Simian Virus 40 Strains with Novel Properties Generated by Replacingthe Viral Enhancer with Synthetic Oligonucleotides

Viola Günther, Till Strassen, Uschi Lindert, Patrizia Dagani, Dominique Waldvogel, Oleg Georgiev, Walter Schaffner,and Tobias Bethge

Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland

Typical enhancers of viral or cellular genes are approximately 100 to 400 bp long and contain several transcription factor bind-ing sites. Previously, we have shown that simian virus 40 (SV40) genomic DNA that lacks its own enhancer can be used as an “en-hancer trap” since it reacquires infectivity upon incorporation of heterologous enhancers. Here, we show that SV40 infectivitycan be restored with synthetic enhancers that are assembled by the host cell. We found that several oligonucleotides, cotrans-fected with enhancerless SV40 DNA into host cells, were incorporated into the viral genome via cellular DNA end joining. Theoligonucleotides tested included metal response elements (MREs), the binding sites for the transcription factor MTF-1, whichinduces gene activity in response to heavy metals. These recombinant SV40 strains showed preferential growth on cells over-loaded with zinc or cadmium. We also cotransfected enhancerless SV40 DNA with oligonucleotides corresponding to enhancermotifs of human and mouse cytomegalovirus (HCMV and MCMV, respectively). In contrast to SV40 wild type, the viruses withcytomegalovirus-derived patchwork enhancers strongly expressed T-antigen in human HEK293 cells, accompanied by viralDNA replication. Occasionally, we also observed the assembly of functional viral genomes by incorporation of fragments of bo-vine DNA, an ingredient of the fetal calf serum in the medium. These fragments contained, among other sites, binding sites forAP-1 and CREB transcription factors. Taken together, our studies show that viruses with novel properties can be generated byintracellular incorporation of synthetic enhancer DNA motifs.

In higher eukaryotes, including mammals, regulatory DNA se-quences for gene transcription can activate transcription over

long distances of thousands of base pairs (bp), independent oftheir orientation and position relative to the transcription unit.These DNA segments, termed enhancers, were first discovered insimian virus 40 (SV40), a member of the Polyomaviridae, and inmouse polyomavirus (2, 6, 18). The first example of a cellularenhancer discovered was the immunoglobulin heavy chain (IgH)enhancer, which was a cell-type-specific regulatory sequence sinceit was active only in B-lymphocyte-type cells and not in other celltypes (1, 9). Thereafter, many more cellular and viral enhancerswere described, including a steroid hormone-responsive segmentin mouse mammary tumor virus (4), the particularly strong en-hancers associated with immediate-early genes of human andmouse cytomegaloviruses (HCMV and MCMV, respectively) (3,7), and zinc-responsive enhancers associated with human andmouse metallothionein genes (28). The typical enhancers of viralor cellular genes are approximately 100 to 400 bp long and containbinding sites for several DNA-binding transcription factors. Forfacilitated identification of enhancers, a selection system termed“enhancer trap” can be used (20, 33). One simple approach is totransfect linearized SV40 genomic DNA lacking its own enhancerinto monkey host cells together with DNA fragments containing aputative enhancer segment. Whenever an enhancerless SV40 ge-nome acquires an active enhancer DNA via cellular DNA end-joining processes, it confers on that recombinant virus the abilityto proliferate (20, 33). Here, we show that this enhancer trap sys-tem, which before was used to select preexisting enhancers, is alsoable to compose new enhancers from a mixture of short DNAsequence motifs, derived either from enhancers of cytomegalovi-ruses or from metal-inducible genes. From the latter we testedbinding sites for the zinc finger protein metal-responsive tran-scription factor 1 (MTF-1; also referred to as metal response ele-

ment [MRE]-binding transcription factor or metal regulatorytranscription factor). MTF-1 induces gene activity in response to aheavy metal load via binding to MREs, short DNA motifs with theconsensus sequence TGCRCNC (35). With this approach, en-hancers were assembled from multiple MRE-containing oligonu-cleotides to yield recombinant SV40 viruses with the novel prop-erty of preferential growth in heavy metal-loaded cells.

