Coronavirus CoV 10 SARS SARS 229E HCoV-229E OC43 HCoV- Yan SARS-CoV OC43 NL63 HCoV-NL63 Vero SARS-CoV 2012 MERS-CoV 1 PEDV porcine epi

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1 ISSN CN / Q http / /cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 1178 DOI /j cnki cjbmb Caspase * PEDV PEDV PEDV P < 0 05 P < 0 01 PLP PEDV caspase-3 PLP caspase-3 PLP caspase-3 Q5 Q93 PEDV Coronavirus Induces Apoptosis through Activation of Caspase-3 by Papain-like Protease ZHAO Qian 1 2 CHEN Xiao-Juan 2 XING Ya-Ling 2 XIE Qing 2 CHEN Zhong-Bin 1 2 * 1 Anhui Medical University Hefei China 2 Beijing Institute of Radiation Medicine Beijing China Abstract Coronavirus is a life threatening virus which may cause severe respiratory infectious disease in human as SARS and MERS But at present the apoptotic mechanisms induced by coronaviruses remain unclear This study was to explore the potential mechanism of apoptosis induced by coronavirus especially taking porcine epidemic diarrhea virus PEDV an animal coronavirus as a model The results showed that the apoptosis rate of PEDV-infected cell was significantly higher than that of mockinfected group and the apoptosis induced by PEDV showed time-and dose-dependent manner P < 0 05 or P < 0 01 Next the role of papain-like protease PLP of coronavirus in apoptosis was further investigated The results showed that coronavirus PLP induced activation of caspase-3 in the HEK 293T when the cells were transfected with PEDV PLP plasmids Moreover PLP2 from other coronaviruses induced apoptosis through activation of caspases 3 implying that the ability to induce apoptosis is a shared attribute of coronavirus PLP2 and its homologs Our results indicated that PLP-induced apoptosis might play a central role in coronavirus-induced apoptosis to inhibit the host antiviral innate immunity and facilitate coronaviral replication and pathogenesis Key words coronavirus porcine epidemic diarrhea virus PEDV papain-like protease PLP apoptosis caspase No No No No * Tel Fax chenzb@ bmi ac cn chenzhongbin2013@ 163 com Received April Reccepted July Supported by National Natural Science Foundation of China No No No No * Corresponding author Tel Fax chenzb@ bmi ac cn chenzhongbin2013@ 163 com

2 Coronavirus CoV 10 SARS SARS 229E HCoV-229E OC43 HCoV- Yan SARS-CoV OC43 NL63 HCoV-NL63 Vero SARS-CoV 2012 MERS-CoV 1 PEDV porcine epidemic diarrhea virus PEDV PLP caspase-3 2 PED PLP 100% PEDV 1 transmissible gastro-enteritis virus TGEV porcine rotavirus PRV 1 1 PEDV HEK 293T 3 PEDV NL63 HCoV NL 63 SARS Vero E6 PEDV CV777 SARS-CoV pcdna3 1-V5 /His B MERS-CoV PEDV V5-PLP1-TM PEDV V5-PLP1 PEDV V5- coronavirinae 4 3 PLP2 MERS V5-PLpro-TM SARS V5-PLpro-TM nonstructural protein3 nsp3 PLP HCoV NL63 V5-PLP2-TM PEDV V5-PLP2-TM 1 zinc finger domain ZF 1 ubiquitin-like domain 5 DMEM 1640 OPTI-MEM 6 PEDV % Gibco papain-like protease 2 PLP2 Lipofectamine TM Invitrogen V5 I MBL β-actin IgG caspase-3 Annexin V-FITC /PI X 7 8 CO 2 Thermo 3 III Thermo DYY-7B FACS Aria III BD HBV HBx 1 2 caspase Vero E6 HEK 293T Fas /FasL TNF-ɑ /TNFR 2% % 9 DMEM 100 U /ml 100 U /ml 37 5% CO 2

