Establishment of the Eukaryotic Cell Lines for Inducible Control of SARS-CoV Nucleocapsid Gene E

Aromatic Hydrocarbon Receptor(AhR)⅐AhR NuclearTranslocator-and p53-mediated Induction of theMurine Multidrug Resistance mdr1Gene by3-Methylcholanthrene and Benzo(a)pyrene in Hepatoma Cells*Received for publication,September18,2000,and in revised form,November10,2000Published,JBC Papers in Press,November28,2000,DOI10.1074/jbc.M008495200Marie-Claude Mathieu,Isabelle Lapierre,Karine Brault‡,and Martine Raymond§From the Institut de Recherches Cliniques de Montre´al,Montre´al,Que´bec H2W1R7,CanadaThe mouse multidrug resistance gene family consists of three genes(mdr1,mdr2,and mdr3)encoding P-gly-coprotein.We show that the expression of mdr1is in-creased at the transcriptional level upon treatment of the hepatoma cell line Hepa-1c1c7with the polycyclic aromatic hydrocarbon3-methylcholanthrene(3-MC). This increase is not observed in the aromatic hydrocar-bon receptor(AhR)-defective TAOc1BP r c1and the AhR nuclear translocator(Arnt)-defective BP r c1variants, demonstrating that the induction of mdr1by3-MC re-quires AhR⅐Arnt.We show that the mdr1promoter (؊1165to؉84)is able to activate the expression of a reporter gene in response to3-MC in Hepa-1c1c7but not in BP r c1cells.Deletion analysis indicated that the re-gion from؊245to؊141contains cis-acting sequences mediating the induction,including a potential p53bind-ing sequence.3-MC treatment of the cells increased the levels of p53and induced p53binding to the mdr1pro-moter in an AhR⅐Arnt-dependent manner.Mutations in the p53binding site abrogated induction of mdr1by 3-MC,indicating that p53binding to the mdr1promoter is essential for the induction.Benzo(a)pyrene,a polycy-clic aromatic hydrocarbon and AhR ligand,which,like 3-MC,is oxidized by metabolizing enzymes regulated by AhR⅐Arnt,also activated p53and induced mdr1tran-scription.2,3,7,8-Tetrachlorodibenzo-p-dioxin,an AhR ligand resistant to metabolic breakdown,had no effect. These results indicate that the transcriptional induc-tion of mdr1by3-MC and benzo(a)pyrene is directly mediated by p53but that the metabolic activation of these compounds into reactive species is necessary to trigger p53activation.The ability of the anticancer drug and potent genotoxic agent daunorubicin to induce mdr1independently of AhR⅐Arnt further supports the proposition that mdr1is transcriptionally up-regulated by p53in response to DNA damage.Multidrug resistance(MDR)1is characterized by cross-resis-tance of the cells to a large number of structurally and func-tionally unrelated cytotoxic agents used in chemotherapy.In cultured cells,MDR is frequently caused by the overexpression of P-glycoprotein(Pgp),an integral membrane protein belong-ing to the ATP-binding cassette superfamily of transporters and which functions as an energy-dependent efflux pump of cytotoxic drugs(1,2).Pgp is encoded by a small family of genes with two members in humans(MDR1and MDR2/MDR3)and three in rodents(mdr1/mdr1b,mdr2,and mdr3/mdr1a)(1,2). Only one human gene(MDR1)and two rodent genes(mdr1/ mdr1b and mdr3/mdr1a)can confer MDR upon overexpression in drug-sensitive cells(1,2).The different mdr genes and Pgp isoforms are expressed in a tissue-specific manner(1,2).In the mouse,mdr1is expressed mostly in the adrenal cortex,kidney,and pregnant uterus, mdr2in the liver at the canalicular face,and mdr3in the intestine and to a lesser extent in the heart,liver,lung,and capillaries of the brain(3).Pgps are localized on the apical membrane of epithelial cells lining luminal spaces,suggesting that they function in normal tissues as transporters of toxic substances and/or specific endogenous cellular products(4). Knockout mice experiments have demonstrated a role for the mdr3gene in the maintenance of the blood-brain barrier and drug elimination and for the mdr2gene in the transport of phospholipids in the bile(5,6).No physiological function has been attributed to the mouse mdr1gene so far,since knockout mdr1(Ϫ/Ϫ)mice display no obvious physiological abnormali-ties(7).However,different experimental evidence indicates that Pgp encoded by mdr1can serve in the transport of steroids(8).A number of factors have been found to modulate the level of mdr gene expression in the liver.For example,high levels of MDR1RNA have been found in human hepatocarcinomas,and overexpression of the mdr1isoforms has also been observed in rodent liver during cholestasis,during regeneration following partial hepatectomy,during chemically induced hepatocarcino-genesis,and following administration of various natural and synthetic xenobiotics(1,2).In particular,it has been shown that expression of the rat mdr1b gene is increased in liver cells in response to treatment with various polycyclic aromatic hy-*This work was supported by a research grant from the Cancer Research Society Inc.(to M.R).The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked“advertisement”in accordance with18 U.S.C.Section1734solely to indicate this fact.‡Supported by a studentship from the Medical Research Council ofCanada.Present address:Dept.of Biological Sciences,Bio-Mega Re-search Division,Boehringer Ingelheim(Canada)Ltd.,Laval,Que´bec H7S2G5,Canada.§Supported by a scholarship from Le Fonds de la recherche en sante´du Que´bec.To whom correspondence should be addressed:Institut de recherches cliniques de Montre´al,110Pine Ave.W.,Montre´al,Que´bec H2W1R7,Canada.Tel.:514-987-5770;Fax:514-987-5764;E-mail: raymonm@ircm.qc.ca.1The abbreviations used are:MDR,multidrug resistance;Pgp,P-glycoprotein;3-MC,3-methylcholanthrene;B(a)P,benzo(a)pyrene; TCDD,2,3,7,8-tetrachlorodibenzo-p-dioxin;DN,daunorubicin;CAT, chloramphenicol acetyl transferase;AhR,aromatic hydrocarbon recep-tor;Arnt,AhR nuclear translocator;EMSA,electrophoretic mobility shift assay;DME,drug metabolizing enzymes;PAH polycyclic aromatic hydrocarbon;XRE,xenobiotic response element;bp,base pair(s);kb, kilobase pair(s).T HE J OURNAL OF B IOLOGICAL C HEMISTRY Vol.276,No.7,Issue of February16,pp.4819–4827,2001©2001by The American Society for Biochemistry and Molecular Biology,Inc.Printed in U.S.A.This paper is available on line at 4819 at ZHEJIANG UNIVERSITY, on November 21, Downloaded fromdrocarbon(PAH)compounds,including3-methylcholanthrene (3-MC),and that this increased expression occurs at the tran-scriptional level(9–11).However,the precise molecular mech-anisms involved in mdr1b regulation in response to3-MC are still unknown.PAHs are carcinogenic compounds arising from the incom-plete combustion of organic matter and are widespread in the environment,including tobacco smoke and tar.PAHs such as 3-MC and benzo(a)pyrene(B(a)P)as well as halogenated aro-matic hydrocarbons such as2,3,7,8-tetrachlorodibenzo-p-di-oxin(TCDD)are specific inducers of genes coding for drug-metabolizing enzymes(DME),including cyp1a1and cyp1a2, that code for cytochromes P450involved in metabolic oxidation (12).PAHs and TCDD bind in the cytoplasm to the aromatic hydrocarbon receptor(AhR),a member of the bHLH-PAS(basic helix-loop-helix Per-Arnt-Sim)family of transcription factors (12,13).The ligand-bound AhR translocates to the nucleus, where it binds as a heterodimer with the AhR nuclear trans-locator(Arnt;another bHLH-PAS protein)to specific cis-acting regulatory DNA sequences located in the promoter of its tar-gets(known as AH-,dioxin-,or xenobiotic-responsive elements (or AHRE,DRE,or XRE,respectively))to enhance their tran-scription(12,13).Given that mdr1b expression is increased in liver cells in response to treatment with various PAHs,it was postulated that mdr1b may be under the control of the AhR(9). However,studies failing to show mdr1induction in the liver of mice treated with TCDD,one of the most potent agonists of the AhR,suggested that mdr1expression was not regulated by AhR(14).The involvement of AhR in the regulation of mdr1 has so far remained controversial.The mouse hepatoma cell lines Hepa-1c1c7(wild type), TAOc1BP r c1(AhR-defective),and BP r c1(Arnt-defective)con-stitute a powerful experimental system to investigate the tran-scriptional regulation of different AhR⅐Arnt targets in response to xenobiotics(12).The two mutant cell lines were derived as B(a)P-resistant variants of Hepa-1c1c7and were identified based on their inability to induce aryl hydrocarbon hydroxylase activity in response to TCDD treatment(15).TAOc1BP r c1cells have a decreased level of AhR(ϳ10%of wild-type cells)and therefore decreased induction of the cyp1a1promoter and lower aryl hydrocarbon hydroxylase activity in response to TCDD and other AhR ligands(15–18).BP r c1cells have a nor-mal cytosolic AhR,which fails to accumulate in the nucleus because of a defective Arnt(15).They have virtually no basal or inducible levels of cyp1a1expression and aryl hydrocarbon hydroxylase activity(15–17).In the present report,we have used this panel of cell lines to investigate the transcriptional regulation of the murine mdr1 gene by3-MC and other xenobiotic compounds.Our results demonstrate that mdr1is transcriptionally induced by3-MC and B(a)P and that this induction is mediated by p53but also requires AhR⅐Arnt.A model for the AhR⅐Arnt-and p53-medi-ated transactivation of mdr1in response to genotoxic stress is proposed.EXPERIMENTAL PROCEDURESCell Culture—Wild-type Hepa-1c1c7and Hepa1–6,AhR-defective TAOc1BP r c1,and Arnt-defective BP r c1cells were obtained from the American Type Culture