Chinese Journal of Biochemistry and Molecular Biology. CHO., 3 hfgl2p ( ) LUC hfgl2p ( - 997) LUC hfgl2p ( - 816) LUC ; hfgl2p (2468)LUC

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 23 (2) : hfgl2 SARS,,,,, (,, ) 3 SARS hfgl2, PCR Western hfgl2 mrna SARS, hfgl2 5, hfgl2, SARS CHO, 3 hfgl2p ( ) LUC hfgl2p ( - 997) LUC hfgl2p ( - 816) LUC ; hfgl2p (2468)LUC, hfgl ( ) SARS ;SARS ; ; R511 Primary Analysis of SARS CoV Nucleocapsid Protein2Response Element ( Region) in hfgl2 Promoter YAO Hua2Ning, HAN Mei2Fang, YAN Wei2Ming, XI Dong, LUO Xiao2Ping, NING Qin 3 ( Department of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China) Abstract Using real2time fluorescence quantitative RT2PCR and Western blot, We demonstrate that the nucleocapsid (N) protein of SARS2CoV induces transcription of the novel fgl2 prothrombinase gene A series of hfgl2 promoterπluciferase reporter constructs containing progressive deletions of hfgl2 promoter within the 5 flanking uncoding region were cotransfected with a nucleocapsid protein expressing construct in Chinese hamster ovary (CHO) cells There was no significant difference in luciferase activity in cells cotransfected with hfgl2p ( ) LUC, hfgl2p ( - 997) LUC and hfgl2p ( ) LUC However, Relative luciferase activity cotransfected with hfgl2p ( - 418) LUC showed a remarkable decline compared with the other three constructs Preliminary mapping of the hf gl2 promoter has defined a region from to ( relative to the transcriptional start site) to be responsive for transcription regulation of hf gl2 gene in response to nucleocapsid protein These results reveal the regulatory elements from to in hf gl2 promoter account for the activation of hf gl2 gene in response to nucleocapsid protein of SARS2CoV and show some light on the relation between SARS2CoV protein and host hf gl2 gene Key words hfgl2πfibroleukin ; SARS coronavirus ; nucleocapsid protein ; gene regulation : , : SARS (No 2003CB514100) SARS ( [2003 ]64 ) 3 Tel : (027) ,Fax : (027) tjmu edu cn Received :August 14,2006 ; Accepted : November 11,2006 Supported by National 973 Project of China for SARS Study(No 2003CB514100) and Ministry of Education of China for SARS Study(No ) 3 Corresponding author :Tel : (027) ,Fax : (027) tjmu edu cn China Academic Journal Electronic Publishing House All rights reserved

2 2 : hfgl2 SARS 131 ( hfgl2) 1995 Ruegg [1 ], DNA Omega,, Lipofectamine 2000 Invitrogen, [2 ], Promega,SSWP,, Pierce [3 5 ] 112 hfgl2, Robertson [6 ] P1 PM166 (severe acute respiratory syndrome, SARS) (pbluescript2m166) hfgl2, hfgl kb DNA ( hfgl2 ) SARS SARS p GL2 p GL22Basic [7 ],, Sma Xho, hfgl2 ( mfgl) mrna hfgl2p ( )2LUC, hfgl2 SARS ( SARS2associated coronaviru,sars2cov) SARS, 5 3 hfgl2, 300,,, 4 SARS (N) hfgl2,hfgl2p ( - 997) :5 2CTT ATG TCT TTC CTG CCT TC2 [8 ] PCR Western 3 ;hfgl2p ( - 816) :5 2GGC AAG AGA AGT TCA GGA hfgl2 mrna C23 ;hfgl2p ( - 468) :5 2AAT ACA GGC TCC CCA ATG, C23 ;hfgl2p ( - 243) :5 2GTG AAT CTT GTT GGC TGT SARS, G23, 5 2TTC GCC CAT CTT hfgl2 5 TAC AGT G23 PCR 50 l, :, hfgl min,94 1 min,50 1 min,72 80 s, 5, 3 25,72 10 min PCR,, SARS hfgl2p ( - 997) hfgl2p ( - 816) PCR PCR3 CHO, 1, hfgl2p ( - 468) hfgl2p ( - 243) PCR, SARS N PCR211, 4, PCR311 ( - 997) PCR311 ( - 816) PCR211 ( - 468) PCR211 ( - 243) Hind Xho hfgl2 hfgl2p2luc hfgl2, PCR211 PCR311 Invitrogen,p GL22Basic hfgl2p ( )2LUC hfgl2p ( )2LUC hfgl2p 2Gal Promega,SARS ( - 468)2LUC hfgl2p ( - 243) LUC ( Fig11) pcdna3112n pcdna3112m pcdna3112s2, CHO 113 THP21 Hfgl2 (McAb) HRP2 IgG Santa Cruz T4 DNA Promega,DNA ( pcdna3112n Taq DNA TaKaRa,Trizol Biotium PCR Qiagen, PCR, hfgl2p ( )2LUC p GL22Basic T4 DNA h, DH5,,, CHO 1 g SARS (N) (M) (S2) pcdna3112m pcdna3112s2), 1 g hfgl2p Invitrogen, EvaGreen PCR ( )2LUC p GL22Basic 015 g 2Gal China Academic Journal Electronic Publishing House All rights reserved

