- Email: [email protected]
Improved EBV-based shuttle vector system: dicistronic mRNA couples the synthesis of the Epstein–Barr nuclear antigen-1 protein to neomycin resistance
Gene 197 (1997) 83–89
Improved EBV-based shuttle vector system: dicistronic mRNA couples the synthesis of the Epstein–Barr nuclear antigen-1 protein to neomycin resistance Anne D. Ramage a, A. John Clark a, Austin G. Smith b, Peter S. Mountford b, David W. Burt a,* a Division of Molecular Biology, Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS, Scotland, UK b Centre for Genome Research, The University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh, EH9 3JQ, Scotland, UK Received 2 December 1996; accepted 17 April 1997; Received by A. Bernardi
Abstract Use of EBV-based vector systems has been limited by the requirement to generate EBNA+ cells which are ’permissive’ for replication of an oriP-vector. In current constructs, selectable marker and EBNA-1 are not always co-expressed. This is a significant problem since the EBNA-1 gene product can be toxic in some cell types and may be selected against. In this paper, we describe a gene construct that overcomes this limitation. We have exploited the piconaviral internal ribosome entry site to allow the genes for Epstein–Barr nuclear antigen-1 and G-418 resistance to be transcribed as a dicistronic fusion mRNA under the control of the phosphoglucokinase promoter. This construct can be routinely integrated into human cell lines. The presence of EBNA-1 protein was reflected by a large increase in transfection frequencies (1000-fold) using an oriP-based vector which was shown to replicate stably in these cells with no apparent gross rearrangements detected after 8 weeks in culture. Using this system, G-418 resistance should directly reflect integration, as well as expression of the EBNA-1 gene, which, in turn, increases transfection frequencies and stability of EBV-based vector systems and should result in its increased use. © 1997 Elsevier Science B.V. Keywords: Epstein–Barr virus; Internal ribosome entry site (IRES ); Eukaryotic episomal vectors; Human cell line; Dicistronic fusion mRNA; PGK promoter
1. Introduction Functional complementation is a powerful method for isolating cDNAs encoding novel proteins in mammalian cells. There is a need for a vector system which can transfect cells with high efficiency, support stable replication at moderate copy number and be re-isolated without rearrangements. Episomal vectors can do this and one such system, currently in use, exploits the EBV replication mechanism which depends on a trans-acting factor, EBNA-1, interacting with the replication origin region * Corresponding author. Tel. +44 131 4402726; Fax +44 131 4400434; e-mail: [email protected] Abbreviations: EBV, Epstein–Barr virus; EBNA-1, Epstein–Barr Nuclear Antigen-1; EMCV, encephalomyocarditis virus; IRES, internal ribosome entry site; neor, gene coding for G418 resistance; hyr, gene coding for hygromycin-B resistance; E. coli, Escherichia coli; PGK, phosphoglucokinase; GAPDH, glyceraldehyde dehydrogenase; PCR, polymerase chain reaction; DNA, deoxyribonucleic acid; RSV LTR, rous sarcoma virus long terminal repeat. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 2 45 - X
(oriP) ( Yates et al., 1984; Sugden et al., 1985; Reisman et al., 1985; Yates et al., 1985). Epstein–Barr virus shuttle systems have been developed on the basis that vectors which contain the EBNA1 and oriP genes can replicate in mammalian cells with a low copy number (Margolskee et al., 1988; Groger et al., 1989) and these systems have been used with some success to clone human cDNAs (Strathdee et al., 1992). One disadvantage of having the two EBV elements on the same vector is that stability may be jeopardized and reports have been made of plasmids undergoing extensive rearrangements (Swirski et al., 1992; Heller et al., 1990). Alternatively, the EBNA-1 gene can first be integrated into the genome of a cell line to render it ‘permissive’ for replication of an oriP-vector. The advantage of integrating EBNA-1 is that this leads both to increased stability of the oriP-vector, as well as an increase in transfection frequencies (Cachianes et al., 1993; Swirski et al., 1992; Carstens et al., 1995; Legerski and Peterson, 1992). Such a system may involve co-transfecting the
84
A.D. Ramage et al. / Gene 197 (1997) 83–89
EBNA-1 gene with a selectable marker, such as the gene conferring G-418 resistance, or using one vector containing both selectable marker and EBNA-1 gene. Many reports (as well as experience in our laboratory) suggest that in such systems expression of the neomycin resistance gene need not necessarily reflect expression or even integration of the EBNA-1 gene (Carstens et al., 1995; Swirski et al., 1992). This is a significant problem since the EBNA-1 gene product may be toxic to some cell types if expression is high ( Vidal et al., 1990; Carstens et al., 1995) and so cells in which EBNA-1 is not expressed have a selective growth advantage. In this paper we describe a novel vector in which the EBNA-1 and G-418 resistance genes are transcribed as a dicistronic fusion mRNA under the control of the PGK promoter. To this end we have exploited the picornaviral IRES which acts as a ribosome binding site and permits the effective internal initiation of translation in mammalian cells (Pelletier and Sonenberg, 1988; Akhteruzzaman et al., 1992) allowing EBNA-1 to be expressed downstream of the neor gene.