Collection(ATCC;Manassas,VA)and main-tained in culture under the conditions recommended by the ATCC. Chinese hamster ovary LR73cell lines stably transfected with plasmid constructs carrying full-length cDNAs for the mouse mdr1,mdr2,or mdr3genes(LR73mdr1,LR73mdr2,and LR73mdr3,respectively;a gift from Dr.Philippe Gros,McGill University,Montre´al,Canada)were grown as described elsewhere(19,20).For inductions,cells atϳ50% confluence were exposed to different concentrations of xenobiotics for various periods of time(the exact conditions for each experiment are indicated in the figure legends).3-MC,B(a)P,and daunorubicin were obtained from Sigma,and TCDD was obtained from the Centre d’expertise en analyze environnementale du Que´bec(Laval,Canada).Stock solutions of3-MC(5m M)and B(a)P(25m M)were prepared in Me2SO,and the stock solutions of daunorubicin(1mg/ml)were pre-pared in water.TCDD was obtained in n-nonane at a concentration of 50␮g/ml and was stored at room temperature.Stock solutions of3-MC, B(a)P,and daunorubicin were stored atϪ80°C.RNA Preparation—Total RNA was prepared from3-MC-treated and untreated hepatocytes as well as from the LR73mdr1,LR73mdr2,and LR73mdr3cell lines by homogenizing the cells in a solution containing guanidium hydrochloride(6M)followed by sequential ethanol precipi-tation,as described previously(21).RNase Protection Assay—The plasmid constructed to detect the mdr1 RNA consisted of a165-bp Bam HI fragment isolated from the mdr1 cDNA(positions1926–2090relative to the ATG initiation codon(22)), blunt-ended with T4DNA polymerase,and cloned into plasmid pGEM-7Z(Promega,Madison,WI)at the Sma I site,giving plasmid pmdr1-G7.This plasmid was linearized with Eco RI and used as a template to synthesize an antisense mdr1probe using SP6RNA polym-erase(Amersham Pharmacia Biotech).The pKX10–3Z plasmid consist-ing of an Xba I–Kpn I mouse␤-actin cDNA fragment(positions724–969 in the␤-actin cDNA)cloned into pGEM-3Z at the Xba I and Kpn I sites (kindly provided by Dr.Rashmi Kothary,Institut du cancer de Mon-tre´al,Montre´al,Canada)was used to generate a control actin probe. pKX10–3Z was linearized with Xba I and used to synthesize an anti-sense actin RNA probe with T7RNA polymerase.The riboprobes were synthesized in the presence of[␣-32P]UTP,and the RNase protection assay was performed according to standard protocols(23).Nuclear Run-on Transcription Assay—The run-on experiment was performed essentially as described by Fisher et al.(24).Nuclei wereisolated from Hepa-1c1c7cells treated with Me2SO or with3-MC(5␮M) for48h and were used to label nascent RNAs with[␣-32P]UTP.Plas-mids pVT101-U/mdr1,carrying the full-length mouse mdr1cDNA(25); pmP1450–3Ј,carrying a1.2-kb Pst I cDNA fragment overlapping part of the mouse cyp1a1cDNA(26)(obtained from the ATCC);and pKX10–3Z were linearized with Stu I,Bam HI,and Xba I,respectively.The linear-ized plasmids were denatured,immobilized in duplicate onto a nylon membrane,and hybridized with the[␣-32P]UTP-labeled RNAs for48h at65°C.The membranes were washed and exposed for7days with two intensifying screens.Slot Blot Analyses—Slot blotting was performed as previously de-scribed(21).RNA samples(10␮g)were denatured in7ϫSSC-7.5% formaldehyde for15min at65°C and applied to a nylon membrane (Zeta-Probe).Detection of specific RNAs was performed by hybridiza-tion at65°C in0.5M NaPO4,pH7.2,1m M EDTA,7%SDS,1%bovine serum albumin,and100␮g/ml salmon sperm DNA with32P-labeled DNA probes.The mdr1probe was a4.2-kb Sph I–Eco RI fragment over-lapping the full-length mouse mdr1cDNA,isolated from plasmid pGEM7/mdr1(a gift from Dr.Philippe Gros,McGill University,Mon-tre´al);the cyp1a1probe was a 1.2-kb Pst I fragment isolated from plasmid pmP1450–3Ј;and the actin probe was a245-bp Xba I–Kpn I fragment isolated from pKX10–3Z.The membranes were washed twiceat65°C with a solution containing40m M NaPO4,pH7.2,5%SDS,1 m M EDTA,0.5%bovine serum albumin and twice with a solutioncontaining40m M NaPO4,pH7.2,5%SDS,and1m M EDTA before autoradiography.Chloramphenicol Acetyl Transferase(CAT)Expression Plasmids—Plasmid pMcat5.9consists of a482-bp DNA fragment containing the dioxin-responsive elements of the cyp1a1gene cloned upstream of the mouse mammary tumor virus promoter and the CAT gene(24)(kindly provided by Dr.Allan Okey,University of Toronto).Plasmids pmdr1, p-452,p-245,p-141,and p-93(previously referred to as pSacICAT, pExo6CAT,pExo2CAT,pExo1CAT,and pAluCAT,respectively)have been described elsewhere(27).The mdr1promoter sequence in these constructs ends at positionϩ84with respect to the transcription start site(27).To produce the p53mutant constructs,pM1and pM2,plasmid pSBM13was used.This plasmid consists of a1.2-kb Sac I–Hin dIII mdr1 promoter fragment(positionsϪ1165toϩ84)cloned into M13mp18. Single-stranded DNA was prepared from pSBM13and used as a tem-plate to perform site-directed mutagenesis of the p53binding site,using the mutant oligonucleotides M15Ј-TACCTGAA T AC A TAAAGACA and M25Ј-CGTAAAGA T AA A TCTATGTA(the base changes are shown in boldface type).The resulting M1and M2mdr1promoter fragments were then excised from pSBM13with Sac I and Hin dIII,blunt-ended with T4DNA polymerase,and cloned into plasmid pCAT at the Hin dIII site also blunt-ended with T4DNA polymerase,yielding plasmids pM1 and pM2.The presence of the mutations in the resulting constructs was confirmed by DNA sequencing.Transient Transfections and CAT Assays—Cells were plated at aInduction of the Mouse mdr1Gene by PAHs4820at ZHEJIANG UNIVERSITY, on November 21, Downloaded fromdensity of 8ϫ105/60-mm plate and transfected on the following day with 10␮g of plasmid DNA,using a standard calcium phosphate pre-cipitation method (28).After incubation with the DNA precipitate for 16h,the cells were washed twice with phosphate-buffered saline and supplied with fresh medium containing the different xenobiotics.After 48h,the cells were collected.Cell extracts were prepared,and protein concentrations were determined by the Bradford method (29).CAT activities were assayed by standard protocols as described previously,using 2␮g of proteins (27).Preparation of Nuclear Extracts—Nuclear extracts were prepared according to Schreiber et al .(30),with some modifications.Cells were harvested in cold phosphate-buffered saline,0.6m M EDTA and col-lected by centrifugation.The cell pellets were resuspended in 400␮l of ice-cold buffer A (10m M Tris,pH 8.0,10m M KCl,0.1m M EDTA,0.1m M EGTA,1m M dithiothreitol)containing 0.5m M phenylmethylsulfonyl fluoride,10␮g/ml aprotinin,1␮g/ml pepstatin,and 5␮g/ml leupeptin and swelled on ice for 15min.Subsequently,25␮l of 10%Nonidet P-40were added,and the tubes were vortexed vigorously.The nuclear pellets were collected by centrifugation and resuspended in 100␮l of cold buffer C (20m M Tris,pH 8.0,400m M NaCl,1m M EDTA,1m M EGTA,1m M dithiothreitol)in the presence of protease inhibitors.The suspen-sions were shaken vigorously at 4°C for 1h and centrifuged for 15min at 4°C,and the supernatants were frozen in aliquots at Ϫ80°C.Proteinconcentrations were determined by the Bradford method (29).ElectrophoreticMobility Shift Assay—Oligonucleotides overlapping the potential p53binding site in the mdr1promoter (5Ј-GAACACGTA-AAGACAAGTCTAT)and the p53consensus sequence in the p21waf1/cip1promoter (5Ј-GAACATGTCCCAACATGTTGAG)(31)were end-labeled with ␥-32P using T4polynucleotide kinase and annealed to their respec-tive in a M M 2.5m M dithiothreitol,4%Ficoll,1␮g of poly(dI-dC),and 20,000cpm of radiolabeled probe.The binding reactions were carried out at room temperature for 15min.Where needed,1␮g of the monoclonal anti-p53antibody pAb421(32)(Calbiochem)or of the polyclonal anti-Jun or anti-Skn-1antibodies (Santa Cruz Biotechnology,Inc.,Santa Cruz,CA)was added,and the incubation was continued for an additional 15min.The complexes were separated on 5%nondenaturing polyacrylamide gels in 1ϫTBE (90m M Tris,65m M boric acid,2.5m M EDTA,pH 8.0)at 200V.The gels were exposed to XAR films (Eastman Kodak Co.)for 16h with two intensifying screens at Ϫ80°C.Western Blotting—Total proteins from 3-MC-or Me 2SO-treated Hepa-1c1c7and BP r c1cells were extracted in ice-cold buffer (10m M Tris-HCl,pH 8.0,150m M NaCl,1m M EDTA,1%Nonidet P-40,and 1%sodium deoxycholate)containing 10␮g/ml leupeptin,10␮g/ml aproti-nin,1␮M sodium orthovanadate,and 1m M phenylmethylsulfonyl flu-oride.Total proteins (75␮g/sample)or nuclear extracts (30␮g/sample)were separated by SDS-polyacrylamide gel electrophoresis on a 10%acrylamide gel,transferred to a nitrocellulose membrane,and analyzed with the monoclonal anti-p53antibody pAb421(32)(Calbiochem)at a concentration of 5␮g/ml.Immune complexes were revealed by incuba-tion with a goat anti-mouse IgG antibody coupled to alkaline phospha-tase (Bio-Rad)and developed with 5-bromo-4-chloro-3-indolylphosphate p -toluidine salt and nitro blue tetrazolium chloride substrates as rec-ommended by the manufacturer (Life Technologies,Inc.).RESULTSTranscriptional Induction of the Mouse mdr1Gene by 3-MC in Hepatoma Cells—We have used an RNase protection assay to study the expression of mdr1in the hepatoma cell line Hepa-1c1c7upon exposure to 3-MC (Fig.1).An mdr1-specific riboprobe was prepared by cloning into pGEM7-Zf a mouse mdr1cDNA fragment overlapping the linker region of the protein,this domain displaying the lowest sequence homology among the three mouse mdr cDNAs (21).When tested with RNA prepared from