Both lines of experiments, with cytomegalovirus-derived mo-tifs and with MREs, show that synthetic enhancers can readily beassembled from double-stranded DNA oligonucleotides contain-ing transcription factor binding sites and that the resulting recom-binant viruses can have novel properties clearly distinct fromthose of the original virus. Interestingly, in a few cases we foundenhancer assemblies not originating from the provided syntheticoligonucleotides but from bovine DNA fragments present in thefetal calf serum (FCS) of the cell culture medium.

MATERIALS AND METHODSOligonucleotides and DNA constructs. All oligonucleotides used in thisstudy were synthesized by Microsynth Co., Balgach, Switzerland. Sinceindividual repeat motifs of cytomegalovirus (CMV) enhancers deviateslightly from each other, we derived an idealized consensus sequence foreach of them. The oligonucleotide sequences with CMV repeat motifs andwith metal response elements are depicted in Fig. 1B and 3A, C, and D,

Received 13 September 2011 Accepted 22 December 2011

Published ahead of print 11 January 2012

Address correspondence to Tobias Bethge, tobias.bethge@uzh.ch.

V. Günther and T. Strassen contributed equally to this article.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.06293-11

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respectively. A single-stranded oligonucleotide representing one copy ofthe 72-bp repeat from the SV40 wild-type (wt) enhancer (see Fig. 1E)(NCBI accession number NC_001669) as well as the separate singlestrands of the 90-bp MCMV (Fig. 1B) was also used. The enhancer trapconstruct, as depicted in Fig. 1A, was described previously (33).

Cell culture and transfection. African green monkey kidney cells(CV-1) and human embryonic kidney cells (HEK293) were maintained inDulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with5% FCS (Biochrom AG, Berlin, Germany), 2 mM L-glutamine, 100 U/mlpenicillin, and 100 �g/ml streptomycin (Invitrogen). Cells were seededinto 10-cm petri dishes 1 day before transfection. At the time of transfec-tion, the cell monolayer was approximately two-thirds confluent. Trans-fections were performed using a standard calcium phosphate coprecipi-tation method (10). One microgram of enhancer trap DNA was mixedwith 1 to 10 �g of double-stranded oligonucleotides (see figures and fig-ure legends), representing a 500- to 1,000-fold molar excess over the SV40genome. Single-stranded oligonucleotides were transfected in a 1,500-fold molar excess over the SV40 genome. To standardize the amount ofDNA, herring sperm DNA was added to a total amount of 20 �g of DNA.Cells were washed 14 to 16 h after transfection with Tris-buffered saline(TBS). Unless indicated otherwise, metal treatments started 1 day aftertransfection by supplementation of the medium with ZnCl2 to a finalconcentration of 100 �M, which was raised to 200 �M after another day.Similarly, cells were conditioned with 3 �M CdCl2, which was subse-quently raised to 6 �M.

T-antigen immunofluorescence. Indirect immunofluorescence wasperformed as described previously (24) with a 1:200 dilution of a mono-clonal mouse anti-T-antigen antibody (Ab-2; product number DP02; On-cogene/Calbiochem) and secondary Alexa Fluor 546 goat anti-mouse an-tibody (A-11030; Molecular Probes/Invitrogen) in a 1:200 dilution.Nuclei were stained by 4=,6-diamidino-2-phenylindol (DAPI) at a con-centration of 1 �g/ml for 1 min.

Analysis of viral DNA. Small, closed circular DNA was extracted frominfected CV-1 cells by an alkaline precipitation procedure according to themethod of G. Magnusson (Department of Medical Biochemistry and Mi-crobiology, Uppsala University, Biomedical Centre, Uppsala, Sweden).First, cells were washed once with TBS and lysed with a NaOH- and SDS-containing buffer (50 mM glucose, 25 mM Tris-HCl, pH 8, 10 mM EDTA,0.2 M NaOH, 1% SDS). After neutralization with 3 M potassium acetate,pH 4.8, and centrifugation of the lysates, viral DNA was purified from thesupernatants by phenol-dichloromethane extraction and isopropanolprecipitation. Purified viral DNA was cloned via a BamHI site into Blue-script plasmid (pBSK). From individual colonies, complete viral genomeswere reclaimed by BamHI digestion and transfected into CV-1 cells toverify their infectivity. In parallel, the enhancer inserts were sequenced.For some experiments, only the enhancer region was isolated, cloned, and,to test for its activity, mixed again with enhancerless SV40 DNA.