2. Materials and methods 2.1. Cells A549 human lung carcinoma cells were from the European Collection of Animal Cell Cultures ( ECACC ). Cells were maintained at 37°C (5% CO ) in 2 Dulbecco’s modified Eagle’s medium with -glutamine, -glucose (100 mg/l ), and sodium pyruvate (Gibco). Fetal bovine serum (10%), penicillin (100 U/ml ) and streptomycin (100 mg/ml ) were added. 2.2. Plasmids pSV2neo (Southern and Berg, 1982), pCMVEBNA (Swirski et al., 1992) and pDR2 were purchased from Clontech Laboratories (Cambridge, UK ). pDR2 can be used for the expression of cloned cDNA under control of the RSV LTR promoter and carries the EBV origin (oriP) which confers episomal maintenance to the vector in the presence of the Epstein–Barr virus nuclear antigen-1 ( EBNA-1). The hyr gene carried by pDR2 functions as a selectable marker in human cell lines. pCMVEBNA and pSCV2neo (Fig. 1) are helper plasmids for use with pDR2. pCMVEBNA carries the EBNA-1 gene and pSV2neo carries a selectable marker (neor). The plasmid 6P-iresneobs is a vector for dicistronic expression in mammalian cells of both the gene of interest and a selectable marker (Mountford and Smith, 1995). This plasmid contains the mouse pgk-I promoter (Adra et al., 1987) 5∞ to the EMCV IRES sequence
which is linked to the neomycin phosphotransferase gene (neo) (Ghattas et al., 1991). The neo gene is followed by intron and termination sequences derived from the rabbit b-globin gene and SV40, respectively (Nicolas and Berg, 1983). The plasmid also contains the interferon response element from the human 6-16 gene (Porter et al., 1988) upstream of the PGK promoter, which allows the induction of increased expression by exposure to a/b interferons. Unique SalI, SacI, NotI and XbaI restriction sites are situated between the promoter and the IRES for cDNA insertion. pBT Bluescript was purchased from Stratagene (Cambridge, UK ). Plasmid pDR2 was isolated from A549 cells with QIAprep spin columns (Qiagen, Crawley, UK ) using 107 cells (Siebenkotten et al., 1995). 2.3. Plasmid construction The pEB plasmid was constructed in two steps: (a) a 2.75 kb HindIII fragment which contains the CMV promoter and EBNA-1 gene was subcloned from the pCMVEBNA-1 vector into pBT Bluescript; and (b) a 2.0 kb XbaI fragment containing the EBNA-1 gene was subcloned from this Bluescript subclone into the the 6P-IresNeo-BS vector. Restriction enzyme digests were carried out on miniprep DNA to identify clones containing the EBNA-1 gene in the sense orientation. A novel 9.89 kb plasmid was recovered and named pEB ( Fig. 1A). 2.4. Transfections Transfections were carried out using the calcium phosphate transfection kit from Promega. Stable cell lines were constructed by transfecting A549 cells with the pEB vector and selecting for growth in G-418 (800 mg/ml ). The presence of the vector pDR2 was selected for by growth in hygromycin (700 mg/ml ). 2.5. Southern blot analysis Isolation of DNA from A549 cells was carried out using the procedure of Blin and Stafford (1976). Digested DNA was electrophoresed on 1% agarose gels and blotted onto nylon Zeta-Probe GT (Bio-Rad ) filters. Probes were radiolabelled by random priming according to the method of Feinberg and Vogelstein (1983, 1984). Hybridizations were carried out at 65°C in 10% PEG, 7% SDS, 1.5×SSPE. Filters were washed with 2×SSC, 0.1% SDS at room temperature followed by 0.1×SSC, 0.1% SDS at 65°C and autoradiographed. 2.6. PCR reactions One hundred nanograms of genomic DNA was placed in a total volume of 25 ml containing 1.5 mM MgCl , 2
A.D. Ramage et al. / Gene 197 (1997) 83–89
85
Fig. 1. Comparison of Epstein–Barr vector systems. (A) pEB, a novel vector system exploiting the IRES, which allows the EBNA-1 and neor genes to be transcribed as a dicistronic fusion mRNA under control of the PGK promoter. (B) Original two-vector system from Clontech. The neor gene is transcribed from the pSV2neo vector, while EBNA-1 is transcribed from pCMVEBNA.
1×PCR buffer, 0.2 mM deoxyribonucleotide triphosphates, 10 pM each of the two EBNA-1 primers 5∞-GGTTTTGAAGGATGCGATTAAG; 3∞-TTTAATACGATTGAGGGCGTCT or GAPDH primers 5∞-GTCAGT GGTGGACCTGACCT; 3∞-TGAGCTTGACAAAGTGGTCG and 1 unit of Taq DNA polymerase. The tubes were cycled 25 times through 94°C for 1 min, 60°C for 2 min and 72°C for 3 min. The PCR products (15 ml ) were electrophoresed on 1% agarose gels and stained with ethidium bromide. Primers were designed using the PRIMER (version 0.5) program (S.E. Lincoln, M.J. Daly and E.S. Lander, Whitehead Institute for Biomedical Research, Boston, MA, USA).
3. Results 3.1. Plasmid construction The 9.83 kb pEB plasmid is shown in Fig. 1A and consists of the entire EBNA-1 gene inserted in the sense orientation into the XbaI site of the 6P-IresNeo-BS plasmid under the control of the PGK promoter. The key property of this vector is that the encephalomyocarditis virus-internal ribosome binding site (EMCV-IRES ) allows the EBNA-1 and neor genes to be translated from a common dicistronic mRNA. G-418 resistance should, therefore, be coupled to expression of the Epstein–Barr Nuclear Antigen. The vector also contains the Col E1
86
A.D. Ramage et al. / Gene 197 (1997) 83–89
ori and ampicillin resistance (apr) genes for replication and selection in E. coli. This differs from the existing Clontech system illustrated in Fig. 1B, where the neor and EBNA-1 genes are transcribed independently on separate plasmids. The system depends on co-integration of both plasmids and co-expression of the neor and EBNA-1 genes. 3.2. Integration of EBNA−1 into A549 cells The pEB vector was linearised with the restriction enzyme MluI and 10 mg was used to transfect the A549 cell line, as described in Section 2. Transfectants were selected in 800 mg/ml G-418 and after 2 weeks six individual colonies were picked and expanded. Cell lines were then propogated in Dulbecco’s modified Eagle’s medium under G-418 selection (400 mg/ml ) for 8 weeks. After this time, genomic DNA was isolated and PCR reactions were carried out on this DNA using GAPDH and EBNA-1 primers. Results are shown in Fig. 2A. The expected EBNA-1 PCR product (244 bp) was found
in all DNA samples, except the untransfected control A549, indicating that the EBNA-1 gene was present in the genomic DNA from each of the neomycin-resistant colonies tested. The expected GAPDH PCR product (212 bp) was present in all samples of DNA tested and is the positive PCR control. We had previously attempted to integrate the EBNA1 gene by cutting the pCMVEBNA vector with HindIII and co-transfecting A549 cells with a selectable marker (neor) on the pSV2neo vector (Clontech). Twenty micrograms of pCMVEBNA and 1 mg pSV2neo were used. Resistant colonies were obtained after 2 weeks under G-418 selection (800 mg/ml ) and these were expanded and propogated for 8 weeks under G-418 (400 mg/ml ) selection. Further analysis of the DNA from 10 of these colonies using PCR as described above showed that none was able to amplify the EBNA-1 gene successfully. Results of six of these are shown in Fig. 2B. This indicates zero percentage co-transfection and suggests that the EBNA-1 gene under the control of the RSV LTR promoter may be toxic to A549 cells. 3.3. A549 cells rendered permissive for high transfection efficiency The six individual A549 EBNA-1+ cell lines selected above (as well as control A549 cells) were transfected with 10 mg pDR2 plasmid (Clontech). Results showed at least a 1000-fold increase in hygromycin-resistant colonies in A549 EBNA-1+ cell lines compared with the A549 control ( Fig. 3). This result shows that the EBNA-1 gene is being expressed in the A549 EBNA-1+, cells rendering them permissive for replication of the pDR2. 3.4. Episomal maintenance and replication of the pDR2 vector in permissive A549 cells
Fig. 2. PCR screen for the presence of the EBNA-1 gene in the genomic DNA of A549 cells. PCR reactions using EBNA-1 and GAPDH primers on genomic DNA derived from A549 cells. Genomic DNA was isolated as described in section 2.5. Lane M: 1 kb ladder; lane 1: no DNA; lane 2: A549 genomic DNA; lanes 3–8: DNA from the six A549 colonies grown under G-418 selection for 8 weeks after transfection with either (A) the pEB vector; or (B) the pSCV2neo and pCMVEBNA vectors (1:20 ratio).