LR73stable transfectants expressing each of the three mouse mdr cDNAs,the mdr1riboprobe was found to recognize the mdr1RNA but not the mdr2or mdr3RNA,thus confirming its specificity (Fig.1,top right ).The mdr1probe was then used with RNA from Hepa-1c1c7cells treated or not with 3-MC (Fig.1,top left ).This experiment showed that the amount of mdr1RNA detected is very low in untreated cells but is strongly increased in 3-MC-treated cells,demonstrating that expression of the mouse mdr1gene is induced by 3-MCtreatment.The use of an actin probe confirmed that equal quantities of RNA were used in the assay (Fig.1,bottom ).A similar experiment performed with mdr2-and mdr3-specific riboprobes showed that the expression of these genes is not induced under such conditions,demonstrating that the induc-tion of mdr1expression by 3-MC is isoform-specific (data not shown).A nuclear run-on experiment was performed to determine whether mdr1induction by 3-MC occurs at the transcriptional level (Fig.2).In addition to the mouse mdr1cDNA,cDNAs for the mouse cyp1a1gene (known to be transcriptionally regu-lated by 3-MC (12))and for the actin gene were also included as positive and negative controls,respectively.The data in Fig.2show that 3-MC induces an increase in the rate of mdr1mRNA synthesis,indicating that 3-MC acts at the transcriptional level to induce mdr1gene expression in Hepa-1c1c7cells.AhR ⅐Arnt-dependent Induction of mdr1Expression by 3-MC—To determine whether the increase in mdr1expression in response to 3-MC exposure is AhR ⅐Arnt-mediated,we ana-lyzed the mdr1RNA levels upon 3-MC treatment in two wild-type hepatoma cell lines Hepa-1c1c7and Hepa 1–6and in two variant cell lines derived from Hepa-1c1c7,TAOc1BP r c1(AhR-defective)and BP r c1(Arnt-defective)(15)(Fig.3).As controls,we also analyzed the level of cyp1a1and actin expression under the same conditions (Fig.3,middle and right ,respectively).This experiment showed that mdr1is expressed at low levels in the four cell lines in the absence of 3-MC induction (Fig.3,left panel ).Upon 3-MC treatment,the expression of mdr1is in-duced in the two wild-type hepatoma cell lines (by ϳ5-fold),this induction being completely abrogated in the AhR-defective or in the Arnt-defective variants (Fig.3,left panel ).The actin control probe confirmed that equal amounts of RNA had been applied to the membrane (Fig.3,right panel ).These data clearly demonstrate that the induction of mdr1in response to 3-MC requires an intact AhR ⅐Arnt complex,like cyp1a1(Fig.3,middle )(12).The Mouse mdr1Promoter Confers 3-MC-regulated Expres-sion in an AhR ⅐Arnt-dependent Manner—To determine if reg-ulatory sequences responsible for mdr1induction by 3-MC are present in the promoter region of the gene,plasmid pmdr1,consisting of a 1.2-kb Sac I–Hin dIII DNA fragment overlapping the mdr1promoter region (positions Ϫ1165to ϩ84with respect to the transcription start site (27))fused to the CAT reporter gene,was analyzed in transient transfection experiments.Plasmid pMcat5.9,which consists of a 482-bp fragment derived from the cyp1a1promoter fused to the mouse mammary tumorF IG .1.Increased mdr1expression in Hepa-1c1c7upon 3-MC treatment.The expression of mdr1was analyzed by RNase protection assay.Total RNAs (45␮g)from Hepa-1c1c7cells treated with 5␮M 3-MC (ϩMC )or with Me 2SO (ϪMC )for 56h and from the control cell lines LR73/mdr1,LR73/mdr2,and LR73/mdr3were analyzed with an mdr1riboprobe,which protects a 169-nt fragment within the mdr1transcript,or with a ␤-actin riboprobe,which protects a 245-nt actin transcript fragment.Autoradiography was for 15h with two intensify-ing screens (mdr1)or for 5h without intensifying screens (actin ).Induction of the Mouse mdr1Gene by PAHs4821at ZHEJIANG UNIVERSITY, on November 21, 2012 Downloaded fromvirus promoter and to the CAT gene (24),as well as the empty pCAT vector were also included as positive and negative con-trols,respectively.The three plasmids were transiently trans-fected into Hepa-1c1c7and BP r c1cells.The cells were treated with 3-MC or with Me 2SO for 48h,and the cellular extracts were prepared and assayed for CAT activity.This experiment showed that the mdr1promoter is transcriptionally active in Hepa-1c1c7cells and BP r c1cells,since it can drive the expres-sion of the CAT gene in both cell lines,albeit at low levels (Fig.4).This result is consistent with the basal level of expression of mdr1detected by slot blot analysis in these cells (Fig.3).3-MC treatment of the Hepa-1c1c7cells transfected with pmdr1re-sulted in a 10-fold induction in CAT activity as compared with untreated cells,reaching levels of CAT activity similar to those detected in the Hepa-1c1c7pMcat5.9transfectants upon 3-MC treatment.However,this induction was completely abrogated in BP r c1cells (Fig.4),consistent with the lack of mdr1induc-tion at the RNA level observed in the slot blot assay (Fig.3).Similar results were obtained upon transfection in TAOc1BP r c1cells (data not shown).These results,showing that the mdr1promoter is able to activate the expression of the reporter gene in response to 3-MC in Hepa-1c1c7but not in BP r c1and TAOc1BP r c1cells,demonstrate that (i)the mdr1promoter is able to confer 3-MC-mediated transcriptional acti-vation;(ii)this activation requires a functional AhR ⅐Arnt com-plex;and (iii)the sequences mediating this induction are lo-cated between positions Ϫ1165and ϩ84in the mdr1promoter.Two Putative XREs Located in the mdr1Promoter Are Dis-pensable for the Induction of mdr1by 3-MC—The AhR ⅐Arnt transcriptional complex binds to a specific DNA sequence,5Ј-(A/T)NGCGTG,known as an XRE to activate transcription (12).XREs render heterologous promoters responsive to xeno-biotics and function in a position-and orientation-independent manner (33,34).Examination of the mdr1promoter sequence indicated the presence of two potential XREs in an inverted orientation in the distal portion of the promoter at positionsϪ1129and Ϫ620(5Ј-CACGCAT and 5Ј-CACGCAA,respective-ly).To identify the cis -acting sequences responsible for the induction of mdr1by 3-MC and to investigate the possible involvement of these putative XREs,we analyzed the tran-scriptional activity of a series of mdr1promoter 5Ј-deletion CAT constructs after transient transfection into Hepa-1c1c7and treatment of the resulting transfectants with 3-MC (Fig.5A ).3-MC treatment of Hepa-1c1c7cells transfected with plas-mids p-452or p-245resulted in a level of CAT induction similar to that observed in cells transfected with plasmid pmdr1car-rying the full-length promoter,indicating that sequences lo-cated within positions Ϫ1165to Ϫ245are dispensable for the induction of mdr1by 3-MC,including the two putative XREs as well as a potential AP-1binding site (5Ј-TGACTCA;positions Ϫ265to Ϫ255(35))(Fig.5,B and C ).However,further deletion of a 104-bp region down to position Ϫ141(p Ϫ141)was found to greatly diminish the induction of CAT activity by 3-MC (Fig.5,B and C ),demonstrating that sequences important for the induction are located between positions Ϫ245and Ϫ141.CAT activity in the absence of 3-MC was reduced in the p Ϫ141transfectants when compared with the p Ϫ245transfectants,indicating that sequences between positions Ϫ245and Ϫ141are also involved in the basal transcriptional activity of the mdr1promoter in hepatoma cells.Finally,we found that alowF IG .2.Nuclear run-on experiment.Nuclei were isolated from Hepa-1c1c7cells treated with 5␮M 3-MC (ϩMC )or with Me 2SO (ϪMC )for 48h.Nascent RNAs were radiolabeled with [␣-32P]UTP and used to probe duplicate nylon membranes on which denatured cDNAs for mdr1,cyp1a1,and actin had been immobilized.The membranes were washed and exposed for 7days with two intensifyingscreens.F IG .3.AhR ⅐Arnt-dependent induction of mdr1expression by 3-MC.Total RNAs (10␮g)from wild-type Hepa-1c1c7and Hepa 1–6,AhR-defective TAOc1BP r c1,and Arnt-defective BP r c1cells treated (ϩMC )or not treated (ϪMC )with 3-MC at 5␮M for 56h were applied onto a nylon membrane.The membrane was hybridized sequentially with an mdr1(left ),a cyp1a1(middle ),and a ␤-actin (right )probe.Autoradiography was for 18h (mdr1and cyp1a1)or for 2h (actin)with two intensifyingscreens.F IG .4.AhR ⅐Arnt-dependent induction of the mdr1promoter by 3-MC.Plasmids pCAT (no promoter),pmdr1(mdr1promoter from position Ϫ1165to ϩ84),and pMcat5.9(pMcat;482-bp fragment from the cyp1a1promoter fused to the mouse mammary tumor virus pro-moter)were transiently transfected into Hepa-1c1c7and BP r c1cells by the calcium phosphate method.The cells were then treated with 3-MC (5␮M )or Me 2SO for 48h.Total cellular extracts were prepared,and equal quantities of proteins (2␮g)were assayed for CAT activity.A ,autoradiogram of a representative CAT assay,showing the activity of plasmids pCAT,pmdr1and pMcat in Hepa-1c1c7and BP r c1cells treated (ϩ)or not treated (Ϫ)with 3-MC (MC ).The position of the [14C]chloramphenicol (CM )and of its acetylated products (AcCM )is indicated on the left .B ,quantitative analysis of CAT activities.The percentage of conversion of [14C]chloramphenicol to its acetylated de-rivatives was quantitated by liquid scintillation counting.Open bars ,ϪMC ;filled bars ,ϩMC .The results presented are the averages of three independent transfections performed in duplicate.S.D.values are rep-resented by the bars .Induction of the Mouse mdr1Gene by PAHs4822 at ZHEJIANG UNIVERSITY, on November 21, 2012 Downloaded from。

