cells were co transfected with cis reporter plasmid containing firefly luciferase under the control of ISRE and an internal control reporter plasmid that constitutively expressed Renilla luciferase After treatment with IFN a for 4 h expression of firefly luciferase was determined and normalized to Renilla luciferase expression Fig 2 a plots vector control and PLpro expressing cells dose dependent transcriptional activity of the ISRE promoter by IFN a ISRE promoter driven luciferase activity in PLpro expressing cells was half of that in vector control cells The mRNA expression of specific ISRE driven gene PKR was analysed in both types of cells in the absence or presence of IFN a using quantitative real time RT PCR assays Fig 2b Induction of PKR by IFN a was approximately sevenfold lower in PLpro expressing cells than in control vector cells Since the endogenous PKR promoter contains not only the ISRE element but also kinase conserved sequence element for both basal and IFN inducible PKR promoter activity Samuel 2001 the other specific ISRE promoter driven gene 29 59 OAS was further analysed Fig 2c Induction of 29 59 OAS by IFN a was sixfold lower in PLpro expressing cells than in vector controls Results confirmed the antagonism of IFN a induced ISRE mediated gene expression by PLpro Subsequently the effect of SARS CoV PLpro on AP 1 mediated responses to IFN a was tested Fig 3 Activity of AP 1 enhancer in response to IFN a was next determined by transient transfection with plasmid vector containing luciferase under the control of the AP 1 enhancer Fig 3 a shows luciferase activity significantly induced in a dose dependent manner in control vector cells by IFN a but induction using the 0 U ml 1 IFN 500 U ml 1 IFN 1500 U ml 1 IFN 3000 U ml 1 IFN 0 U ml 1 IFN 3000 U ml 1 IFN 0 U ml 1 IFN 3000 U ml 1 IFN Fig 2 Effect of PLpro on ISRE mediated gene expression in response to IFN a a Vector control cells and PLpro expressing cells were transiently co transfected with reporter plasmid containing firefly luciferase under the control of the ISRE and an internal control reporter pRluc C1 that constitutively expressed Renilla luciferase After 4 h IFN a treatment firefly luciferase and Renilla luciferase were measured and firefly luciferase activity normalized to Renilla luciferase activity as reported Each bar is the mean of three independent experiments error bar is SEM The mRNA expressions of ISRE driven gene PKR b and 29 59 OAS c in vector control cells and SARS PLpro expressing cells untreated or treated was measured by quantitative real time PCR Relative fold levels of PKR or 29 59 OAS mRNA level appear as ratio of PKR or 29 59 OAS mRNA GAPDH mRNA Each bar graph is the mean of three independent experiments error bars represent SEM S W Li and others 1130 Journal of General Virology 92 same level of IFN a totally absent in PLpro expressing cells These results indicate SARS CoV PLpro mediated suppression and AP 1 mediated promoter activity in response to IFN a Upon stimulation with IFN a a 15 fold increase in IL 6 mRNA was induced in vector control cells no significant induction occurred in PLpro expressing cells Fig 3b Since the AP 1 element was also required for the IL 8 expression Hoffmann et al 2002 thus IL 8 mRNA levels in response to IFN a were also measured Fig 3c Levels of IL 8 mRNA were 3 5 fold higher in both unstimulated and stimulated vector controls than in unstimulated and stimulated PLpro expressing cells Fig 3c suggesting interference by PLpro with basal level IL 8 mRNA transcription AP 1 promoter activity and driven gene expression indicated SARS CoV PLpro as signifi cantly inhibiting mRNA expression of AP 1 mediated genes Downregulation of IFN a induced ERK1 mediated signalling by PLpro For a global perspective mechanism of type I IFN antagonism by SARS CoV PLpro differential protein expression in vector control and PLpro expressing cells in the absence or presence of IFN a was analysed by two dimensional 2D gel electrophoresis and nanoscale capillary liquid chromatography electrospray ionization Q TOF MS to identify differentially regulated proteins In Fig 4 a downregulated protein ERK1 and upregulated ubiquitin conjugating enzyme UBC E2 25K appeared in 2D gels of IFN a treated PLpro expressing cells and then identified by trypsin digestion and NanoLC Trap Q TOF MS analysis ERK1 showed a Mascot score of 109 sequence coverage of 14 and two matched peptides UBC E2 25K showed a Mascot score of 248 sequence coverage of 59 and four