The six A549 EBNA-1+(pDR2) cell lines above were propagated in DMEM with neomycin (400 mg/ml ) and hygromycin (400 mg/ml ) for 8 weeks before testing for the presence and stability of the pDR2 vector. Free episomal DNA was isolated using QIAprep spin columns, as described in Section 2.2. Lysates were used to transform E. coli cells, 24 colonies were picked, expanded in 5 ml LB-amp and mini-preps prepared. Plasmid DNA from each of the 24 colonies was cut separately with BamHI and PstI and run on a 1% agarose gel to test for vector stability. All samples showed the same banding pattern as pDR2 control DNA, indicating no gross rearrangements had occurred to the pDR2 vector ( Fig. 4). To test for episomal replication of the pDR2 vector in ’permissive’ A549 cells, Southern hybridization was carried out. Total DNA was isolated from five of the A549 EBNA-1+ (pDR2) cell lines and probed with a
A.D. Ramage et al. / Gene 197 (1997) 83–89
87
Fig. 3. High-frequency transformation of a ‘permissive’ A549 cell line (A549 EBNA-1+). Cell lines (A) control A549; or (B) A549 EBNA-1+ were transfected with 10 mg of the pDR2 vector and selected for 14 days under (A) hygromycin (700 mg/ml ) only; (B) hygromycin (700 mg/ml ) and neomycin (800 mg/ml ). Plates were fixed with ethanol and stained with haemotoxylin and eosin.
1.2 kb EcoRV-BamHI fragment from the pDR2 vector which contains part of the ori gene, as well as the entire RSV LTR. Fig. 5 shows the results from one of these cell lines. Control A549 DNA spiked with pDR2 vector and A549 EBNA-1+(pDR2) both gave a 10.7 kb band when cut with BamHI, whereas XhoI digestion (which should leave the pDR2 vector uncut) gave banding patterns indicative of an intact episomal DNA in both samples. From this, we can confirm that the pDR2 vector has not integrated into the host genome in A549 EBNA-1+(pDR2) cells but is present in the free state. The enzymes MboI and DpnI cut the same GATC site but are dependent on different methylation patterns on the adenine residues. MboI sensitivity provides evidence of a methylation pattern characteristic of DNA which has replicated in mammalian cells. Fig. 5 shows that the pDR2 DNA has indeed replicated in the ’permissive’ A549 cells, and is rendered sensitive to MboI digestion.
4. Discussion
Fig. 4. Restriction fragment analysis of pDR2 recovered from human A549 EBNA-1+ cells. Lysates were recovered from cells with QIAprep spin columns using 107 cells which had been grown under hygromycin selection for 8 weeks (section 2.2) and used to transform DH5a cells. Nine independent E. coli ampicillin-resistant colonies are shown: (A) cut with BamHI; (B) cut with PstI, and run on a 1% agarose gel. Lane M: l HindIII molecular weight markers; lane 1: control pDR2 vector; lanes 2–10: minipreps.
A recent advance in the construction of expression vectors is the introduction of the IRES to produce dicistronic RNAs (Mountford et al., 1994; Kaufman et al., 1991; Mountford and Smith, 1995). Here we describe the construction of a novel vector (pEB,) in which the IRES element enables a single transcription unit to efficiently produce both EBNA-1 protein and a selectable marker (neomycin phosphotransferase) under the control of the PGK promoter. Attempts to make ‘permissive’ A549 cell lines using Clontechs co-transfection system (pSCV2neo/
88
A.D. Ramage et al. / Gene 197 (1997) 83–89
reflect EBNA-1 integration and expression. The availability of this new system should faciliate the construction of ’permissive’ cell lines and encourage the use of EBV-based vectors to construct cDNA libraries for expression cloning.
Acknowledgement This work was supported by the Biotechnology and Biological Science Research Council (BBSRC ).