Porcine reproductive and respiratory syndrome virus as a vector:Immunogenicity of green fluorescent protein and porcine circovirus type 2capsid expressed from dedicated subgenomic RNAsYanlong Pei a ,Douglas C.Hodgins a ,Jiaqiang Wu a ,1,Siao-Kun W.Welch b ,Jay G.Calvert b ,Gang Li c ,Yijun Du d ,Cheng Song a ,d ,Dongwan Yoo d ,⁎aDepartment of Pathobiology,University of Guelph,Guelph,Ontario,Canada N1G 2W1bP fizer Animal Health,Kalamazoo,MI 49001,USA cInstitute of Animal Health and Husbandry,Chinese Academy of Agricultural Sciences,Beijing,China dDepartment of Pathobiology,University of Illinois at Urbana-Champaign,2001South Lincoln Ave,Urbana,IL 61802,USAa b s t r a c ta r t i c l e i n f o Article history:Received 31January 2009Returned to author for revision 3March 2009Accepted 31March 2009Available online 6May 2009Keywords:PRRSVReverse geneticsForeign gene expression vector Vaccine vector Nidovirus ArterivirusPorcine reproductive and respiratory syndrome virus (PRRSV)is the causative agent of PRRS,which is characterized by late-term abortions in sows and respiratory disease in young ing an infectious cDNA clone of North American PRRSV strain P129,the viral genome was engineered to transcribe an additional subgenomic RNA initiating between non-structural and structural genes.Two unique restriction sites and a copy of the transcription regulatory sequence for ORF6(TRS6)were inserted between ORFs 1b and 2a,yielding a general purpose expression vector.The enhanced green fluorescent protein (GFP)gene was cloned between the unique sites such that the inserted gene was transcribed from TRS2which was located upstream within ORF1b,while the copy of TRS6drives ORF2a/b transcription.Upon transfection of cells with this plasmid,PRRSV infection was initiated and progeny virus “P129-GFP ”was obtained.Cells infected with P129-GFP showed fluorescence and the inserted gene was phenotypically stable for at least 37serial in vitro passages.Subsequently,a capsid (C)protein gene was cloned from porcine circovirus type 2(PCV2)recovered from an outbreak of porcine multisystemic wasting syndrome (PMWS)and inserted into the PRRSV infectious clone vector,generating virus “P129-PCV ”.To determine the immunogenicity of the recombinant viruses,pigs were immunized intramuscularly with P129-WT (wild-type),P129-GFP,or P129-PCV2.By 5weeks post-infection,speci fic antibody responses to GFP and PCV2capsid were elicited.This is the first report of foreign gene expression using PRRSV from dedicated subgenomic RNAs and demonstrates the potential use of PRRSV as a vaccine vector for swine pathogens.©2009Elsevier Inc.All rights reserved.IntroductionPorcine reproductive and respiratory syndrome (PRRS)is an emerged and re-emerging disease in pigs.The disease was first recognized in Germany and the US almost simultaneously in the late 1980s and has since spread globally to most pork producing countries (Keffaber,1989;Ben field et al.,1992;Albina,1997).PRRS is characterized by abortions and mummi fied fetuses in sows,and respiratory distress with poor growth in young pigs.The disease is mild in gilts and boars,but the virus (PRRSV)persists in semen and thus can be transmitted widely by arti ficial insemination.Since its emergence,PRRS has become one of the most economically important diseases in the swine industry.Modi fied-live vaccines are available,but safety and limited ef ficacy areongoing concerns.No speci fic treatment is available for PRRS,and thus the economic losses are enormous.Numerous isolates of PRRSV representing many geographical regions have been sequenced,revea-ling the existence of two distinct genotypes of PRRSV:European (type I)and North American (type II).The two genotypes share overall sequence identity of 63%and differ antigenically as well as genetically (Nelson et al.,1993;Meng et al.,1995;Wootton et al.,2000).PRRS virus is an enveloped,single-stranded,positive-sense RNA virus belonging to the family Arteriviridae and along with the Coronaviridae family,forms the order Nidovirales .The PRRSV genome is approximately 15kb in size and includes the 5′cap structure and 3′polyadenylated tail (Sagripanti et al.,1986;Wootton et al.,2000).The genome consists of nine genes:open reading frames (ORFs)1a,1b,2a,2b,3,4,5,6,and 7.The 5′three-quarters of the genome consists of two slightly overlapping ORFs,1a and 1b,and they are translated directly from the genome-sense RNA.ORF1b is expressed as a fusion protein with ORF1a by a frame-shifting mechanism,and the ORF1a and ORFla/b proteins are auto-cleaved by viral proteases into at least 13cleavage products that are theVirology 389(2009)91–99⁎Corresponding author.E-mail address:dyoo@ (D.Yoo).1Current address:Shandong Key Laboratory of Animal Disease Control and Breeding,Shandong Academy of Agricultural Sciences,Jinan,Shandong,China.0042-6822/$–see front matter ©2009Elsevier Inc.All rights reserved.doi:10.1016/j.virol.2009.03.036Contents lists available at ScienceDirectVirologyj o u r n a l ho m e p a g e :w w w.e l s ev i e r.c o m /l o c a t e /y v i r onon-structural proteins Nsp1α,Nsp1β,and Nsp2through Nsp12.The Nsps are thought to be involved in genome replication and subgenome transcription(van Dinten et al.,1999;van Marle et al.,1999).The viral structural proteins are encoded by ORFs2a,2b,and3through7which are located downstream of ORFs1a and1b.These genes are expressed as a3′-coterminal nested set of subgenomic(sg)RNAs(de Vries et al., 1990).The5′untranslated leader sequences of the sgRNAs are derived from the5′end of the viral genome and fused to the body segments of the sgRNAs at conserved hexanucleotide motifs[5′UCAAC(U/C)3′] located immediate upstream of every transcription unit(de Vries et al., 1990;den Boon et al.,1996).The conserved hexanucleotide motif and poorly conservedflanking sequences form secondary structures in the sgRNAs that make up the transcriptional regulatory sequences(TRS)that are necessary for sgRNA formation(Pasternak et al.,2000).With the exception of sgRNA7,sgRNAs are structurally polycistronic but,with the exception of sgRNA2a/b,functionally monocistronic as only the5′most proximal gene of each sgRNA is translated.ORFs2a,2b,and3through7 code for GP2(glycoprotein2),E(envelope),GP3,GP4,GP5,M (membrane)and N(nucleocapsid)proteins,respectively,and they make up virion particles(Meulenberg et al.,1995).The recent development of infectious clones for PRRSV has allowed specific alterations of viral genomes and generation of mutant viruses(Yoo et al.,2004).However,manipulation of arterivirus genomes is complicated by the condensed organization of the viral genome.In the case of the European genotype of PRRSV,each gene overlaps slightly except for ORFs 1b and2a(Meulenberg et al.,1993),although the overlap of genes seems less important for virus replication and growth for equine arteritis virus (de Vries et al.,2000).For the North American genotype,structuralgenes Fig.1.Genomic organization of P129-WT,P129-GFP,and P129-PCV2.(A)Genomic organization and sequence of the region surrounding the ORF1b/ORF2junction of the unmodified “wild-type”P129strain of PRRSV(P129-WT).The boxed sequence indicates the core hexanucleotide of TRS2.Amino acids indicate translated sequences of polyprotein1a/b(ORF1b),the GP2protein(ORF2a),and the E protein(ORF2b).The initiation codons are indicated in bold,and the E protein sequence is italicized.Numbers in parenthesis indicate genomic sequence positions.Stars indicate translation stops.(B)P129-GFP.Unique AflII and Mlu I sites and a copy of TRS6were introduced into the non-coding region between ORF1b and ORF2a,creating expression vector pCMV-S-P129-1bMCS2.The GFP gene was amplified by PCR and inserted between the AflII and Mlu I restriction sites.