matched peptides Peptide peaks from Q TOF MS analysis from two representative spots of ERK1 and UBC 0 U ml 1 IFN 500 U ml 1 IFN 3000 U ml 1 IFN 0 U ml 1 IFN 3000 U ml 1 IFN 0 U ml 1 IFN 3000 U ml 1 IFN Fig 3 Effect of PLpro on AP 1 mediated gene expression in response to IFN a a Vector control and PLpro expressing cells were transiently co transfected with reporter plasmid containing AP 1 driven firefly luciferase and an internal control reporter pRluc C1 that constitutively expressed Renilla luciferase After 4 h treatment with IFN a AP 1 driven firefly luciferase and Renilla luciferase were measured and firefly luciferase activity normalized to Renilla luciferase activity is reported Each bar is the mean of three independent experiments error bar is SEM Inaddition themRNAexpressionsof AP 1 driven genes IL 6 b and IL 8 c in vector control cells and SARS PLpro expressing cells untreated or treated was measured by quantitative real time PCR Relative fold levels of IL 6 or IL 8 mRNA level are presented as the ratio of IL 6 or IL 8 mRNA GAPDH mRNA Each bar on the graph is the mean of three independent experiments error bars represent SEM SARS CoV PLpro suppressed ERK1 STAT1 signalling http vir sgmjournals org 1131 E2 25K Fig 4b c respectively ERK1 in particular is reported in several biological pathways mitogen activated protein kinase kinase cytokine mediated inflammation IFN signalling pathways and thus could play an important role in the mechanism of IFN a antagonism by PLpro Upregulation of UBC E2 25K of ubiquitin proteasome pathways by PLpro Quantitative RT PCR was employed to determine expres sion levels of ERK1 and UBC E2 25K in PLpro expressing and vector control cells in the absence or presence of IFN a Fig 5 Amount of ERK1 mRNA showed no difference between vector control and PLpro expressing cells whether treated with IFN a or not Fig 5a Relative level of UBC E2 25K mRNA in PLpro expressing cells was markedly higher than that in vector controls with or without IFN a treatment Fig 5b proving that SARS CoV PLpro activates the ubiquitin proteasome system in human promonocyte cells To compare ERK1 protein levels in vector control and PLpro expressing cells in the presence or absence of IFN a ERK1 and ERK2 were measured by Non IFN IFN Fig 4 Effect of SARS CoV PLpro on protein profiles of vector control cells and PLpro expressing cells in response to IFN a Total protein 100 mg from control vector cells in the absence or presence of IFN a or PLpro expressing cells in the absence or presence of IFN a was resolved by 2D electrophoresis a Enlarged images of 2D gel electrophoresis of protein expression in PLpro expressing cells and vector control cells in response to IFN a treatment b Nanoelectrospray mass spectrum of triply charged ion m z 1514 77 for ERK1 is shown ITVEEALAHPYLEQYYDPTDEPVAEEPFTFAM ox ELDDLPK amino acid sequence was determined from mass differences in y and b fragment ions series and matched residues 319 357 of ERK1 MAPK3 c Nanoelectrospray mass spectrum of the doubly charged ion m z 725 41 for UBC E2 25k is shown Amino acid sequence VDLVDENFTELR was determined from mass differences in y and b fragment ions series and matched residues 29 40 of ubiquitin conjugating enzyme E2 25k Only y and b fragment ions are labelled in the spectrum S W Li and others 1132 Journal of General Virology 92 Western blots with anti p44 p42 ERK1 2 mAb Fig 6a Western blotting showed 42 kDa ERK2 protein levels roughly similar in vector control and PLpro expressing cells whereas the protein level of 44 kDa ERK1 in PLpro expressing cells was near 50 of that in controls determined by densitometry normalized to b actin protein control in each sample Fig 6a lanes 1 2 IFN a treatment caused time dependent reduction of ERK1 but not ERK2 in PLpro expressing cells Fig 6a lanes 4 and 6 Results confirmed data of 2D MALDI TOF MS which showed definite reduction of ERK1 in PLpro expressing cells in response to IFN a Since PLpro expressing cells have no difference in mRNA amount but a significant reduction of ERK1 protein levels by IFN a we suggest that upregulation of UBC E2 25k in PLpro expressing cells could increase ubiquitination on ERK1 enhancing ERK1 degradation by IFN a treatment To test the hypothesis ERK1 immunoprecipitation fol lowed by Western blot probed with anti ubiquitin antibodies was conducted in the absence or presence of IFN a Fig 6b revealing that ERK1 conjugated with different sizes of poly ubiquitin chains i e molecular sizes of 52 60 68 76