References
Fig. 5. Southern blot illustrating extrachrosomal maintenance and replication of pDR2 plasmid in ‘‘permissive’’ A549 cell lines. Total DNA was isolated as described in section 2.5 and probed with the 1.2 kb EcoRV–BamHI fragment from pDR2. Lanes 1–2: control A549 EBNA-1+ DNA spiked with pDR2 and cut with (1) BamHI; (2) XhoI; lanes 3–6: A549 EBNA-1+(pDR2) total DNA cut with (3) BamHI; (4) XhoI; (5) DpnI; (6) MboI.
pCMVEBNA) were unsuccessful. G-418-resistant colonies were recovered but the EBNA-1 gene did not co-integrate into the genome of these cells. The pEB vector was used to transfect A549 cells to G-418 resistance and all resistant colonies contained the EBNA-1 gene and were shown to be ‘permissive’ for episomal replication of the oriP-vector pDR2. This was characterized by an increase in transfection frequencies of at least a thousand-fold compared with the parental A549 cell line. pDR2 was readily recovered from the ‘permissive’ A549 cells after 8 weeks of hygromycin selection and no integrations or rearrangements were observed. The pEB vector has worked successsfully in A549 cells and we wish to extend our results to other cell types, particularly those that have shown EBNA-1 sensitivity in the past ( Vidal et al., 1990). Toxicity caused by EBNA-1 may have been reduced by using the relatively weaker PGK promoter present on the pEB vector but any effects of EBNA-1 on cell survival can be readily checked by including the control vector (6P-IresNeo-BS) when transfecting different cell types. In summary, the novel vector pEB is a significant improvement to existing EBV-based shuttle systems where expression of selectable marker does not always
Adra, C.N., Boer, P.H., McBurney, M.W., 1987. Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter. Gene 60, 65–74. Akhteruzzaman, M., Jang, S.K., Paul, A.V., Reuer, Q., Wimmer, E., 1992. Cardioviral internal ribosomal entry site is functioning in a genetically engineered dicistronic poliovirus. Nature 356, 255–257. Blin, N., Stafford, D.W., 1976. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3, 2303 Cachianes, G., Ho, C., Weber, R.F., Williams, S.R., Goeddel, D.V., Leung, D.W., 1993. Epstein–Barr virus-derived vectors for transient and stable expression of recombinant proteins. BioTechniques 15, 255–259. Carstens, C.-P., Gallo, J.C., Maher, V.M., McCormick, J.J., Fahl, W.E., 1995. A system utilizing Epstein–Barr virus-based expression vectors for the functional cloning of human fibroblast growth regulators. Gene 164, 195–202. Feinberg, A.P., Vogelstein, B., 1983. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13. Feinberg, A.P., Vogelstein, B., 1984. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity: Addendum. Anal. Biochem. 137, 266–267. Ghattas, I.R., Sanes, J.R., Majors, J.E., 1991. The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. Mol. Cell. Biol. 11, 5848–5859. Groger, R.K., Morrow, D.M., Tykocinski, M.L., 1989. Directional antisense and sense cDNA cloning using Epstein–Barr virus episomal expression vectors. Gene 81, 285–294. Heller, R.A., Song, K., Villaret, D., Margolskee, R., Dunne, J., HayakawaRingold, G.M., 1990. Amplified expression of tumor necrosis factor receptor in cells transfected with Epstein–Barr virus shuttle vector cDNA libraries. J. Biol. Chem. 265, 5708–5717. Kaufman, R.J., Davies, M.V., Wasley, L.C., Michnick, D., 1991. Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. Nucleic Acids Res. 19, 4485–4490. Legerski, R., Peterson, C., 1992. Expression cloning of a human DNA repair gene involved in xeroderma pigmentosum group C. Nature 359, 70–73. Margolskee, R.F., Kavathas, P., Berg, P., 1988. Epstein–Barr virus shuttle vector for the stable episomal replication of cDNA expression libraries in human cells. Mol. Cell. Biol. 8, 2837–2847. Mountford, P.S., Smith, A.G., 1995. Internal ribosome entry sites and dicistronic RNAs in mammalian transgenics. Trends in Genetics 11, 179–184. Mountford, P.S., Zevnick, B., Duwel, A., Nichols, J., Li, M., Dani, C., Robertson, M., ChambersSmith, A., 1994. Dicistronic targeting
A.D. Ramage et al. / Gene 197 (1997) 83–89 constructs: reporters and modifiers of mammalian gene expression. Proc. Natl. Acad. Sci. USA 91, 4303–4307. Nicolas, J.F., Berg, P., 1983. Regulation of expression of genes transduced into embryonal carcinoma cells. In Silver, L.M., Martin, G.R. Strickland, S. ( Eds), Teratocarcinoma Stem Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 469–486. Pelletier, J., Sonenberg, N., 1988. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334, 320–325. Porter, A.C.G., Chernajovsky, Y., Dale, T.C., Gilbert, C.S., Stark, G.R., Kerr, I.M., 1988. Interferon response element of the human 6-16 gene. EMBO J. 7, 85–92. Reisman, D., Yates, J., Sugden, B., 1985. A putative origin of replication of plasmids derived from Epstein–Barr virus is composed of two cis-acting components. Mol. Cell. Biol. 5, 1822–1832. Siebenkotten, G., Leyendeckers, H., ChristineRadbruch, A., 1995. Isolation of plasmid DNA from mammalian cells with QIAprep. QIAGEN News 2, 11–12. Southern, P.J., Berg, P., 1982. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1, 327–341. Strathdee, C.A., Gavish, H., Shannon, W.R., Buchwald, M., 1992.
89
Cloning of cDNAs for Fanconi’s anaemia by functional complementation. Nature 356, 763–767. Sugden, B., Marsh, K., Yates, J., 1985. A vector that replicates as a plasmid and can be efficiently selected in B-lymphocytes transformed by Epstein-Barr virus. Mol. Cell. Biol. 5, 410–413. Swirski, R.A., Van Den Berg, D., Murphy, A.J.M., Lambert, C.M., Friedberg, E.C., Schimke, R.T., 1992. Improvements in the Epstein–Barr-based shuttle vector system for direct cloning in human tissue culture cells. Methods: A Companion to Methods in Enzymology 4, 133–142. Vidal, M., Wrighton, C., Eccles, S., Burke, J., Grosveld, F., 1990. Differences in human cell lines to support stable replication of Epstein–Barr virus-based vectors. Biochim. Biophys. Acta 1048, 171–177. Yates, J., Warren, N., Reissman, D., Sugden, B., 1984. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids with latently infected cells. Proc. Natl. Acad. Sci. USA 81, 3806–3810. Yates, J.L., Warren, N., Sugden, B., 1985. Stable replication of plasmids derived from Epstein–Barr virus in various mammalian cells. Nature 313, 812–815.
