Improved EBV shuttle vectors

Mutation Research, 220 (1989) 125-132

125

Elsevier MTR 02611

Improved EBV shuttle vectors Steven B. Haase, Scott S. Heinzel, Patrick J. Krysan and Michele P. Calos Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.)

(Accepted 27 September 1988)

Keywords: Shuttle vector; EBV; SV40; Origin of replication

Summary Shuttle vectors based on Epstein-Barr virus (EBV) replicate autonomously in the nuclei of human cells. These vectors represent reasonable models for chromosomes, have low background mutation frequencies, and have been useful for studying induced mutation in human cells. Two improvements in the EBV vector system are discussed. Attempts are described to increase vector copy number per cell by using a limited period of replication driven by the simian virus 40 (SV40) origin of replication. Isolation of h u m a n sequences that can replace the viral origin o f replication in providing for autonomous replication of-the vectors is also described. These improvements are leading toward shuttle vectors that are more efficient and more closely resemble authentic chromosomes.

Shuttle vectors permit the rapid analysis of mutations generated in mammalian cells, since shortly after mutagenesis vector D N A can be transferred to bacteria for scoring and analysis of mutations. If the shuttle vector is derived from E p s t e i n - B a r r virus (EBV), it has the attractive properties of a replication mode resembling that of bona fide chromosomes and a low spontaneous mutation frequency. The vectors appear to replicate only once per cell cycle, like Epstein-Barr virus itself (Adams, 1987). They replicate autonomously in the nuclei of human cells using the host replication apparatus, the EBV latent origin of replication, and one viral gene product, EBNA-1 (Yates et al., 1984, 1985). The EBV latent origin of

Correspondence: Dr. Steven B. Haase, Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.).

replication, oriP, and the gene encoding EBNA-1 are present on our EBV shuttle vectors (DuBridge et al., 1987). Alternatively, EBNA-1 can be supplied in trans (Yates et al., 1984, 1985; Drinkwater and Klinedinst, 1986). Because EBV replication is compatible with long-term survival of the host cells, clonal lines carrying the vector can be selected. This feature allows us to readily screen for and obtain cell lines free of the mutation associated with transfection of vector D N A into m a m m a l i a n cells (Calos et al., 1983; Razzaque et al., 1983). Such lines do not increase in mutation frequency over time, indicating that EBV replication is not associated with a high mutation frequency (DuBridge et al., 1987). The mutation frequency for lacI carried on EBV vectors is approximately 10 -5, which is similar to the mutation frequency for the gene in E. coli and for genes in h u m a n cells (DuBridge et al., 1987; Heinzel et al., 1988; Lewin, 1980). This

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126 mutation frequency can be raised at least 10-100fold by exposure of the cell lines to mutagens such as ethyl nitrosourea (Drinkwater and Klinedinst, 1986) and N-nitroso-N-methylurea (DuBridge et al., 1987). These induced mutations have been readily analyzed at the molecular level. Since vector molecules are individually assayed in E. coli for mutation, it is an inherent property of shuttle vector systems that forward mutation can be scored even when the target gene is present in multiple copies in the human cell. This is not possible if the mutations are scored in the m a m malian cell, since the mutants are generally recessive to the remaining wild-type copies of the gene. The ability to score forward mutation in a multicopy situation creates the potential for a highly sensitive mutation detection system. In the presence of a mutagen, mutations in the target gene would have a greater likelihood of occurring before a lethal mutation to the cell occurred. Furthermore, if each h u m a n cell carried m a n y vector copies, a great deal of mutational information could be obtained from relatively few human cells. Currently available EBV vectors exist at a low copy number per cell. Consequently, the generation of sufficient mutant plasmids is relatively labor-intensive. Our efforts to raise vector copy number are described below. The formation and fixation of mutations is intimately connected with D N A replication. For this reason, the ideal shuttle vector should replicate using the host replication apparatus exclusively. In contrast, viral origins of replication require at least one viral protein for initiation of replication. Therefore, replacement of the viral origin with an origin of replication derived from a h u m a n chromosome would be desirable. Since such sequences are not currently available, we set out to devise a genetic assay that could be used to isolate functional origins of replication from human D N A . Our strategy, described below, has been successful in producing a large set of human fragments that mediate autonomous replication of our vectors when substituted for the viral origin. We are studying these sequences in order to characterize replication and its control in human cells. This information will be useful in designing a new generation of shuttle vectors that could be described as shuttle chromosomes.