Detection of viral DNA by Southern blotting. Total DNA of HEK293cells that were transfected with wild-type or recombinant SV40 viral DNAwas isolated and purified as described above under “Analysis of viralDNA.” Five micrograms of DNA was digested by EcoRI, and viral DNAwas detected by Southern blotting with a 32P-end-labeled oligonucleotideprobe (5=-GCACACTCAGGCCATTGTTTGCAGTACATTGCATCAACACCAGG-3=). As an internal control, pBluescript-KS was cotransfectedand duplicates were run on a parallel gel and detected with the followingoligonucleotide: 5=-CCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCT-3=. Signals were visualized using the fluorescent im-age analyzer FLA-7000.

Semiquantitative PCR. Reactions were performed with 28 cycles us-ing the following primers: 5=-GCCAAGCAACTCCAGCCATC-3= and 5=-TGAGGAAAGTTTGCCAGGTG-3= to detect SV40 and 5=-GCGGATAAAGTTGCAGGACCAC-3= and 5=-CTGCTGGAAGCCAGTTACCTTC-3=to detect cotransfected pBluescript as a control.

EMSA. Electrophoretic mobility shift assays (EMSAs) of transcriptionfactor binding sites were performed as described before (25). Briefly, 10

�g of nuclear extract from CV-1 cells was mixed with binding buffer (20mM PIPES [piperazine-N,N=-bis(2-ethanesulfonic acid)], pH 6.8, 50 mMNaCl, 1 mM dithiothreitol [DTT], 0.25 mg/ml bovine serum albumin[BSA], 100 �M ZnSO4, 0.05% NP-40, 4% Ficoll) and approximately 5fmol of 32P-labeled, duplexed oligonucleotide in a final volume of 20 �l.Nuclear extracts were prepared by scraping cells off 10 cm-dishes withice-cold phosphate-buffered saline (PBS) and collecting them in Eppen-dorf tubes. After centrifugation, cells were swollen in hypotonic buffer (10mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 2.5 mM DTT) and lysedby addition of NP-40 (final concentration, 0.5%). After centrifugation ofthe nuclei, proteins were extracted in nuclear extract buffer (20 mMHEPES, pH 7.9, 25% glycerol, 400 mM NaCl, 1 mM EDTA, 2.5 mMDTT). Nuclear extracts with activated CREB were prepared after cellswere treated for 1 h with 10 �M forskolin (Sigma-Aldrich). Competing,unlabeled oligonucleotides were used in 200-fold excess. After the mix-ture was incubated for 30 min on ice, samples were separated on a native4% polyacrylamide gel with 0.25� Tris-borate-EDTA (TBE) as a runningbuffer. The detection of �-decay within the samples was carried out withFujifilm Imaging Plates and a Fujifilm FLA-7000 Image Plate reader.

RESULTSCytomegalovirus-derived oligonucleotides are integrated intothe SV40 enhancer trap to restore enhancer activity. The verystrong enhancer associated with the immediate-early transcrip-tion unit of human cytomegalovirus (HCMV) harbors four majortypes of transcription factor-binding DNA sequence motifs, re-ferred to as the 17-bp repeat, 18-bp repeat, 19-bp repeat, and21-bp repeat. These repeats are embedded within several hundredbase pairs of nonrepetitive enhancer sequence (3). Similar motifsare present also in the mouse cytomegalovirus (MCMV) en-hancer, which is organized into several nested repeat units of ap-proximately 90 bp (7). To find out whether a synthetic transcrip-tion enhancer that is able to substitute for SV40’s own enhancercan be generated from double-stranded oligonucleotides, we usedthe SV40 enhancer trap, an enhancerless viral genome that hasbeen shown to reacquire infectivity upon incorporation of a het-erologous viral or cellular enhancer (33). SV40 DNA was liberatedfrom the vector plasmid by cleavage with XbaI and KpnI to yieldthe enhancerless viral genome of 5,040 bp (Fig. 1A). This genomewas cotransfected with either a mixture of all four repeated se-quence motifs of the HCMV enhancer or with oligonucleotidesrepresenting individual repeats (Fig. 1B). In a parallel experiment,a synthetic 90-bp consensus repeat from the MCMV enhancer,which among other sites contains binding sites for NF-�B andAP-1, was also tested (Fig. 1B). In all cases, infectious viruses werereadily obtained.