Improved EBV-based shuttle vector system: dicistronic mRNA couples the synthesis of the Epstein–Barr nuclear antigen-1 protein to neomycin resistance Anne D. Ramage a, A. John Clark a, Austin G. Smith b, Peter S. Mountford b, David W. Burt a,* a Division of Molecular Biology, Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS, Scotland, UK b Centre for Genome Research, The University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh, EH9 3JQ, Scotland, UK Received 2 December 1996; accepted 17 April 1997; Received by A. Bernardi
Abstract Use of EBV-based vector systems has been limited by the requirement to generate EBNA+ cells which are ’permissive’ for replication of an oriP-vector. In current constructs, selectable marker and EBNA-1 are not always co-expressed. This is a significant problem since the EBNA-1 gene product can be toxic in some cell types and may be selected against. In this paper, we describe a gene construct that overcomes this limitation. We have exploited the piconaviral internal ribosome entry site to allow the genes for Epstein–Barr nuclear antigen-1 and G-418 resistance to be transcribed as a dicistronic fusion mRNA under the control of the phosphoglucokinase promoter. This construct can be routinely integrated into human cell lines. The presence of EBNA-1 protein was reflected by a large increase in transfection frequencies (1000-fold) using an oriP-based vector which was shown to replicate stably in these cells with no apparent gross rearrangements detected after 8 weeks in culture. Using this system, G-418 resistance should directly reflect integration, as well as expression of the EBNA-1 gene, which, in turn, increases transfection frequencies and stability of EBV-based vector systems and should result in its increased use. © 1997 Elsevier Science B.V. Keywords: Epstein–Barr virus; Internal ribosome entry site (IRES ); Eukaryotic episomal vectors; Human cell line; Dicistronic fusion mRNA; PGK promoter
1. Introduction Functional complementation is a powerful method for isolating cDNAs encoding novel proteins in mammalian cells. There is a need for a vector system which can transfect cells with high efficiency, support stable replication at moderate copy number and be re-isolated without rearrangements. Episomal vectors can do this and one such system, currently in use, exploits the EBV replication mechanism which depends on a trans-acting factor, EBNA-1, interacting with the replication origin region * Corresponding author. Tel. +44 131 4402726; Fax +44 131 4400434; e-mail: [email protected] Abbreviations: EBV, Epstein–Barr virus; EBNA-1, Epstein–Barr Nuclear Antigen-1; EMCV, encephalomyocarditis virus; IRES, internal ribosome entry site; neor, gene coding for G418 resistance; hyr, gene coding for hygromycin-B resistance; E. coli, Escherichia coli; PGK, phosphoglucokinase; GAPDH, glyceraldehyde dehydrogenase; PCR, polymerase chain reaction; DNA, deoxyribonucleic acid; RSV LTR, rous sarcoma virus long terminal repeat. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 2 45 - X
(oriP) ( Yates et al., 1984; Sugden et al., 1985; Reisman et al., 1985; Yates et al., 1985). Epstein–Barr virus shuttle systems have been developed on the basis that vectors which contain the EBNA1 and oriP genes can replicate in mammalian cells with a low copy number (Margolskee et al., 1988; Groger et al., 1989) and these systems have been used with some success to clone human cDNAs (Strathdee et al., 1992). One disadvantage of having the two EBV elements on the same vector is that stability may be jeopardized and reports have been made of plasmids undergoing extensive rearrangements (Swirski et al., 1992; Heller et al., 1990). Alternatively, the EBNA-1 gene can first be integrated into the genome of a cell line to render it ‘permissive’ for replication of an oriP-vector. The advantage of integrating EBNA-1 is that this leads both to increased stability of the oriP-vector, as well as an increase in transfection frequencies (Cachianes et al., 1993; Swirski et al., 1992; Carstens et al., 1995; Legerski and Peterson, 1992). Such a system may involve co-transfecting the
84
A.D. Ramage et al. / Gene 197 (1997) 83–89
EBNA-1 gene with a selectable marker, such as the gene conferring G-418 resistance, or using one vector containing both selectable marker and EBNA-1 gene. Many reports (as well as experience in our laboratory) suggest that in such systems expression of the neomycin resistance gene need not necessarily reflect expression or even integration of the EBNA-1 gene (Carstens et al., 1995; Swirski et al., 1992). This is a significant problem since the EBNA-1 gene product may be toxic to some cell types if expression is high ( Vidal et al., 1990; Carstens et al., 1995) and so cells in which EBNA-1 is not expressed have a selective growth advantage. In this paper we describe a novel vector in which the EBNA-1 and G-418 resistance genes are transcribed as a dicistronic fusion mRNA under the control of the PGK promoter. To this end we have exploited the picornaviral IRES which acts as a ribosome binding site and permits the effective internal initiation of translation in mammalian cells (Pelletier and Sonenberg, 1988; Akhteruzzaman et al., 1992) allowing EBNA-1 to be expressed downstream of the neor gene.