3 11 Caspase PEDV Vero E6 Vero E6 2% / ml 6 24 h 0 25% Annexin V - /PI - Annexin V + / PI - 2% ml 37 5% AnnexinV + CO 2 24h MOI = 2 PEDV MOI = 5 PEDV CdCl 2 60 μmol /L 0 05 MOI = 5 Vero E h PEDV 36 h 1 4 HEK 293T 6 Vero E6 36 h MOI = 2 MOI = 5 80% Lipofectamine TM μg PEDV PLP1 PEDV PLP2 PEDV PLP1-TM PEDV PLP2-TM NL63 PLP2-TM SARS PLpro-TM MERS PLpro-TM pcdna3 1-V5 /HisB DNA CdCl 2 60 μmol /L 1 5 EDTA PBS PEDV PLP r /min 4 5 min /ml 500 μl binding buffer 5 μl Annexin V-FITC 5 μl PI 10 min Ex = 488 nm Em = 530 nm PEDV PLP 1 6 Western Fig 2 A 1 μl BCA PLP PLP 10 min SDS-PAGE PEDV PLP1 PLP2 PVDF 5% 1 h TM caspase β- HEK 293T 24 h Western actin V TBST h TBST ECL 1 7 PLP2 PEDV PLP1-TM PEDV PLP2-TM GraphPad Prism PEDV PLP x 珋 ± s t PEDV PEDV Vero E6 Annexin V-FITC /PI /PI + MOI = 2 PEDV Vero E6 6 h P < P < % ± 0 33% PEDV PEDV Vero E6 Fig 1 PEDV PEDV RNA 28 kb PLP1 PLP2 nsp3 ppla /pplab N 12 PEDV PLP1 PEDV caspase-3 Fig 2 B PEDV PLP caspase-3 PLP PEDV PLP Fig 3 A PEDV PLP1 PEDV PLP2 PEDV PLP1-TM PEDV PLP2-TM 24 h

4 Fig 1 PEDV induced apoptosis in dose-and time-dependent manner A Cell death analysis by FCM with dual Annexin V-PI cell labeling Cells collected from different time periods and different dose were dually labeled with AnnexinV and PI and analyzed by FCM The lower left quadrants represent intact cells Annexin V negative /PI negative the lower right quadrants represent early apoptotic cells Annexin V positive /PI negative the upper right quadrants indicate late apoptotic and /or necrotic cells Annexin V positive /PI negative the upper left quadrants indicate necrotic cells Annexin V negative /PI positive The normal cells were treated as blank control The cells treated by 60 μmol /L CdCl 2 were analyzed to serve as a positive control for induction of apoptosis B Statistical analysis showed more apoptosis of cells induced by PEDV x 珋 ± s n = 3 * P < 0 05 ** P < 0 01 compared to the control group The cell apoptosis was increased with the infected hours

5 11 Caspase Fig 2 Caspase-3 was activated by PEDV papain-like proteases A A schematic diagram of PEDV-CoV ployprotein cleavage was showed PLP1 /PLP2 was the papain-like protease domain TM was the transmembrane domain B Caspase-3 was activated by PEDV papain-like proteases The PLP constructs were transfected into HEK293T cells At the indicated time the cellular lysates were collected resolved by SDS-PAGE transferred to a nitrocellulose membrane and immunoblotted using an antibody recognized caspase-3 top panel or PEDV PLpro protein third panel The second panel was imaged after longer exposure to confirm the presence of activated caspase-3 The blot was also reacted with mouse MAb against the β-actin to verify equal protein loading bottom panel The cells treated by 60μmol /L CdCl 2 were analyzed to serve as a positive control PEDV PLP2-TM HEK 293T 2 4 PLP P < % ± 1 24% 6 77% ± 0 11% PEDV PLP2- TM P < 0 01 PEDV PLP2-TM P < % ± 2 16% PEDV PLP2-TM PLP DUB PLP HEK 293T MERS PLpro- Fig 3 C PEDV PLP2-TM 24 h TM SARS PLpro-TM PEDV PLP2-TM NL63 PLP2- TM 24 h 39% PEDV PLP2-TM 18 85% ± 1 45% HEK 293T PEDV P < 0 01 PLP PLP