Various enhancer lengths and compositions result in similarlevels of virus propagation. To characterize the synthetic en-hancer sequences acquired by enhancerless SV40, viral DNA wasisolated from cell lysates and subcloned. After a sequencing step,selected clones were reanalyzed by retransfecting cells to identifyopportunistic genomes, which had replicated without having in-corporated a functional enhancer of their own. In the case ofHCMV oligonucleotides, we found all four enhancer motifs invarious combinations and orientations in different isolates (Fig.1C), which upon retesting were similarly infective (first signs ofcytopathic effects [CPE] at around day 10 and full infection ataround day 14 after transfection). Thus, although the lengthsand compositions of the isolated enhancers were quite variable,our experimental setup apparently selected for viral enhancerswith similar activities. Of note, the 18-bp repeat, which harborsa previously described binding site for NF-�B (23), was inte-

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grated with the highest frequency: in 10 representative cloneswith enhancer inserts (Fig. 1C), which on average incorporated6.9 oligonucleotides, the 18-bp fragment appeared with an av-erage frequency of 2.8 copies, or about 40%. This finding isconsistent with previous studies, which showed that NF-�B isimportant for enhancer activation in CMV-infected humanfibroblasts (23, 29), and suggests that under stress conditions oftransfection/viral infection, transcriptional activation byNF-�B is particularly robust. The second-most-abundant oli-gonucleotide was the 17-bp repeat, which contains a bindingsite for the housekeeping factor NF-1 (12, 19) and occurredwith a frequency of 1.7 oligonucleotides per insert. The lowestin abundance, with an average of 1.2 oligonucleotides per in-sert, were the 19-bp repeat with a CREB site (13) and the 21-bprepeat with an Sp1 site (3). The potency of NF-�B sites wasconfirmed by offering individual HCMV oligonucleotides,rather than a mixture, to the enhancer trap. When CV-1 cellswere transfected with enhancer trap vector and the 18-bp re-peat, the first signs of CPE could be observed after an average of22 days. In direct comparison, this was as efficient as the use ofthe oligonucleotide mixture with all four sites. An intermediate

progression of viral infectivity was observed with the 21-bp or17-bp fragment (33 days or 35 days until first signs of CPE,respectively). Consistent with the integration ratios seen withthe oligonucleotide mixture, the poorest enhancer activationand viral growth seemed to be conferred by the 19-bp oligonu-cleotide with its CREB site, leading, on average, to first signs ofCPE after 41 days (data not shown). Regarding the discrepan-cies of timelines between different experiments, it has to benoted here that the reinfection of cells with isolated virusesleads to a faster progression of viral infection and visible CPEthan the transfection with enhancer trap and oligonucleotidessince the initial number of functional viruses is much lower inthe latter case.

Assembly of recombinant viruses by cellular end-joiningprocesses. Even though the oligonucleotides had 5= protruding“sticky ends,” these were not necessarily used by the cell. For ex-ample, in head-to-tail or head-to-head fusions of oligonucleotideswhere the ends did not match each other, the new enhancers weremost likely assembled by a trimming and ligation process, termednonhomologous end joining (NHEJ) (5, 11, 15, 30, 34). An exten-sively trimmed junction between the XbaI site of the enhancerless

FIG 1 Enhancer trap and CMV oligonucleotides. (A) Junctions of enhancerless SV40. Linear SV40 DNA of 5,040 bp was liberated from the pBSK vector plasmidby XbaI and KpnI. Numbers refer to the positions in the SV40 wt genome. Ori, viral origin of replication. (B) Oligonucleotides harboring the four HCMV-derivedrepeat motifs of 17 bp (blue), 18 bp (yellow), 19 bp (red), 21 bp (green), and MCMV 90 bp (gray). Respective transcription factor binding sites are underlined.(C) Ten representative SV40 isolates and their composition. (D) Example for enhancer assembly by NHEJ. Cotransfected was the 18-bp repeat derived fromHCMV with sticky 3= GG and CC overhangs (18 bp; yellow box). (E) Example for enhancer assembly from a single-stranded 72-nt SV40 wild-type enhancersegment (red) by MMEJ. SV40 enhancer trap sequence (black) attaches to the oligonucleotide, noncomplementary overhangs are trimmed (blue), and cellularenzymes fill the gaps and synthesize the complementary strand (green).