(C)P129-PCV2.The PCV2 capsid gene was amplified by PCR and cloned between the AflII and Mlu I restriction sites.92Y.Pei et al./Virology389(2009)91–99also overlap with the exceptions of ORFs 1b and 2a,and ORFs 4and 5(Nelsen et al.,1999;den Boon et al.,1991;Snijder and Meulenberg,1998).Thus,the entire genome of the North American type PRRSV possesses only 4short non-coding regions:191nucleotides of 5′untranslated region (UTR),1nucleotide between ORF1b and ORF2a,10nucleotides between ORF4and ORF5,and 151nucleotides of 3′UTR upstream of the polyadenylation tail.The 5′and 3′UTRs contain genome replication and transcription signals,and therefore offer limited sites for gene insertion and manipulation.The presence of overlapping genes hampers mutational analysis of the N-and C-termini of the structural proteins and also makes it dif ficult to insert heterologous genes into the viral genome.In the present study,we used a genomic cDNA clone of PRRSV (Lee et al.,2005)to generate a vector for foreign gene expression from a dedicated subgenomic transcription unit inserted in the region between the structural and non-structural genes.The vector was used to express GFP in vitro and in vivo .The inserted gene was tolerated by the virus,stable for at least 37passages in cell culture,and induced antibodies to GFP in young pigs.Using this approach,we also generated a recombinant PRRSV expressing the capsid protein gene of porcine circovirus type 2(PCV2).PCV2is a small DNA virus in the Circoviridae family,with a genome ofonly 1.76kb.PCV2ORF1is essential for viral DNA replication (Mankertz et al.,1998;Fenaux et al.,2000),while ORF2encodes the capsid protein containing type-speci fic epitopes that are believed to be important for virus neutralization (Nawagitgul et al.,2000;Fenaux et al.,2004).Accumulating evidence suggests a major role for PCV2in postweaning multisystemic wasting syndrome (PMWS)and porcine dermatitis and nephropathy syndrome (PDNS)(Hasslung et al.,2005).These syndromes cause serious economic impacts in the swine industry today (Chae 2005).We showed the recombinant PRRSV P129-PCV2induced an anti-PCV2antibody response in immunized pigs in the presence of maternal antibodies to PCV2.Our vector construction may be applicable not only for PRRSV,but also for other members of the families Arteriviridae and Coronaviridae .ResultsDevelopment of PRRSV as an expression vector for GFPTo explore the possibility of developing PRRSV as a gene expression vector,the GFP gene was inserted between the stop codon ofORF1bFig.2.(A)Restriction patterns of P129-WT (lane 1),P129-GFP (lane 2),and P129-PCV2(lane 3)genomic clones generated by Sma I digestion.Fragments of 590bp,4736bp and 10,779bp are expected from all three clones.The fourth fragment varies in size depending on the gene inserted at the non-structural and structural gene junction.Relative to the P129-WT fragment (2787bp,lane 1),the GFP-containing fragment from P129-GFP is 766bp larger (3553bp,lane 2)and the PCV2capsid-containing fragment from P129-PCV2is 754bp larger (3541bp,lane 3).(B –E)Recovery of recombinant PRRSV foci from full-length genomic clones.(F)Integration of the GFP gene in P129-GFP viral genome.Genomic RNA was extracted from P129-GFP virus and digested with DNase I prior to reverse transcription.Without reverse transcription,no products were ampli fied from P129-GFP using ORF7-speci fic PCR primers P129-7F and P129-7R (lane 1)or primers P129-1bF and P129-2aR that span the insertion site (lane 2).With reverse transcription,primers P129-7F and P129-7R ampli fied the ORF 7fragment (534bp)from both P129-WT (lane 3)and P129-GFP (lane 4).Using P129-1bF and P129-2aR,a product of 766bp larger (lane 6)than the product from P129-WT (lane 5)was ampli fied from P129-GFP.(G)Integration of the PCV2C gene in PRRSV.Genomic RNA from P129-WT (lanes 9,10,12)and P129-PCV2(lanes 7,8,and 11)was ampli fied using primers P129-1bF and pared to P129-WT (lane 9)a product that is 754bp larger was ampli fied from P129-PCV2(lane 7).Using PCV2C gene speci fic primers PCV2-F and PCV2-R,the expected 497bp product was generated from P129-PCV2(lane 8)but not from P129-WT (lane 10).Using PRRSV ORF4-speci fic primers P129-4F and P129-4R,the expected 567bp product was ampli fied from both P129-PCV2and P129-WT (lanes 11and 12,respectively).93Y.Pei et al./Virology 389(2009)91–99and the start codon of ORF2a (Fig.1A).This non-coding region is extremely short,comprising only one adenosine nucleotide.The TRS associated with ORFs 2a and 2b (TRS2;TGAACC)is positioned 26nucleotides upstream from the start of ORF2a and is embedded in ORF1b.Upon insertion of GFP to the region,TRS2will drive transcription of the GFP gene instead of ORFs 2a and 2b.Thus,a synthetic TRS (TTAACC)with flanking sequences derived from TRS6was introduced 22nucleotides downstream of GFP and 17nucleotides upstream from the ORF2a start (Fig.1B).TRS6was chosen because RNA secondary structure suggested that it was shorter than other PRRSV TRS elements and because the distance between the copy of TRS6driving ORF2a/b and the authentic TRS6ensured that potential intramolecular homologous RNA recombination would result in a non-viable (ORF2-5deleted)virus.The PRRSV genomic clone containing GFP was designated P129-GFP,and the insertion was con firmed by restriction patterns (Fig.2A,lane 2)and sequencing.MARC-145cells were transfected with P129-GFP and the production of virus was monitored daily for development of cytopathic effect (CPE)(Figs.2B,C,D).CPE was observed 4days post-transfection and the development of CPE was one day slower than for P129-WT.After three consecutive passages,P129-GFP virus was re-examined for GFP sequence integration in the viral genome (Fig.2F).While ORF7ampli fication products were identical in size for P129-WT (lane 3)and P129-GFP (lane 4),1bF and 2aR primers produced a larger size product from P129-GFP (lane 6)than from P129-WT (lane 5).The larger product was the expected size for correct insertion of GFP gene intothe viral genome.Replication of P129-GFP was slightly slower than that of P129-WT at passages 1to 3.However,plaques were comparable in size and morphology for P129-WT and P129-GFP,and the titers at passage 3were 5×105plaque forming units (PFU)/ml and 1×105PFU/ml,respectively.Fluorescence was evident in P129-GFP-infected cells (Fig.3A),demonstrating the expression of GFP during infection.To examine the genetic stability of recombinant PRRSV,P129-GFP was passaged 37times in MARC-145cells and GFP expression was monitored by fluorescence microscopy.Individual plaques of 20formed by P129-GFP were randomly chosen and examined for fluorescence.The selected plaques were all positive for fluorescence (Table 1),and sequencing of the viral RNA con firmed stability of the insert (data not shown).This data showed the genetic stability of P129-GFP after serial passages in cell culture and the stable expression of GFP.It also demonstrates that the region between ORF1b and ORF2a is a suitable site for foreign gene insertion for PRRSV.This was the first demonstration of the use of a nidovirus as an expression vector wherein the foreign gene is inserted in the region between the non-structural and structural protein codingsequences.Fig.3.Expression of GFP or PCV2capsid protein by P129-GFP and P129-PCV2in MARC-145cells.