132 23 100 l OPTI2MEM, 100 l OPTI2 CTC TTG GTC AGA AG23 MEM ( 5 l Lipofectamine 2000), 115 Western hfgl2 20 min Western SARS2CoV 200 l 6 90 % 95 % (pcdna3112n, pcdna3112m pcdna3112 CHO, 800 l,37, S2)

3 l OPTI2MEM, 100 l OPTI2 CTC TTG GTC AGA AG23 MEM ( 5 l Lipofectamine 2000), 115 Western hfgl2 20 min Western SARS2CoV 200 l 6 90 % 95 % (pcdna3112n, pcdna3112m pcdna3112 CHO, 800 l,37, S2) THP21, 5 % CO h,,, 12 %, SDS2PAGE, 30 l 2Gal ( 3 (NC), S ), 20 l ( TD220) NC, 5 % 2 h 2gal,, hfgl2 (1 500) 37 1 h, 5, IgG ( ),37 1 h, NC 5 min, 10 min, hfgl2 PCR311 ( - 997) PCR311 ( - 816) PCR211 ( - 468) PCR211 ( - 243) Hind Xho ( p GL22Basic), PCR Hind Xho, ( Fig12) GenBank (AH007300) Fig 1 Analysis of a series of hfgl2 promoterπluciferase reporter constructs 1 g SARS N hfgl2 015 g 2gal 0145 g, CHO, 114 RT2PCR hfgl2 mrna Fig 2 Electrophoretic analysis and identification of a,hfgl2 series recombinant plasmids of hfgl2 promoterπluciferase THP21 reporter gene SARS2CoV hfgl2 Trizol SARS (N) (M) (S2) THP2 1 RNA, EvaGreen PCR hfgl2 mrna 5 2ACT GTG ACA TGG AGA CCA TG23, 5 2TCC TTA M:DL2000 marker ; 1 :hfgl2p ( - 997) LUCΠHind + Xho ;2 :hfgl2p ( - 816)LUCΠHind + Xho ;3 :hfgl2p ( - 468) LUCΠHind + Xho ;4 :hfgl2p ( - 243)LUCΠHind + Xho 212 SARS N hfgl2 SARS hfgl China Academic Journal Electronic Publishing House All rights reserved

2 : hfgl2 SARS 133, CHO, hfgl2 2Gal, 4 p GL22Basic 1,, CHO, pcdna3112n 416 SPSS 4,N pcdna311 P < 0101,, N hfgl2 M S2 pcdna311 ( P > 0105) (Fig13), M S2 hfgl2 Fig 4 Real2time PCR analysis of

4 2 : hfgl2 SARS 133, CHO, hfgl2 2Gal, 4 p GL22Basic 1,, CHO, pcdna3112n 416 SPSS 4,N pcdna311 P < 0101,, N hfgl2 M S2 pcdna311 ( P > 0105) (Fig13), M S2 hfgl2 Fig 4 Real2time PCR analysis of expression of hfgl2 in response to SARS2CoV N, M and S2 proteins THP1 cells were transfected with pcdna3 12N, pcdna3 12M, pcdna3 12S2 and pcdna23 1 empty vector respectively, and untranfected cells as control group Values represent the means SD of three separate experiments done in triplicate with control group 3 P < 0 01 compared Fig 3 Effects of SARS- CoV protein on transcriptional activation of hfgl2 in CHO cells pcdna3 1-N,pcDNA3 1-M or pcdna3 1- S2 was cotransfected with hfgl2p-luc or p GL2-Basic in CHO cells for hours,and the cells were harvested for measurement of luciferase activity Values represent the means S E M of four separate experiments 3 P < 0 01 compared with cells contransfected with empty pcdna3 1 vector ( 2actin), pcdna3112n, 62 kd, hfgl2, pcdna3112m pcdna3112s2 pcdna311 ( + ) ( Fig15), pcdna2n pcdna3112m pcdna3112s2 THP21 40 kd 3 RT2PCR hfgl2 mrna RT2PCR pcdna3112n, pcdna3112m pcdna3112s2 THP21 hfgl2, THP21 ( Fig14) Fig 5 Western blot analysis of hfgl2 expression in response to SARS2CoV N, M and S2 proteins THP1 cells were transfected with pcdna3 12N, pcdna3 12M, pcdna3 12S2 and pcdna23 1 empty vector, respectively 1 : pcdna3 12N ; 2 : pcdna3 12M; 3 : pcdna3 12S2 ; 4 : pcdna23 1 empty vector 312 hfgl2 311 Western hfgl2 hfgl2 SARS2CoV THP21, CHO Western, pcdna311 ( + ) China Academic Journal Electronic Publishing House All rights reserved