Results and discussion Raising vector copy number

The mechanism controlling the copy number of EBV vectors is not understood. EBV-based vectors typically have copy numbers of approximately 1 - 1 0 0 / c e l l in a variety of human cell fines (Yates et al., 1985; Drinkwater and Klinedinst, 1986; Kioussis et al., 1987; DuBridge et al., 1987). When selection for the vector is maintained, copy number is relatively stable over time (Yates et al., 1984). We would like to raise this copy number by 1 - 2 orders of magnitude to obtain human cell lines stably carrying several hundred to several thousand vectors per cell. A straightforward means of increasing vector copy number is to take advantage of the high copy number, multiple-round replication of SV40 vectors. By including the SV40 origin of replication on our EBV vectors and arranging for transient provision of SV40 T-antigen, which is necessary to activate the SV40 origin, we can increase vector copy number. We first demonstrated the feasibility of this approach using chimeric bovine papilloma virus-SV40 vectors (DuBridge et al., 1985). We have recently applied a similar approach using EBV-SV40 chimeras (Heinzel et al., 1988). Plasmid pMCi5, an l l - k b EBV vector carrying the SV40 origin of replication, was introduced into human cells, where it replicated stably at a low copy number using its EBV origin of replication. This plasmid carries the EBNA-1 gene and oriP for EBV replication (see Fig. 1). The SV40 origin of replication is present on the vector, but cannot function in the absence of SV40 T-antigen. In order to switch on SV40 replication, a non-replicating vector expressing T-antigen, p R T A K (Fig. 2), was then transfected into 5.4, a clonal cell line carrying pMCi5. During the next few days pMCi5 vector copy number underwent a dramatic increase of at least 10-100-fold, reaching a peak approximately 4 days after transfection with p R T A K . By this time p R T A K D N A and T-antigen protein (detected by immunofluorescence) had essentially disappeared from the cells. Analysis of the copy number increase is hindered by the variability inherent in a transfected population. Since only a fraction of the cells in a transfection receive D N A , only a portion

127 SV40 ori lacZct

SV4O ori

lacZa

/acl

EBV

,

E

/ac/

~,

V

~

sv4o

~ J

ori

pMCi4

k

B

pMCi5

/

/

Fig. 1. EBV shuttle vectors. The 300 bp that form direct and indirect repeats of amp sequences are shown in black with arrows to indicate orientation. Borders between lacI and selected or screened sequences are shown as bars to indicate flanking. The unlabeled segment contains the pBR322 origin of replication and the gene for hygromycin resistance.

of the cells actually express T-antigen and increase their copy number by SV40 replication. Also, among the cells that do take up DNA, variability exists in the quanitity of DNA that each cell receives. Thus, the expression of T-antigen may vary from cell to cell. For this reason we have developed a strategy that allows us to identify cells which have received the T-antigen vector and to fractionate them on the basis of expression using the Fluorescence Activated Cell Sorter (FACS). The assay involves tagging the vector p R T A K with the gene encoding the human class I surface molecule HLA-A2 and sorting cells after staining with anti-HLA-A2 antibody followed by a fluorescein-conjugated second antibody. For these experiments the HLA-A2 gene

(Koller and Orr, 1985) was added to p R T A K , producing p H O R T A (Fig. 2). After transfection of 5.4 with pHORTA, an increase in copy number was obtained, as shown in Fig. 3. Lane 1 shows the copy number after introduction of pRTAK, to demonstrate the average increase in copy number of the unfractionated population. We call the process of increasing copy number by introduction of T-antigen a 'boost'. The copy number of 5.4 prior to the boost is indicated in lane 2. (The vector copy number is too low to be visible on this exposure, but is visible upon longer exposure of the film.) Using the FACS we sorted the cells which received low, medium, and high amounts of pHORTA, as judged by the amount of HLA-A2