(A)Live cells infected with P129-GFP passage 3;(B)live cells infected with P129-GFP passage 37;(C)uninfected cells;(D)P129-PCV2passage 3fixed and stained with PCV2-speci fic antibody conjugated with FITC at 48h post-infection;(E and F)P129-PCV2infected cells fixed and co-stained with PCV2-speci fic antibody conjugated with FITC (E)or GP4protein-speci fic monoclonal antibody 169(F).Table 1Stability of GFP expression in P129-GFP virus.Virus Titer (PFU/ml)GFP expressing plaques Passage 38.3×10e620positive/20plaques Passage 372.4×10e720positive/20plaques94Y.Pei et al./Virology 389(2009)91–99Construction of PRRSV expressing PCV2capsidPCV2is associated with porcine multisystemic wasting disease (PMWS,now termed PCVAD [PCV-associated disease])and porcine dermatitis and nephropathy syndrome (PDNS).PCV2is transmitted by the oro-nasal route and shed in the bronchial secretions and feces,thus the transmission route is similar to that of PRRSV.The capsid (C)protein is the major antigen able to elicit protective immunity against PCV2.Thus,using the PRRSV vector system described above,an additional recombinant PRRSV was constructed to carry the PCV2C gene.A 702bp C gene was cloned by PCR from a lung tissue positive for PCV2.Sequencing of the C gene showed 99–100%amino acid identity to published sequences available in the GenBank database.The P129-PCV2clone was constructed by inserting the C gene into PRRSV in the same way as the GFP gene insertion for P129-GFP (Fig.1C).The insertion of the C gene was con firmed by restriction digestion pattern (Fig.2A,lane 3)and sequencing.The P129-PCV2recombinant virus was recovered from MARC-145cells by transfection (Fig.2E),and the insertion of the C gene in the viral genome was con firmed by RT-PCR of the viral RNA (Fig.2G).The titer of passage 3virus was 2×105PFU/ml and the plaque morphology was indistinguishable from P129-WT.Infection of cells with P129-PCV2and staining with PCV2-speci fic antibody produced distinct fluorescence (Fig.3E),which shows the expression of the C protein by P129-PCV2.The capsid protein of PCV2is arginine-rich and normally shuttles into the nucleus during PCV2replication (Liu et al.,2001),and similarly,the PRRSV N has also been shown to localize in the nucleus and nucleolus (Lee et al.,2006;Pei et al.,2008).Thus,in cells infected with P129-PCV2,the synthesis of legitimate PCV2capsid should be evident by the translocation of capsid into the nucleus.Thus,the C protein expression by P129-PCV2was con firmed by co-staining of virus-infected cells with PRRSV N protein-speci fic antibody and PCV2-speci fic antiserum (Fig.4).While the PRRSV N protein was found in the both cytoplasm and the nucleolus as usual (panel A),the PCV2capsid protein was speci fically localized to the nucleus and nucleolus (panel C)in the same cell,clearly demonstrating the expression of PCV2capsid protein by the recombinant PRRSVP129-PCV2.Fig.4.Co-expression of the PRRSV N protein (green)and the PCV2capsid protein (red)during infection of P129-PCV2.MARC-145cells were infected with P129-PCV2and stained 24h post-infection with PRRSV N-speci fic MAb SDOW-17(A and B)or PCV2-speci fic pig serum (C and D).Arrows indicate the PRRSV N protein in the nucleolus (panel A)in addition to the cytoplasm and the PCV2capsid protein in the nucleus and nucleolus (panel C).Panel E shows the merge of A and C.Panel F shows the merge of B and D.Table 2PCR primers and their genomic Sequence (5′–3′)aGenomic position b PurposePCV2-F cacggatattgtagtcctggt 1093–1114PCV2PCR test PCV2-R ccgcaccttcggatatactgtc1565–1586PCV2PCR testPCV2-orf2F gatgcttaagatgacgtatccaaggtggcg 1715–1734PCV2ORF2ampli fication PCV2-orf2R gtacacgcgtcattaagggttaagtcccccc 1031–1050PCV2ORF2ampli ficationP129-F1F aacagaagagttgtcgggtccac11,699–11,721P12911,783–12,055,ampli fication P129-F1R gctttcacgcgtccccacttaagttcaattcaggcctaaagttggttca 12,031–12,055Introduction of A flII and Mlu IP129-F2F gcgacgcgt gttccgtggcaacccctttaaccagagtttcagcggaaga atgaaatggggtctatacaaagcctcttcgaca 12,056–12,089P12912,056–12,697,ampli fication and introduction of Mlu I and TRS6P129-F2R aacagaacggcacgatacaccacaaa 13,819–13,844P12912,056–12,679,ampli fication P129-7F tcatccgattgcggcaaatg 14,724–14,743P129ORF7ampli fication P129-7R agaatgccagcccatca 15,242–15,258P129ORF7ampli fication P129-4F gtttcacctagaatggctg 13,213–13,231ORF4ampli fication P129-4R ccccaacatacttgaacattc 13,750–13,770ORF4ampli ficationP129-1bF ggtgaggactgggaggattac 11,921–11,941ORF1b –ORF2a,region ampli fication P129-2aRcagtacgtagcattggaacc12,758–12,777ORF1b –ORF2a,region ampli ficationa Restriction sites are underlined.TRS6and surrounding sequences are indicated in bold.bGenomic positions for PCV2primers were based on GenBank accession AF027217.Genomic position for P129primers were based on GenBank accession AF494042.95Y.Pei et al./Virology 389(2009)91–99Infection of pigs and antibody responses to GFP and PCV2capsid protein To determine antibody responses to GFP and the PCV2capsid protein,pigs were immunized with the recombinant PRRS viruses.Fifteen PRRSV-free pigs at 4weeks of age were randomly allotted to 3groups of 5pigs each.Animals were immunized twice on days 0and 21by intramuscular injection of 5×105PFU per animal with either P129-WT,P129-GFP,or P129-PCV2.Following inoculation,the animalswere maintained for 5weeks for clinical observation and serum collection.Clinical signs of PRRS were minimal and comparable in all 3groups (data not shown).Mild clinical signs were not unexpected,since the infectious cDNA clone used in these studies was not attenuated.Tonsil samples were collected at necropsy on day 35and assessed by RT-PCR for the presence of PRRSV ORF7using primers P129-7F and P129-7R (Table 2)as well as the GFP and PCV2capsid inserts using primers P129-1bF and P129-2aR.All 15pigs were positive for ORF7,indicating infection and persistence of PRRSV in the tonsils.PCR products from the ORF1b/ORF2a junction were not detected in these tonsil samples,possibly due to the much lower molar ratio of ORF1b-containing RNA template relative to ORF7-containing RNA in infected cells.Alternatively,it is possible that the GFP and PCV2capsid genes might have been unstable in vivo and lost in the inoculated pigs over time.To examine antibody responses in these pigs,ELISAs were conducted for PRRSV,GFP,and PCV2C protein.All pigs produced good levels of antibodies to PRRSV (Fig.5A),and the antibody titers were comparable among groups.P129-GFP elicited speci fic antibodies to GFP,whereas pigs immunized with P129-PCV2or P129-WT were negative for GFP (Fig.5B).The GFP antibodies increased following first immunization,and the second immunization at day 21boosted the antibody response somewhat (Fig.5B).Similarly,antibodies for PCV2C protein were detected in pigs immunized with P129-PCV2(Fig.5C).In these pigs,however,PCV2antibodies were detected at day 0in all 3treatment groups and tended to wane over time.These antibodies likely represent maternal antibodies taken up in colostrum shortly after farrowing at the farm of origin and prior to experimental infection.However,an increase in anti-PCV2antibodies was observed at 28days in the P129-PCV2group only (Fig.5C)due to boosting effects from the second immunization at day 21.In contrast,anti-PCV2antibodies in the other two treatment groups waned gradually from day 0until at least until day 35.To further determine the speci fic antibody responses to GFP and the PCV2C protein,Western blots were conducted using sera from these pigs.Serum from the P129-GFP group was reactive with GFP (Fig.6A),consistent with the ELISA data (Fig.5B).Similarly,serum from the P129-PCV2group showed a strong reaction with C pro-tein (Fig.6B).Weaker reactions were observed using sera from the P129-WT and P129-GFP groups,consistent with the presence of maternalantibodies.Fig.5.ELISA showing induction of speci fic antibodies in sera from pigs inoculated with P129-GFP,P129-PCV2,or P129-WT viruses.