5 N, hfgl2 hfgl2p ( - 468)LUC, 3 ( Fig14) hfgl2 hfgl2p ( ) LUC hfgl2p ( ) LUC hfgl2p ( ) ( - 816)LUC, Fig 6 Effects of SARS2CoV nucleocapsid protein on transcriptional activation of a series of hfgl2 promoter in CHO cells pcdna3 12N was cotransfected with a series of hfgl2 promoterπluciferase reporter gene containing progressive deletions of the base pair fragment4 or pgl22 Basic in CHO cells for hours, and cells were harvested for measurement of luciferase activity 4 ( hfgl2), 7q11123,, X ( ), [3 5, ] SARS, SARS, [9 12 ] Ning [13,14 ],MHV23 (N) mfgl2, Basic, ( ATG ), hfgl2 PCR Western, hfgl2 mrna p GL22, 4 (HNF4), HBc HBx hfgl2,,, [15 ] hfgl2,, hfgl2 : SARS2CoV, : hfgl China Academic Journal Electronic Publishing House All rights reserved

6 2 : hfgl2 SARS ( ) SARS N, hfgl , AP24 HSF SKn21 CEB22 LEF21 c2 Ets22 Dfd CΠEBP CdxA, fgl2 ( References) [ 1 ] Ruegg C, Pytela R Sequence of a human transcript expressed in T2 lymphocytes and encoding a fibrinogen2like protein[j ] Gene, 1995, 160(2) : [ 2 ] Levy G A, Liu M F, Ding J W, et al Molecular and functional analysis of the human prothrombinases gene ( HFGL2) and its role in viral hepatitis[j ] Am J Pathol,2000, 156 (4) : [ 3 ] Marazzi S, Blum S, Hartmann R, et al Characterization of human fibroleukin, a fibrinogen2like protein secreted by T lymphocytes[j ] J Immunol, 1998, 161(1) : [ 4 ] Ding J W, Ning Q, Liu M F, et al Fulminant hepatic failure in murine hepatitis virus strain 3 infection : tissue2specific expression of a novel fgl2 prothrombinase[j ] J Virol, 1997,71(12) : [ 5 ] Ning Q, Brown D, Parodo J, et al Ribavirin inhibits viral2induced acrophage production of TNF, IL21, the procoagulant fgl2 prothrombinase and preserves Th1 cytokine production but inhibits Th2 cytokine response[j ] J Immunol, 1998,160 (7) : [ 6 ] Robertson M Fgl2 : link between hepatitis B and SARS[J ] Drug Discov Today, 2003, 8( 17) : [ 7 ] Yan W M, Zhu C L, Luo X P, et al Establishment of a substitutive SARS murine model and its application in SARS study [J ] J Clin Immunol, 2005, Suppl (1) : [ 8 ],,, SARS N hfgl2 [J ] ( Yao Hua2Ning, Wang Zhan2Hui, Han Mei2Fang, et al Nucleocapsid protein of SARS2 associated coronavirus upregulates the transcription of hfgl2 prothrombinase Πfibroleukin gene [ J ] Virol Sin), 2006, 21 ( 4 ) : [ 9 ] Marsden P A, Ning Q, Fung L S, et al The fgl2πfibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis[j ] J Clin Invest, 2003,112 (1) :58266 [10 ] Ning Q, Sun Y, Han M, et al Role of fibrinogen2like protein 2 prothrombinaseπfibroleukin in experimental and human allograft rejection[j ] J Immunol, 2005,174(11) : [11 ],,, [J ] ( Chen Yue, Ning Qin, Wang Bao2Ju, et al Expression of human fibroleukin gene acute on chronic hepatitis B and its clinical significance[j ] Natl Med J China), 2003,83 (6) : [12 ] Clark D A, Ding J W, Chaouat G, et al The emerging role of immunoregulation of fibrinogen2related procoagulant fgl2 in the success or spontaneous abortion of early pregnancy in mice and humans[j ] Am J Reprod Immunol, 1999,42( 1) :37243 [13 ] Ning Q, Liu M, Kongkham P, et al The nucleocapsid protein of murine hepatitis virus type 3 induces transcription of the novel fgl2 prothrombinase gene[j ] J Biol Chem, 1999,274 (15) : [14 ] Ning Q, Lakatoo S, Liu M, et al Induction of prothrombinase fgl2 by the nucleocapsid protein of virulent mouse hepatitis virus is dependent on host hepatic nuclear factor24 alpha [J ] J Biol Chem, 2003, 278 (18) : [15 ],,, [J ] ( Han Mei2 Fang, Xi Dong, Luo Xiao2Ping, et al Hepatitis B virus proteins promote transcription of fibroleukin gene [J ] Chin J Biochem Mol Biol), 2006,22( 1) : China Academic Journal Electronic Publishing House All rights reserved

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Supplemental file 3. All 306 mapped IDs collected by IPA program. Supplemental file 6. The functions and main focused genes in each network.

LIST OF SUPPLEMENTAL FILES Supplemental file 1. Primer sets used for qrt-pcr. Supplemental file 2. All 1305 differentially expressed genes. Supplemental file 3. All 306 mapped IDs collected by IPA program.