2. Materials and methods 2.1. Cells A549 human lung carcinoma cells were from the European Collection of Animal Cell Cultures ( ECACC ). Cells were maintained at 37°C (5% CO ) in 2 Dulbecco’s modified Eagle’s medium with -glutamine, -glucose (100 mg/l ), and sodium pyruvate (Gibco). Fetal bovine serum (10%), penicillin (100 U/ml ) and streptomycin (100 mg/ml ) were added. 2.2. Plasmids pSV2neo (Southern and Berg, 1982), pCMVEBNA (Swirski et al., 1992) and pDR2 were purchased from Clontech Laboratories (Cambridge, UK ). pDR2 can be used for the expression of cloned cDNA under control of the RSV LTR promoter and carries the EBV origin (oriP) which confers episomal maintenance to the vector in the presence of the Epstein–Barr virus nuclear antigen-1 ( EBNA-1). The hyr gene carried by pDR2 functions as a selectable marker in human cell lines. pCMVEBNA and pSCV2neo (Fig. 1) are helper plasmids for use with pDR2. pCMVEBNA carries the EBNA-1 gene and pSV2neo carries a selectable marker (neor). The plasmid 6P-iresneobs is a vector for dicistronic expression in mammalian cells of both the gene of interest and a selectable marker (Mountford and Smith, 1995). This plasmid contains the mouse pgk-I promoter (Adra et al., 1987) 5∞ to the EMCV IRES sequence
which is linked to the neomycin phosphotransferase gene (neo) (Ghattas et al., 1991). The neo gene is followed by intron and termination sequences derived from the rabbit b-globin gene and SV40, respectively (Nicolas and Berg, 1983). The plasmid also contains the interferon response element from the human 6-16 gene (Porter et al., 1988) upstream of the PGK promoter, which allows the induction of increased expression by exposure to a/b interferons. Unique SalI, SacI, NotI and XbaI restriction sites are situated between the promoter and the IRES for cDNA insertion. pBT Bluescript was purchased from Stratagene (Cambridge, UK ). Plasmid pDR2 was isolated from A549 cells with QIAprep spin columns (Qiagen, Crawley, UK ) using 107 cells (Siebenkotten et al., 1995). 2.3. Plasmid construction The pEB plasmid was constructed in two steps: (a) a 2.75 kb HindIII fragment which contains the CMV promoter and EBNA-1 gene was subcloned from the pCMVEBNA-1 vector into pBT Bluescript; and (b) a 2.0 kb XbaI fragment containing the EBNA-1 gene was subcloned from this Bluescript subclone into the the 6P-IresNeo-BS vector. Restriction enzyme digests were carried out on miniprep DNA to identify clones containing the EBNA-1 gene in the sense orientation. A novel 9.89 kb plasmid was recovered and named pEB ( Fig. 1A). 2.4. Transfections Transfections were carried out using the calcium phosphate transfection kit from Promega. Stable cell lines were constructed by transfecting A549 cells with the pEB vector and selecting for growth in G-418 (800 mg/ml ). The presence of the vector pDR2 was selected for by growth in hygromycin (700 mg/ml ). 2.5. Southern blot analysis Isolation of DNA from A549 cells was carried out using the procedure of Blin and Stafford (1976). Digested DNA was electrophoresed on 1% agarose gels and blotted onto nylon Zeta-Probe GT (Bio-Rad ) filters. Probes were radiolabelled by random priming according to the method of Feinberg and Vogelstein (1983, 1984). Hybridizations were carried out at 65°C in 10% PEG, 7% SDS, 1.5×SSPE. Filters were washed with 2×SSC, 0.1% SDS at room temperature followed by 0.1×SSC, 0.1% SDS at 65°C and autoradiographed. 2.6. PCR reactions One hundred nanograms of genomic DNA was placed in a total volume of 25 ml containing 1.5 mM MgCl , 2
A.D. Ramage et al. / Gene 197 (1997) 83–89
85
Fig. 1. Comparison of Epstein–Barr vector systems. (A) pEB, a novel vector system exploiting the IRES, which allows the EBNA-1 and neor genes to be transcribed as a dicistronic fusion mRNA under control of the PGK promoter. (B) Original two-vector system from Clontech. The neor gene is transcribed from the pSV2neo vector, while EBNA-1 is transcribed from pCMVEBNA.
1×PCR buffer, 0.2 mM deoxyribonucleotide triphosphates, 10 pM each of the two EBNA-1 primers 5∞-GGTTTTGAAGGATGCGATTAAG; 3∞-TTTAATACGATTGAGGGCGTCT or GAPDH primers 5∞-GTCAGT GGTGGACCTGACCT; 3∞-TGAGCTTGACAAAGTGGTCG and 1 unit of Taq DNA polymerase. The tubes were cycled 25 times through 94°C for 1 min, 60°C for 2 min and 72°C for 3 min. The PCR products (15 ml ) were electrophoresed on 1% agarose gels and stained with ethidium bromide. Primers were designed using the PRIMER (version 0.5) program (S.E. Lincoln, M.J. Daly and E.S. Lander, Whitehead Institute for Biomedical Research, Boston, MA, USA).
3. Results 3.1. Plasmid construction The 9.83 kb pEB plasmid is shown in Fig. 1A and consists of the entire EBNA-1 gene inserted in the sense orientation into the XbaI site of the 6P-IresNeo-BS plasmid under the control of the PGK promoter. The key property of this vector is that the encephalomyocarditis virus-internal ribosome binding site (EMCV-IRES ) allows the EBNA-1 and neor genes to be translated from a common dicistronic mRNA. G-418 resistance should, therefore, be coupled to expression of the Epstein–Barr Nuclear Antigen. The vector also contains the Col E1
86
A.D. Ramage et al. / Gene 197 (1997) 83–89
ori and ampicillin resistance (apr) genes for replication and selection in E. coli. This differs from the existing Clontech system illustrated in Fig. 1B, where the neor and EBNA-1 genes are transcribed independently on separate plasmids. The system depends on co-integration of both plasmids and co-expression of the neor and EBNA-1 genes. 3.2. Integration of EBNA−1 into A549 cells The pEB vector was linearised with the restriction enzyme MluI and 10 mg was used to transfect the A549 cell line, as described in Section 2. Transfectants were selected in 800 mg/ml G-418 and after 2 weeks six individual colonies were picked and expanded. Cell lines were then propogated in Dulbecco’s modified Eagle’s medium under G-418 selection (400 mg/ml ) for 8 weeks. After this time, genomic DNA was isolated and PCR reactions were carried out on this DNA using GAPDH and EBNA-1 primers. Results are shown in Fig. 2A. The expected EBNA-1 PCR product (244 bp) was found
in all DNA samples, except the untransfected control A549, indicating that the EBNA-1 gene was present in the genomic DNA from each of the neomycin-resistant colonies tested. The expected GAPDH PCR product (212 bp) was present in all samples of DNA tested and is the positive PCR control. We had previously attempted to integrate the EBNA1 gene by cutting the pCMVEBNA vector with HindIII and co-transfecting A549 cells with a selectable marker (neor) on the pSV2neo vector (Clontech). Twenty micrograms of pCMVEBNA and 1 mg pSV2neo were used. Resistant colonies were obtained after 2 weeks under G-418 selection (800 mg/ml ) and these were expanded and propogated for 8 weeks under G-418 (400 mg/ml ) selection. Further analysis of the DNA from 10 of these colonies using PCR as described above showed that none was able to amplify the EBNA-1 gene successfully. Results of six of these are shown in Fig. 2B. This indicates zero percentage co-transfection and suggests that the EBNA-1 gene under the control of the RSV LTR promoter may be toxic to A549 cells. 3.3. A549 cells rendered permissive for high transfection efficiency The six individual A549 EBNA-1+ cell lines selected above (as well as control A549 cells) were transfected with 10 mg pDR2 plasmid (Clontech). Results showed at least a 1000-fold increase in hygromycin-resistant colonies in A549 EBNA-1+ cell lines compared with the A549 control ( Fig. 3). This result shows that the EBNA-1 gene is being expressed in the A549 EBNA-1+, cells rendering them permissive for replication of the pDR2. 3.4. Episomal maintenance and replication of the pDR2 vector in permissive A549 cells
Fig. 2. PCR screen for the presence of the EBNA-1 gene in the genomic DNA of A549 cells. PCR reactions using EBNA-1 and GAPDH primers on genomic DNA derived from A549 cells. Genomic DNA was isolated as described in section 2.5. Lane M: 1 kb ladder; lane 1: no DNA; lane 2: A549 genomic DNA; lanes 3–8: DNA from the six A549 colonies grown under G-418 selection for 8 weeks after transfection with either (A) the pEB vector; or (B) the pSCV2neo and pCMVEBNA vectors (1:20 ratio).