128 HLA

Bam

,~

~'~

Bam

Bam

vector. amp

Pst

TsA1609

Another explanation may be that cells carrying a high vector copy number are at a selective disadvantage, either because of the overreplication of the vectors or some other toxic effect of the vectors, such as a gene product synthesized by the

pRTAK kan

To further understand the vector copy number loss we could put a marker than can be scored on the FACS onto the EBV vector itself. In this way, it would be possible to sort and follow the fate of individual cells carrying a high copy number, well after the T-antigen plasmid has disappeared. If the marker were also selectable over a broad dose range of the selective agent, we could exert genetic selection for cells carrying a high vector copy number. In this way, we could obtain mutants in

Pst RSV LTR

amp/orl

Fig. 2. The structures of p R T A K and p H O R T A . The construction of p R T A K has been described elsewhere (Heinzel et aL, 1988). SV40 T-antigen is expressed from a promoter in the long terminal repeat of Rous sarcoma virus. A gene conferring kanamycin resistance was inserted into the pBR322 sequences, rendering p R T A K ampicillin sensitive and kanamycin resistant.

they expressed on their surface. Since T-antigen and HLA-A2 are expressed from the same p H O R T A vector, we assume that T-antigen expression will be correlated with HLA-A2 expression. As shown in Fig. 3, lanes 3-5, the magnitude of the copy number increase is correlated with the amount of T-antigen the cells received. An interesting aspect of this phenomenon is indicated in lanes 9-11, which show the same populations 12 days after the introduction of p H O R T A . The populations retain the same relative order in terms of vector copy number, but copy number has dropped in each case. The explanation for this drop is under investigation. It is possible that vectors may have some problem in resuming replication using their EBV origin following the boost. It is also possible that EBV replication requires a host component that is present in limiting amounts, and that unless this limitation is overcome there is an upper limit in the number of EBV vectors that can be carried.

O

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O

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2

3

4

5

6

7

8

9 10 11 12

Fig. 3. Copy n u m b e r boost of 5.4 with p H O R T A . The parent cell line 293s (Graham et al., 1977; Stillman and Gluzman, 1985) was used for all the work reported in this study. 10 ~g of p H O R T A or p R T A K D N A per 100-mm dish were transfected into cell line 5.4 (carrying a low copy n u m b e r of pMCi5). 3 days after transfection the cells were stained with anti-HLA-A2 monoclonal antibody CRl1351 obtained from R. Salter and sorted into 3 groups expressing low, medium, and high levels of HLA-A2. Cells were plated in non-selective media and grown until Hirt extraction at 4 and 12 days. The Hirt extracts were digested with Sail. Extract from approximately 5 x 104 cells was loaded in each lane of a 0.7% agarose gel. The gel was subjected to blotting and hybridization, using 32p-labeled pMCi5 D N A as a probe. Markers: 1 ng of p H O R T A and pMCi5 digested with SalI. Lanes 1: Unsorted population receiving p R T A K ; 2: untransfected 5.4; 3, 4 and 5 : 5 . 4 plus p H O R T A , sorts with low, medium, and high HLA-A2 expression. Lanes 7-11: same samples as 1-5, but harvested 12 days after transfection. Lanes 6 and 12 show 293s plus p H O R T A , 4 and 12 days after transfection.

129

either the cell or vector that overcome the limitation on copy number. Such mutants may be informative about the nature of the limitation. In any case, they would permit us to reach our goal of obtaining human cell lines carrying a stable high vector copy number. A suitable selectable marker for this purpose appears to be the gene coding for multiple drug resistance, mdrl. This gene encodes a membrane pump which exports a variety of toxic drugs, such as colchicine, from the cell. Unlike commonly used drug markers such as the genes for hygromycin or G418 resistance, the amount of mdrl expression correlates with the level of drug resistance shown by the cells over a broad range. We have obtained the human mdrl gene (Roninson et al., 1986) and corresponding antibodies (Hamada and Tsuruo, 1986) and plan to add the gene to our vectors. In this way, we hope to acquire a means both for following vector copy number by FACS analysis and for genetically selecting cells which carry a high vector copy number. Mutation and recombination Because a high mutation frequency associated with transfection of SV40-based shuttle vectors had previously been observed (Calos et al., 1983; Razzaque et al., 1983), we wanted to measure whether the period of SV40 replication involved in copy number increase induced mutations at a high frequency. The boost experiment nicely separates the two most likely causes of the high mutation