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SV40 and an 18-bp repeat oligonucleotide is depicted in Fig. 1D.Apparently, before or in the course of integration, the 5= end of theoligonucleotide as well as the 3= end of the SV40 genome wastrimmed by cellular nucleases, resulting in the removal of thesticky ends and the XbaI site, and this was followed by blunt-endligation.

Interestingly, the enhancer trap system also works with single-stranded substrate oligonucleotides, such as the separate singlestrands of the 90-bp MCMV enhancer segment (Fig. 1B) or a72-nucleotide (nt) repeat of the genuine SV40 enhancer (Fig. 1E).However, in these cases incorporation into the viral genome seemsto be less efficient than with the use of duplexed oligonucleotides.To achieve assembly of functional viruses, the 72-nt repeat had tobe provided in larger molar excess over the SV40 genome(1,500-fold instead of 500- to 1,000-fold double-stranded oli-gonucleotides). First signs of CPE could be observed after 23days. Sequence analysis suggested that integration had not hap-pened via NHEJ but by a variant mechanism, termedmicrohomology-mediated end joining (MMEJ) (30), referringto the fact that prior to repair-ligation, single-stranded DNA orsingle-stranded overhangs can attach to sticky ends with thehelp of one or more complementary bases. In the exampleshown in Fig. 2E, the XbaI site as well as the KpnI site of theenhancer trap was able to pair a G or C with a terminal base ofthe oligonucleotide, followed by the removal of noncomple-mentary overhangs (blue sequences), gap filling, and synthesisof the complementary strand (green sequences).

T-antigen expression and viral DNA replication in humancells as a novel property of CMV-SV40 recombinants. To com-pare the performance of CMV enhancer-containing recombinantviruses with the SV40 wild type (wt), CV-1 cells were transfectedwith a mixture of viral DNA from cell culture supernatants. Whilecells transfected with SV40 wt showed the first CPE after 2 daysand full infection after 4 days, progression of infection with theCMV segment-containing viruses was delayed by approximately 2days. Interestingly, although the ability of the recombinant virusesto replicate in monkey CV-1 cells was not equivalent to that of theSV40 wt, they gained an advantage over SV40 wt by acquiringCMV elements. That is, if transfected into immortalized humanembryonic kidney (HEK293) cells, SV40 with its genuine en-hancer failed to produce T antigen at a detectable level, which is aprerequisite for a successful infection. In contrast, human andeven mouse CMV segment-containing viruses readily expressedthe early gene region, as determined by T-antigen immunofluo-rescence (Fig. 2A and B). This is further testimony of the strongpromiscuous activity of HCMV and MCMV enhancers (3, 7, 8)and demonstrates that enhancer composition is a determinant ofhost cell tropism. In agreement with T-antigen expression of therecombinants, Southern blotting and semiquantitative PCR re-vealed that in HEK293 cells the synthetic CMV-derived enhanc-ers, especially the enhancer from HCMV, boosted viral replica-tion, while only a small amount of SV40 wild-type DNA wasproduced (Fig. 2C and D).

FIG 2 Cytomegalovirus-derived synthetic enhancers drive SV40 early gene expression and DNA replication in human cells. Recombinant SV40 viral genomeswhich contain HCMV-derived or MCMV-derived enhancers or its wild-type enhancer (SV40) were used to transfect monkey CV-1 cells or human HEK293 cells.(A) Immunofluorescence stainings using anti-T-antigen antibody; nuclei were stained with DAPI. (B) Columns represent the percentages of T-antigen-positivecells of two independent experiments. (C) Southern blotting with total DNA isolated from HEK293 cells that were transfected 8 days prior to harvesting with amixture of viral DNA and pBluescript (plasmid) as a control (ctrl), run on parallel gels. (D) Semiquantitative PCR with template DNA isolated from thetransfected HEK293 cells described for panel C. �, anti.