(A)antibodies to PRRSV N protein;(B)antibodies to GFP;(C)antibodies to PCV2Cprotein.Fig. 6.Western blots showing induction of speci fic antibodies in sera from pigs inoculated with P129-GFP,P129-PCV2,or P129-WT at day 0or day 35post-inoculation.Blots contain GFP protein (A)or PCV2virions (B).Arrows indicate positions of GFP and PCV2capsid protein.C denotes positive control (anti-GFP monoclonal antibody).M indicates molecular weight markers.The virus used to infect pigs that contributed to the serum pools is indicated above each lane.96Y.Pei et al./Virology 389(2009)91–99DiscussionThe primary target cell of PRRSV is the alveolar macrophage,and pigs are the only animal species known to be susceptible to PRRSV infection.Therefore,development of PRRSV as a vaccine vector would be useful for the delivery of porcine pathogen genes to the respiratory tract of the pig.Arterivirus genomes are organized in a complex way. Most genes overlap one another in different reading frames,making it difficult to engineer the genome for foreign gene insertion.An early approach involved engineering the3′terminal region of the N gene (Groot Bramel-Verheije et al.,2000).Modification of the3′terminal sequence of N gene was possible and caused only minimal effects on virus replication and growth.However,no more than7amino acids were inserted.More recently,Nsp2,a large product of proteolytic cleavage of the ORF1a and ORF1a/b polyproteins,was found to be remarkably heterogeneous in sequence,with several hypervariable regions.Therefore,the GFP gene was inserted in-frame into or between the hypervariable regions to create nsp2-GFP fusion proteins (Fang et al.,2006;Kim et al.,2007).This approach was successful and allowed for the production of recombinant viruses.However,in all cases the inserted GFP gene was not phenotypically stable and lost greenfluorescence after several passages in cell culture(Fang et al., 2006;Kim et al.,2007;Han et al.,2007).In the present study,we inserted GFP and the PCV2capsid genes into the short region separating the non-structural protein genes from the structural protein genes.In contrast to the nsp2-GFP fusion proteins described above,we expressed GFP as a separate transcription unit resulting in an additional sgRNA.This approach has the advantage of eliminating the need to alter the coding sequence of a viral gene,and also minimizes effects on expression and post-translational modification of viral gene products.As a result,our recombinant virus was stable for at least37cell culture passages without loss of the gene or the greenfluorescent phenotype.PRRSV is known to induce immune suppressive effects in pigs (Charerntantanakul et al.,2006)and can persist up to6months in infected pigs(Wills et al.,1997).For these reasons,co-infection of PRRSV with other pathogen such as PCV2can result in much more severe clinical outcome than either agent alone(Harms et al.,2001; Kim et al.,2003).Therefore,a dual-purpose vaccine capable of protecting pigs against both PRRS and PCV2would be advantageous. Our study demonstrates the potential of PRRSV as a viral vaccine vector.Prior to the purchase of animals for infection studies using P129-PCV2,all pigs were screened for PCV2by PCR.PCR is the gold standard for detection of PCV2,whereas antibody screening is not routinely conducted in diagnostic laboratories due to cross-reactivity between PCV2and PCV1which is ubiquitous and widely distributed in thefield (Magar et al.,2000).Although all pigs entering the present study tested negative for the presence of PCV2by PCR,antibodies were detected in sera collected on day0.These antibodies were most likely of maternal origin,since they decreased in concentration over the duration of the experiment in pigs receiving P129-WT or P129-GFP. Serological studies show that maternal antibodies for PCV2decay during thefirst2months of life(Rodríguez et al.,2002;Larochelle et al.,2003).Western blots and ELISA gave comparable results in this regard.In the current study,antibodies to PCV2only increased after day21of the study and did so only in pigs receiving P129-PCV2, suggesting that the increases were most likely specific responses to P129-PCV2vaccination.At the termination of the study(day35post-infection),tonsil samples in all3groups were positive for the PRRSV N gene by RT-PCR.The presence of the inserted GFP and PCV2genes in these same samples could not be confirmed using primersflanking the region of the gene insertion.No products were amplified,even from pigs infected with the P129-WT virus.Failure to amplify the ORF1b/ORF2a junction is likely the result of template RNA concentra-tions below the level of detection of the PCR assay,and is consistent with the observation that the copy number of ORF1b(present only on genomic RNA)is much lower than the copy number of ORF7(present on all sgRNAs as well as genomic RNA).In conclusion,a PRRSV gene expression vector was generated, capable of expressing a foreign gene from an additional transcription unit located in the region between the non-structural and structural genes of the virus.The recombinant PRRSVs expressing the GFP or PCV2capsid genes were generated and shown to replicate well in cell culture.The addition of766nt(GFP)or754nt(PCV2capsid)of foreign genetic material,representing approximately5%of the PRRSV genome,was tolerated with no evidence of compensatory deletions or rearrangements elsewhere in the genome.Pigs inoculated with these recombinant PRRSVs produced foreign gene specific antibodies.Our study demonstrates the potential of PRRSV to function as a vector for development of multivalent vaccines against swine diseases.Materials and methodsCells and virusesMARC-145African green monkey kidney cells(Kim et al.,1993) were maintained as previously described(Lee et al.,2003).Dulac porcine kidney cells,kindly provided by L.Babiuk(Vaccine and Infectious Disease Organization,SK,Canada),were grown in Modified Eagle's Medium(MEM)supplemented with5%fetal bovine serum (FBS;Gibco BRL),penicillin(100U/ml),and streptomycin(50μg/ml). Cells were maintained at37°C with5%CO2.Stocks of recombinant viruses derived from infectious clones were prepared by passaging three times on MARC-145cells.Titers of PRRSV were determined by standard plaque assays on MARC-145cells using6-well plates(35mm diameter)in duplicate.Plaques were stained with0.01%neutral red. For isolation of PCV2,lung tissues were obtained from a PCR-positive pig(Ontario18099)submitted to Animal Health Laboratory of the University of Guelph(Guelph,ON,Canada).The tissues were homogenized in PBS and thefiltrate was used to infect Dulac cells. At3days post-infection,cells were stained using a porcine circovirus hyperimmune serum(VMRD,Pullman,WA,USA)to confirm infection. On day4post-infection,cells were harvested and freeze–thawed three times.Cell debris was removed by centrifugation at5000×g, and the supernatant was stored at−80°C until use.Construction of a PRRSV expression vector and recombinant PRRSVsFor construction of the PRRSV expression vector,the regions flanking the ORF1b/ORF2a junction were amplified by PCR using the shuttle plasmid p2-7D4(containing genomic positions11,504to 15,395)as template.Two DNA products corresponding to positions 11,783to12,055and12,056to12,697were amplified.The primer set P129F1-F(containing an Eco47III site)and P129F1-R(containing AflII and Mlu I sites)was used for the upstream product(Table2).The primers set P129F2-F(containing Mlu I site and TRS6)and P129F2-R (containing a Bsr GI site)was used to amplify the downstream product(Table2).The twoflanking products were digested with Eco 47III–Mlu I and Mlu I–Bsr GI,respectively,and included in a three-way ligation with Eco47III–Bsr GI-digested full-length genomic cDNA clone pCMV-S-P129(Lee et al.,2003).The resulting construct pCMV-S-P129-1bMCS2contained a complete PRRSV genome with unique AflII and Mlu I sites and a copy of TRS6inserted between ORF1b and ORF2a.Transfection of MARC-145cells with this construct produced viable virus that replicated normally(data not shown).For insertion of foreign genes,pCMV-S-P129-1bMCS2was digested with AflII–Mlu I and ligated to either the GFP gene or the PCV2capsid gene into which AflII and Mlu I sites were introduced during PCR (Fig.1).Recombinant genomic clones were screened by Sma I digestion(Fig.2),and selected clones were sequenced to confirm the presence of insertions.The PCV2capsid protein gene was cloned97Y.Pei et al./Virology389(2009)91–99。