6 Fig 3 Flow cytometry anaysis of cell apoptosis in PEDV PLP-transfected HEK 293 T cells A The apoptosis condition of HEK 293Tcells were detected by FCM after transfected with PEDV PLP1 PEDV PLP1-TM PEDV PLP2 and PEDV PLP2-TM After 24 hours cells were analyzed for apoptosis rate by flow cytometry The apoptosis condition of HEK 293Tcells were detected by FCM after transfected with PEDV PLP2-TM for 24 hours After transfected for 24 hours cells were analyzed for apoptosis rate by FCM The normal cells were treated as blank control The cells treated by 60 μmol /L CdCl 2 were analyzed to serve as a positive control for induction of apoptosis B Statistical analysis showed that the apoptosis rate of cells transfected by PEDV s PLP plasmids was pretty higher than that of the control group x 珋 ± s n = 3 *** P < 0 01 C The apoptosis of HEK 293Tcells were detected by FCM after transfected with PEDV PLP2-TM for 24 hours After transfected for 24 hours cells were analyzed for apoptosis rate by FCM The cells treated by 60 μmol /L CdCl 2 were analyzed to serve as a positive control for induction of apoptosis D Statistical analysis showed more apoptosis of cells transfected with PEDV PLP2-TM x 珋 ± s n = 3 ** P < 0 05 *** P < 0 01 compared to the blank control group Fig 4 Cell apoptosis was induced by PLP-TM from other coronaviruses A The apoptosis rate of HEK 293Tcells were detected by FCM after transfected with MERS PLpro-TM SARS PLpro-TM PEDV PLP2-TM and NL63 PLP2-TM After 24 hours cells were analyzed for apoptosis rate by flow cytometry The cells treated by 60 μmol /L CdCl 2 were analyzed to serve as a positive control for induction of apoptosis B Statistical analysis showed the apoptosis condition of cells transfected by CoV s PLP plasmids was pretty higher than that of the black control group 珋 x ± s n = 3 *** P < 0 01

7 11 Caspase Fig 4 PLP PLP2-TM HEK 293T 24 h Western caspase-3 Fig 5 PLP MERS PLP caspase-3 PLpro-TM SARS PLpro-TM PEDV PLP2-TM NL63 Fig 5 Coronaviruses PLP induced apoptosis through activation of caspases-3 V5-tagged NL63 PLP2-TM SARS PLpro- TM MERS PLpro-TM and PEDV PLP2-TM were transfected into HEK 293T cells for 24 hours At the indicated time the cellular lysates were collected resolved by SDS-PAGE transferred into a nitrocellulose membrane and immunoblotted using an antibody recognized caspase-3 top panel or CoV PLP protein third panel The second panel was imaged after longer exposure to confirm the presence of activated caspase-3 The blot was also reacted with mouse MAb against the β-actin to verify equal protein loading bottom panel The cells treated by 60 μmol /L CdCl 2 were analyzed to serve as a positive control 3 baculoviruses poxviruses PEDV PLP1 PLP2 14 TM PEDV PLP1 PEDV PLP2 PEDV PLP1-TM PEDV PLP2-TM HEK 293T PEDV PLP PLP2-TM PEDV PLP 24 h caspase-3 caspase-3 PEDV caspase-3 PLP2-TM caspase-3 PEDV caspase Vero E6 PEDV PEDV Vero E6 caspase 15 caspase-3 Vero E6 16 PEDV PLP2 PEDV Vero E6 P < PEDV PLP2 PEDV VeroE6 PLP 12 MERS PLpro-TM SARS herpesviruses C PLpro-TM PEDV PLP2-TM HCoV NL63 PLP2-TM

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