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MREs can be assembled to generate metal-dependent SV40recombinant viruses. In a second series of experiments, we used amixture of oligonucleotides representing established metal re-sponse elements (MREs) (21, 27). These were derived from me-tallothionein genes and other known cellular target genes ofMTF-1 and were chosen because of their avid binding to MTF-1(22, 36). Most of these MRE-containing oligonucleotides had pro-truding single-stranded ends which did not, however, fit eachother (Fig. 3A). Nevertheless, by using a culture medium supple-

mented with 150 �M ZnCl2, a number of viruses were readilyobtained in independent culture dishes. Upon cloning and se-quence analysis, they turned out to have incorporated a mix ofdifferent MRE sequences, similar to the situation with the se-quence elements from the human cytomegalovirus enhancer (Fig.3B). Also here, as observed for the CMV-derived segments, therewas no obvious preference regarding the orientation of the MREsfor enhancer activation. Note that only two MRE oligonucleotideshave sticky ends that might facilitate self-ligation: the artificialconsensus MREs (Fig. 3B, red) and the MRE of selenoproteinSepW1 (Fig. 3B, gray). The fact that no overrepresentation of mul-tiple tandem arrangements of these two MREs was observed sup-ports the notion that compatible sticky ends are not a prerequisitefor the intracellular assembly of enhancers. Again, the aforemen-tioned repair via NHEJ led to the trimming and ligation ofincompatible ends. To expand on the observation that the MRE-mediated activation of transcription was independent not only ofthe orientation but also of the spacing between individual MREs,we decided to test preformed arrays of tandem oligonucleotideswith defined spacings of 15 and 20 bp between the beginning ofone core MRE sequence and the next [MRE(15) and MRE(20),respectively] (Fig. 3C). With both spacings, severe cytopathic ef-fects were observed at about day 10 postinfection. Recombinantviruses were sequenced, and in all cases the predetermined spacingwas maintained, indicating that spacing is not critical (data notshown).

In a further experiment we sought to examine whether asingle MRE, the well-characterized MREd (metal response el-ement d) of the mouse metallothionein 1 gene promoter,could, alone or in multiple copies, substitute for the SV40 en-hancer. For this experiment, an oligonucleotide with compat-ible sticky ends was used, resulting in functional viruses thatgrew well in zinc-supplemented medium (Fig. 3D). To confirmthe novel properties of the MRE-containing viral isolates, CV-1cells were reinfected with viruses from supernatants of previ-ous experiments and kept in either medium supplemented with200 �M ZnCl2, or control medium. Here, a clear preference forviral growth in zinc-containing medium was evident sincecomplete lysis of host cells was observed after 18 days in thepresence of ZnCl2, while cells in unsupplemented medium didnot even show signs of CPE at day 23 when the experiment wasstopped (Fig. 4A). In contrast, the SV40 wild type did notmultiply any better in zinc-loaded versus control cells (datanot shown). In a separate experiment with another MRE-containing virus, CV-1 cells were infected with either SV40 wtvirus or recombinant viruses containing 14 tandem copies ofthe MRE(15) motif. After treatment with or without 150 �Mzinc for 17 days, the viral DNAs were extracted and analyzed bygel electrophoresis (Fig. 4B). In the cases of de novo DNA rep-lication, a band was visible that migrated at the speed of a 3-kblinear marker DNA, which represents supercoiled, covalentlyclosed circular viral DNA (Fig. 4B, ccc). As expected, the yieldof DNA from MRE-containing recombinant viruses correlatedwith the zinc treatment (Fig. 4B). The zinc-treated cells, in-fected with SV40-MRE, showed viral replication (lane 6 to 8),while the untreated cells (lanes 1 to 3) did not produce super-coiled viral DNA. In contrast, the amount of viral DNA fromSV40 wt-infected cells was independent of zinc (Fig. 4B, lane 4versus lane 9). Regardless of the zinc treatment or enhancer, inall lanes with infected cells a band which migrated at approxi-