Vol.41No.3Mar.2021上海交通大学学报（医学版）JOURNAL OF SHANGHAI JIAO TONG UNIVERSITY (MEDICAL SCIENCE)构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究赵艳娜1*，邱荣2*，沈南1，唐元家11.上海交通大学医学院附属仁济医院风湿病科，上海市风湿病学研究所，上海200127；2.中国科学院上海营养与健康研究所，上海200031［摘要］目的·结合Dox 诱导型单链导向RNA （single guide RNA ，sgRNA ）表达载体和Cas9转基因小鼠，构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究。

方法·根据四环素诱导表达系统原理，基因合成U6-TetO-sgRNA 和EF1α-T2A-Puro-BFP-T2A-TetR 片段。

通过同源重组将2个片段组装进反转录病毒载体骨架，获得Dox 诱导型sgRNA 反转录病毒载体。

为了验证系统有效性，分离Cas9转基因小鼠骨髓细胞并诱导其向巨噬细胞方向分化。

设计对照（non-targeting control ，NC ）组和实验组（靶向F4/80）的sgRNA ，利用反转录病毒感染细胞，分化条件设置添加Dox 组（Dox +）和不添加Dox 组（Dox -）。

通过流式细胞术和T7核酸内切酶Ⅰ（T7endonuclease Ⅰ，T7E Ⅰ）实验检测基因敲除效果。

结果·①成功构建Dox 诱导型sgRNA 反转录病毒表达载体和Cas9转基因小鼠。

②流式结果显示，在NC Dox -组、NC Dox +组和F4/80Dox -组中，几乎无F4/80阴性细胞群体；而在F4/80Dox +组中，F4/80阴性细胞群体高达50%。

③T 7E Ⅰ结果显示，在F4/80Dox -组中，DNA 条带完整，而在F4/80Dox +组中发生基因突变，DNA 条带被切开。

Alb-cre／DTR 小鼠可诱导性肝损伤模型的建立任晓楠;任蓉蓉;刘雪;杨华;秦波音;周晓辉【摘要】目的：建立Alb-cre／DTR双转基因小鼠模型并进行相关表型分析，在此基础上建立可诱导性肝损模型，用于肝脏疾病的相关研究。

方法将引进的Alb-cre和DTR小鼠扩繁后，通过杂交的方式获得双转基因小鼠。

提取小鼠尾部组织DNA，利用PCR方法进行基因型鉴定。

在双转基因小鼠上腹腔注射白喉毒素，之后在不同时间点进行称重、采血，检测血清ALT、AST水平。

结果将Alb-cre和DTR小鼠杂交、筛选后获得了Alb-cre／DTR双转基因小鼠，对该小鼠使用0.625 ng／g剂量的白喉毒素，可使小鼠血清中ALT与AST水平显著升高，解剖小鼠后观察到肝整体变白，HE染色结果显示肝细胞明显坏死。

结论成功建立可诱导特异性肝损伤小鼠模型。

%Objective To analyze the Alb-cre/DTR mouse phenotype, and establish a model of induced liver damage to serve basic researches of liver diseases.Methods The introduced Alb-cre and DTR mice were crossed to obtain Alb-cre/DTR mice and the genomic DNAs were extracted from the tail tissue of the mice for genotying by PCR.Diphtheria toxin was intraperitoneally(i.p.)injected into the Alb-cre/DTR mice, then the body weights were monitored and the sera were collected for the detection of serum ALT and AST levels.Results By crossing Alb-cre and DTR mice we obtained the Alb-cre and DTR double transgenic mouse.The intraperitoneal injection of diphtheria toxin in a dose of 0.625 ng/g body weight significantly induced liver injury in these mice, as showed by the elevated levels of ALT and AST, the gross appearance of liver damage and the pathological changes such as necrosis in the liver tissue.ConclusionsWe have ob-tained a novel mouse strain of Alb-cre/DTR by crossing Alb-cre and DTR mice.Liver damages in those Alb-cre/DTR mice can be induced by injection of diphtheria toxin.This established mouse model of inducible liver damage is a useful platform for the studies of liver damage and recovery, as well as liver transplantation.【期刊名称】《中国实验动物学报》【年(卷),期】2016(024)002【总页数】5页(P134-138)【关键词】Alb-cre;DTR;转基因;特异性肝损伤;小鼠【作者】任晓楠;任蓉蓉;刘雪;杨华;秦波音;周晓辉【作者单位】上海市公共卫生临床中心，上海201508;上海市公共卫生临床中心，上海201508;上海市公共卫生临床中心，上海201508;上海市公共卫生临床中心，上海201508;上海市公共卫生临床中心，上海201508;上海市公共卫生临床中心，上海 201508【正文语种】中文【中图分类】Q95-33肝疾病是对人类威胁最大的疾病之一［1-2］，除病毒性肝炎外［3-4］，肝脏疾病中肝硬化、肝癌等危害也相当严重；肝脏移植手术对于解决肝系统疾病和肝代谢紊乱等来说是一种非常重要的手段［5］，但是肝移植手术仍然面临很多尚未解决的问题，需要更多的相关研究来提高移植后肝细胞的再生和增殖问题。