frequency, namely, transfection or SV40 replication. Since the lacI target gene being scored for mutation is already in the cell and replicating stably on an EBV vector, only mutations due to SV40 replication will be detected in the boost experiment. We performed boosts on cell lines containing pMCil and pMCi5 (Fig. 1) and measured the resultant mutation frequencies, as reported in Heinzel et al. (1988). This experiment indicated that tlaere was no detectable increase in point mutation frequency associated with SV40 replication. No overall increase in mutation frequency was found for pMCi5 (Table 1). (The reason for the higher background mutation frequency for pMCi5 in bacteria is not known.) A 15-fold increase, entirely accounted for by deletions, was measured for pMCil (Table 1). An explanation for this difference between p M C i l and pMCi5 is that deletions extending out of lacl will be scored as I - mutants in pMCil, but cannot be recovered in pMCi5, where lacl is flanked on both sides by selected or screened sequences (see below). We also observed that 300-bp direct repeats present on pMCil gave rise to a high frequency of intramolecular recombination. This recombination might also be involved in stimulating deletion formation. To test this idea we established clonal human cell lines stably carrying 2 additional EBV shuttle vectors, pMCi2 and pMCi4. As shown in Fig. 1, these plasmids allow us to distinguish between the

TABLE 1 LacI M U T A N T F R E Q U E N C I E S Plasmid

pMCil

pMCi2

pMC i 4

pMCi5

Recombination

yes

no

yes

no

Lacl flanked

no

no

yes

yes

Bacterial LacI -

2.1 × 1 0 - 5 ( 2 / 9 5 257)

1.7 × 1 0 - 5 ( 2 / 1 2 0 721)

3.4 × 1 0 - 5 ( 5 / 1 4 7 847)

7.0 × 1 0 - ~ (20/ 288 799)

Human LacI-

2.0 × 1 0 - 5 ( 5 / 2 4 9 455)

< 4.6 × 1 0 - 5 (0/21954)

5.7 × 10- 5 ( 2 / 3 4 803)

9.1 x 1 0 - 5 ( 7 / 7 6 556)

SV40 boost L a c I - a

3.2 x 10 - 4 (116/365000)

5.5 x 1 0 - 4 (107/194197)

6.3 × 1 0 - 5 (9/ 142784)

9.6 × 1 0 - 5 (118/1 225094)

N u m b e r of mutants detected per number of colonies examined is shown in parentheses below the Lacl mut a nt frequency. a p R T A K was used to boost copy number. Vector was recovered from human cells 4 days after transfection with pR TA K .

130 effects of flanking and recombination. This series of plasmids has the following features: plasmids pMCi2 and pMCi5 lack direct repeats that would give rise to recombination. [We have shown that repeats in an inverted orientation do not give rise to a high recombination frequency (Heinzel et al., 1988).] In contrast, pMCil and pMCi4 have direct repeats that would be expected to recombine. That p M C i l and pMCi4 do undergo intramolecular recombination at a high frequency while pMCi2 and pMCi5 do not was verified by transient replication experiments. Recombination was assayed by the appearance of additional bands on a Southern blot. The presence and nature of recombinant forms was verified by examining the plasmid D N A from individual rescued colonies. In the case of p M C i l and pMCi4, the majority of the rescued colonies carried the recombinant form. Another difference in the series pertains to the sequences adjacent to the lacI target gene. Both pMCi4 and pMCi5 carry lacl closely flanked on both sides by sequences selected or screened in the experiment. These sequences are the SV40 origin, required for replication in human cells, and the alpha portion of lacZ, required to score a blue colony (see Calos et al., 1983). Because of this close flanking, plasmids containing large deletions extending out of lacl would not be scored in our assay. By contrast, pMCil and pMCi2 contain essential sequences on only one side of lacI. Deletions could extend for several kilobases out of the 5' end of lacl (into EBV sequences in the case of p M C i l and into the hygromycin resistance gene in the case of pMCi2) and still allow the plasmids to replicate during the SV40 boost and be rescued in E. coli as blue colonies. Therefore, large deletions can be recovered in pMCil and pMCi2. Comparison of the mutational behavior of these 4 plasmids should elucidate the contributions of recombination and flanking to the SV40-induced mutation frequency. The mutation frequencies for pMCi2 and pMCi4 in bacteria, in clonal lines of human cells, and after boosting of such lines with T-antigen are shown in Table 1. As for pMCil and pMCi5, the mutation frequencies for pMCi2 and pMCi4 when carried as EBV vectors in human cells resemble their mutation frequencies as plasmids in E. coli. This result again indicates that EBV replication