The six A549 EBNA-1+(pDR2) cell lines above were propagated in DMEM with neomycin (400 mg/ml ) and hygromycin (400 mg/ml ) for 8 weeks before testing for the presence and stability of the pDR2 vector. Free episomal DNA was isolated using QIAprep spin columns, as described in Section 2.2. Lysates were used to transform E. coli cells, 24 colonies were picked, expanded in 5 ml LB-amp and mini-preps prepared. Plasmid DNA from each of the 24 colonies was cut separately with BamHI and PstI and run on a 1% agarose gel to test for vector stability. All samples showed the same banding pattern as pDR2 control DNA, indicating no gross rearrangements had occurred to the pDR2 vector ( Fig. 4). To test for episomal replication of the pDR2 vector in ’permissive’ A549 cells, Southern hybridization was carried out. Total DNA was isolated from five of the A549 EBNA-1+ (pDR2) cell lines and probed with a
A.D. Ramage et al. / Gene 197 (1997) 83–89
87
Fig. 3. High-frequency transformation of a ‘permissive’ A549 cell line (A549 EBNA-1+). Cell lines (A) control A549; or (B) A549 EBNA-1+ were transfected with 10 mg of the pDR2 vector and selected for 14 days under (A) hygromycin (700 mg/ml ) only; (B) hygromycin (700 mg/ml ) and neomycin (800 mg/ml ). Plates were fixed with ethanol and stained with haemotoxylin and eosin.
1.2 kb EcoRV-BamHI fragment from the pDR2 vector which contains part of the ori gene, as well as the entire RSV LTR. Fig. 5 shows the results from one of these cell lines. Control A549 DNA spiked with pDR2 vector and A549 EBNA-1+(pDR2) both gave a 10.7 kb band when cut with BamHI, whereas XhoI digestion (which should leave the pDR2 vector uncut) gave banding patterns indicative of an intact episomal DNA in both samples. From this, we can confirm that the pDR2 vector has not integrated into the host genome in A549 EBNA-1+(pDR2) cells but is present in the free state. The enzymes MboI and DpnI cut the same GATC site but are dependent on different methylation patterns on the adenine residues. MboI sensitivity provides evidence of a methylation pattern characteristic of DNA which has replicated in mammalian cells. Fig. 5 shows that the pDR2 DNA has indeed replicated in the ’permissive’ A549 cells, and is rendered sensitive to MboI digestion.
4. Discussion
Fig. 4. Restriction fragment analysis of pDR2 recovered from human A549 EBNA-1+ cells. Lysates were recovered from cells with QIAprep spin columns using 107 cells which had been grown under hygromycin selection for 8 weeks (section 2.2) and used to transform DH5a cells. Nine independent E. coli ampicillin-resistant colonies are shown: (A) cut with BamHI; (B) cut with PstI, and run on a 1% agarose gel. Lane M: l HindIII molecular weight markers; lane 1: control pDR2 vector; lanes 2–10: minipreps.
A recent advance in the construction of expression vectors is the introduction of the IRES to produce dicistronic RNAs (Mountford et al., 1994; Kaufman et al., 1991; Mountford and Smith, 1995). Here we describe the construction of a novel vector (pEB,) in which the IRES element enables a single transcription unit to efficiently produce both EBNA-1 protein and a selectable marker (neomycin phosphotransferase) under the control of the PGK promoter. Attempts to make ‘permissive’ A549 cell lines using Clontechs co-transfection system (pSCV2neo/
88
A.D. Ramage et al. / Gene 197 (1997) 83–89
reflect EBNA-1 integration and expression. The availability of this new system should faciliate the construction of ’permissive’ cell lines and encourage the use of EBV-based vectors to construct cDNA libraries for expression cloning.
Acknowledgement This work was supported by the Biotechnology and Biological Science Research Council (BBSRC ).