induces no detectable increase in mutation frequency. The mutation frequency of the pMCi2 clonal line increased by approximately an order of magnitude after SV40 replication. The majority of the mutations examined (16 of 21) were deletions. Therefore, the frequency and nature of the mutations induced during SV40 replication of pMCil and pMCi2 are similar, despite the fact that pMCi2 does not undergo intramolecular recombination. This data argues against a major role for recombination in stimulating the deletion formation observed. This conclusion is further supported by the behavior of pMCi4 upon boosting. Even though pMCi4 shows recombination, there is no significant increase in mutation frequency during the SV40 replication period. Most of the mutations (6 of 9) were of the point mutation class. The lacI target gene in pMCi4 is flanked and large deletions cannot be recovered. To summarize the mutation data, we find that SV40 replication is not highly mutagenic for point mutations, nor does intramolecular recombination on the vector have a profound effect on mutation frequency. On vectors where large deletions can be scored we do find an increase of about an order of magnitude in the frequency of deletions recovered. Because of the replicative advantage exhibited by smaller SV40 vectors over large ones documented by us and others (Santangelo and Cole, 1983; Calos, 1986; Heinzel et al., 1988), it is difficult to specify how much of this increase is due to an inherent increase in deletion frequency and how much is due to the replicative advantage enjoyed by the vectors bearing deletions. In any case, it is clear that for vectors in which the target gene is closely flanked by selected or screened sequences, there is no measurable increase in mutation frequency associated with the transient SV40 replication period. Therefore, our boost protocol can increase the copy number of such EBV vectors without increasing their mutation frequency. Origin of replication We have developed a novel strategy for the isolation of human sequences which can mediate autonomous replication in human cells (Krysan et al., 1989). We believe that two requirements must be met in order to obtain an autonomously repli-

131

cating plasmid in mammalian cells. In addition to an initiation site where D N A synthesis can begin, a means of retaining the plasmid in the nucleus may also be required. EBV vectors must possess such a means, since they are stably maintained in the nuclei over long periods of time. The EBV latent origin of replication, oriP, consists of a family of repeat units and a region of dyad symmetry (Reisman et al., 1985). It is known that EBNA-1 can bind to both portions of oriP (Rawlins et al., 1985). By analogy with other origins, the dyad is hypothesized to be the region where DNA synthesis begins. Reisman et al. (1985) showed that both the family of repeats and the dyad are required for replication of EBV vectors. We have found that EBNA-1 plus the family of repeats without the dyad mediate prolonged retention of non-replicating D N A in human cells (Krysan et al., 1989). This interaction could potentially be used to stabilize human origins of replication and formed the basis of our cloning strategy. We deleted the dyad symmetry region from oriP of the EBV vector p220.2 (DuBridge et al., 1987), to create p D Y - . This vector has little or no replication activity in human cells. Random human DNA was cloned into p D Y - . This human library was transfected into 293s cells and placed under hygromycin selection. After 2 months of growth, vector D N A was recovered from the human cells, digested with Dpnl to remove unreplicated input DNA, and transformed into E. coli (see Lebkowski et al., 1984). Several hundred colonies were recovered from the extracts indicating that the p D Y - plasmids containing human inserts had the ability to replicate stably. We examined the inserts by restriction mapping and found that they contained a heterogeneous set of human fragments, most of which ranged in size from 10 to 20 kb. Several of the plasmids containing human inserts were chosen for further study and shown to replicate stably when reintroduced into human cells (Krysan et al., 1989). The copy number of the plasmids containing human inserts is similar to the copy number of EBV vectors, approximately 1-50/cell. Further evidence that the human inserts contain origins of replication was obtained by moving them to a plasmid lacking all EBV sequences and testing for replication. This plasmid and its deriva-