References
Fig. 5. Southern blot illustrating extrachrosomal maintenance and replication of pDR2 plasmid in ‘‘permissive’’ A549 cell lines. Total DNA was isolated as described in section 2.5 and probed with the 1.2 kb EcoRV–BamHI fragment from pDR2. Lanes 1–2: control A549 EBNA-1+ DNA spiked with pDR2 and cut with (1) BamHI; (2) XhoI; lanes 3–6: A549 EBNA-1+(pDR2) total DNA cut with (3) BamHI; (4) XhoI; (5) DpnI; (6) MboI.
pCMVEBNA) were unsuccessful. G-418-resistant colonies were recovered but the EBNA-1 gene did not co-integrate into the genome of these cells. The pEB vector was used to transfect A549 cells to G-418 resistance and all resistant colonies contained the EBNA-1 gene and were shown to be ‘permissive’ for episomal replication of the oriP-vector pDR2. This was characterized by an increase in transfection frequencies of at least a thousand-fold compared with the parental A549 cell line. pDR2 was readily recovered from the ‘permissive’ A549 cells after 8 weeks of hygromycin selection and no integrations or rearrangements were observed. The pEB vector has worked successsfully in A549 cells and we wish to extend our results to other cell types, particularly those that have shown EBNA-1 sensitivity in the past ( Vidal et al., 1990). Toxicity caused by EBNA-1 may have been reduced by using the relatively weaker PGK promoter present on the pEB vector but any effects of EBNA-1 on cell survival can be readily checked by including the control vector (6P-IresNeo-BS) when transfecting different cell types. In summary, the novel vector pEB is a significant improvement to existing EBV-based shuttle systems where expression of selectable marker does not always
Adra, C.N., Boer, P.H., McBurney, M.W., 1987. Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter. Gene 60, 65–74. Akhteruzzaman, M., Jang, S.K., Paul, A.V., Reuer, Q., Wimmer, E., 1992. Cardioviral internal ribosomal entry site is functioning in a genetically engineered dicistronic poliovirus. Nature 356, 255–257. Blin, N., Stafford, D.W., 1976. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3, 2303 Cachianes, G., Ho, C., Weber, R.F., Williams, S.R., Goeddel, D.V., Leung, D.W., 1993. Epstein–Barr virus-derived vectors for transient and stable expression of recombinant proteins. BioTechniques 15, 255–259. Carstens, C.-P., Gallo, J.C., Maher, V.M., McCormick, J.J., Fahl, W.E., 1995. A system utilizing Epstein–Barr virus-based expression vectors for the functional cloning of human fibroblast growth regulators. Gene 164, 195–202. Feinberg, A.P., Vogelstein, B., 1983. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13. Feinberg, A.P., Vogelstein, B., 1984. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity: Addendum. Anal. Biochem. 137, 266–267. Ghattas, I.R., Sanes, J.R., Majors, J.E., 1991. The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. Mol. Cell. Biol. 11, 5848–5859. Groger, R.K., Morrow, D.M., Tykocinski, M.L., 1989. Directional antisense and sense cDNA cloning using Epstein–Barr virus episomal expression vectors. Gene 81, 285–294. Heller, R.A., Song, K., Villaret, D., Margolskee, R., Dunne, J., HayakawaRingold, G.M., 1990. Amplified expression of tumor necrosis factor receptor in cells transfected with Epstein–Barr virus shuttle vector cDNA libraries. J. Biol. Chem. 265, 5708–5717. Kaufman, R.J., Davies, M.V., Wasley, L.C., Michnick, D., 1991. Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. Nucleic Acids Res. 19, 4485–4490. Legerski, R., Peterson, C., 1992. Expression cloning of a human DNA repair gene involved in xeroderma pigmentosum group C. Nature 359, 70–73. Margolskee, R.F., Kavathas, P., Berg, P., 1988. Epstein–Barr virus shuttle vector for the stable episomal replication of cDNA expression libraries in human cells. Mol. Cell. Biol. 8, 2837–2847. Mountford, P.S., Smith, A.G., 1995. Internal ribosome entry sites and dicistronic RNAs in mammalian transgenics. Trends in Genetics 11, 179–184. Mountford, P.S., Zevnick, B., Duwel, A., Nichols, J., Li, M., Dani, C., Robertson, M., ChambersSmith, A., 1994. Dicistronic targeting
A.D. Ramage et al. / Gene 197 (1997) 83–89 constructs: reporters and modifiers of mammalian gene expression. Proc. Natl. Acad. Sci. USA 91, 4303–4307. Nicolas, J.F., Berg, P., 1983. Regulation of expression of genes transduced into embryonal carcinoma cells. In Silver, L.M., Martin, G.R. Strickland, S. ( Eds), Teratocarcinoma Stem Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 469–486. Pelletier, J., Sonenberg, N., 1988. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334, 320–325. Porter, A.C.G., Chernajovsky, Y., Dale, T.C., Gilbert, C.S., Stark, G.R., Kerr, I.M., 1988. Interferon response element of the human 6-16 gene. EMBO J. 7, 85–92. Reisman, D., Yates, J., Sugden, B., 1985. A putative origin of replication of plasmids derived from Epstein–Barr virus is composed of two cis-acting components. Mol. Cell. Biol. 5, 1822–1832. Siebenkotten, G., Leyendeckers, H., ChristineRadbruch, A., 1995. Isolation of plasmid DNA from mammalian cells with QIAprep. QIAGEN News 2, 11–12. Southern, P.J., Berg, P., 1982. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1, 327–341. Strathdee, C.A., Gavish, H., Shannon, W.R., Buchwald, M., 1992.
89
Cloning of cDNAs for Fanconi’s anaemia by functional complementation. Nature 356, 763–767. Sugden, B., Marsh, K., Yates, J., 1985. A vector that replicates as a plasmid and can be efficiently selected in B-lymphocytes transformed by Epstein-Barr virus. Mol. Cell. Biol. 5, 410–413. Swirski, R.A., Van Den Berg, D., Murphy, A.J.M., Lambert, C.M., Friedberg, E.C., Schimke, R.T., 1992. Improvements in the Epstein–Barr-based shuttle vector system for direct cloning in human tissue culture cells. Methods: A Companion to Methods in Enzymology 4, 133–142. Vidal, M., Wrighton, C., Eccles, S., Burke, J., Grosveld, F., 1990. Differences in human cell lines to support stable replication of Epstein–Barr virus-based vectors. Biochim. Biophys. Acta 1048, 171–177. Yates, J., Warren, N., Reissman, D., Sugden, B., 1984. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids with latently infected cells. Proc. Natl. Acad. Sci. USA 81, 3806–3810. Yates, J.L., Warren, N., Sugden, B., 1985. Stable replication of plasmids derived from Epstein–Barr virus in various mammalian cells. Nature 313, 812–815.