tive containing a human insert were transfected into human cells, extracted 4 days later, and digested with DpnI, which does not cut D N A replicated in eukaryotic cells. The vector D N A alone was completely digested by DpnI, indicating no replication activity in human cells. However, the vector with a human insert showed DpnI-resistant DNA, indicating replication (Krysan et al., 1989). This result suggests that replication is initiating in the human DNA fragment. We believe that the human inserts are sufficient to provide for initiation of replication, but that the EBV sequences are required to ensure maintenance of the replicating plasmids in the nucleus over many generations. Presumably, cellular origins do not need such sequences, since they are part of large chromosomes which are retained and segregated by virtue of centromeres. The human sequences we have isolated will replace the EBV viral origin of D N A synthesis in the new generation of shuttle vectors currently under construction. We will also attempt to isolate human sequences, such as those derived from centromeres, that can replace the viral sequences that appear to be involved in nuclear retention. We are in the process of determining whether the copy number of vectors replicating using human sequences can be increased using the SV40 strategy described above. If so, we will have created high copy number shuttle vectors using human sequences for replication. We anticipate that the further development of such vectors will lead to high fidelity shuttle systems for the study of mutation in human cells at the molecular level.

Acknowledgements We thank Charles Andre and Robert DuBridge for helpful discussions, Gilbert Chu, Russell Salter, and Peter Parham for HLA-A2 sequences and monoclonal antibodies, and Jo Ann Katheiser for secretarial assistance. This work was supported by grants CA-33056, CA-45365, and CA-09302 from the National Cancer Institute.

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M.P. Calos (1984) Transfected DNA is mutated in monkey, mouse, and human cells, Mol. Cell. Biol., 4, 1951-1960. Lewin, B. (1980) Eukarytotic chromosomes, in: Gene Expression, Vol. 2, 2nd Edn., Wiley, New York, pp. 142-188. Rawlins, D.R., G. Milman, S.D. Hayward and G.S. Hayward (1985) Sequence-specific DNA binding of the Epstein-Barr nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region, Cell, 42, 859-868. Razzaque, A., H. Mizusawa and M. Seidman (1983) Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells, Proc. Natl. Acad. Sci. (U.S.A.), 80, 3010-3014. Reisman, D., J. Yates and B. Sugden (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. Roninson, I.B., J.E. Chin, K. Choi, P. Gros, D.E. Housman, A. Fojo, D.W. Shen, M.M. Gottesman and I. Pastan (1986) Isolation of human mdr DNA sequences amplified in multidrug-resistant KB carcinoma ceils, Proc. Natl, Acad. Sci. (U.S.A.), 83, 4538-4542. Santangelo, G.M., and C.N. Cole (1983) Preparations of a "functional library" of African green monkey DNA fragments which substitute for the processing/polyadenylation signal in the herpes simplex virus type I thymidine kinase gene, Mol. Cell. Biol., 3, 643-653. Stillman, B.W., and Y. Gluzman, (1985) Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells, Mol. Cell. Biol., 5, 2051-2060. Wigler, M., S. Silverstein, L.-S. Lee, A. Pellicer, Y.-C. Cheng and R. Axel (1977) Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells, Cell, 11, 223-232. Yates, J., N. Warren, D. Reisman and B. Sugden (1984) A cis-acting element from the Epstein-Burr viral genome that permits stable replication of recombinant plasmids in latently infected cells, Proc. Natl. Acad. Sci. (U.S.A.), 81, 3806-3810. Yates, J.L., N. Warren and B. Sugden (1985) Stable replication of plasmids derived from Epstein-Burr virus in various mammalian cells, Nature (London), 313, 812-815.