FIG 3 Substitution of the SV40 enhancer by synthetic MREs. (A) Oligonucle-otides representing MREs from several metal-inducible genes were cotrans-fected with enhancer trap DNA into CV-1 cells. Csrp1 MRE2, cysteine andglycine-rich protein 1 MRE2 (green); Ndr1 MRE3,4, N-myc downstream-regulated 1 protein MRE3/MRE4 (blue); Ndr1 MRE2, N-myc downstream-regulated gene 1 protein MRE2 (yellow); MRE-s, synthetic MRE consensusoligonucleotide (red); SepW1 MRE, selenoprotein W1 MRE (gray); Slc30a10,zinc transporter MRE2 (orange). Underlined are the MRE core sequences. (B)Two examples of MRE-derived enhancers. The color for each MRE block isaccording to the scheme in panel A. (C) MRE(15) and MRE(20), MRE oligo-nucleotides with 15-bp and 20-bp spacing, respectively, between oligomerizedoligonucleotides. (D) Enhancer assembled from a single type of motif, theMREd from the mouse metallothionein 1 promoter (shown without the stickyends). All motifs were found to be arranged in the same direction, either asshown here or in the opposite orientation (not shown).

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mately 5 kb can be observed (Fig. 4B, rc), which most likelyoriginates from viral input DNA. Other than the newly synthe-sized supercoiled DNA, the input DNA persists as nicked, re-laxed circles due to nuclease-mediated damage.

Cadmium is a nonessential, toxic heavy metal that, like zinc,strongly induces transcription via MTF-1/MRE motifs. We alsotested medium with 6 �M cadmium salt in the same series ofexperiments. While higher concentrations of cadmium are partic-ularly strong inducers of MTF-1 in transient transfection assays,we found that for long-term culture of CV-1 cells, 6 �M cadmiumwas close to the upper tolerated limit. MRE-containing recombi-nant viruses that proliferated well in zinc also did so in cadmium-supplemented medium, but the first signs of infection appearedwith a delay of a few days (data not shown).

Taken together, in both lines of experiments, with cyto-megalovirus-derived motifs and with metal response elements, vi-ruses with novel properties could be generated.

Spontaneous generation of SV40 recombinants by incorpo-ration of bovine DNA fragments from cell culture medium. In-terestingly, in some enhancer trap experiments, we found insertswhich did not match with the oligonucleotide sequences provided inthat experimental setup. Sequence analysis revealed that the SV40enhancer trap constructs had acquired 85- to 100-bp-long fragmentsof bovine DNA. The likely source for these fragments was the fetal calfserum used to supplement the cell culture medium. With the help ofthe Transcription Element Search System (TESS [http://www.cbil.upenn.edu/cgi-bin/tess/tess), we looked for potential transcriptionfactor binding sites within the bovine sequences to explain the en-hancer activity and designed oligonucleotides carrying several of thepromising sites (Fig. 5A); among them were a CRE element (Bo1-CREB) and an AP-1 site (Bo2-AP-1). By subjecting these oligonucle-otides to electrophoretic mobility shift assays (EMSAs), we could ver-ify the binding of the aforementioned transcription factors (Fig. 5B).In the case of Bo1-CREB, the amount of CREB and, hence, CREBbinding could be increased by stimulating the CV-1 cells with forsko-lin (14, 26), while an unlabeled oligonucleotide containing the CREBbinding site from the somatostatin promoter (17) or the HCMV

19-bp oligonucleotide (HCMV19) (Fig. 1B) could be used to com-pete with the binding of Bo1-CREB. Likewise, the Bo2-AP-1 oligo-nucleotide showed binding to multiple transcription factors, amongthem AP-1, as demonstrated by competition with oligonucleotides

FIG 4 Growth of MRE-containing SV40 in the presence of zinc. (A) Reinfection of MREd-containing, recombinant SV40 to compare growth in zinc-supplementedmedium (200 �M ZnCl2) and normal medium (without zinc). Arrows mark days of inspection. (B) DNA agarose gel after treatment with or without 150 �M ZnCl2 ofcells with SV40 wt or SV40 containing tandem copies of MRE(15) inserts. Seventeen days after infection of CV-1 cells, DNA was extracted and loaded onto an agarosegel. M, phage � DNA fragments as markers; arrows mark covalently closed circular (supercoiled) SV40 DNA (ccc) and nicked, relaxed circular SV40 input DNA (rc).

FIG 5 EMSA with oligonucleotides derived from bovine sequences. (A) Oligo-nucleotides derived from isolated bovine sequences. Underlined are potentialbinding sites for CREB and AP-1. (B) EMSA gels. CV-1 ex, nuclear extracts fromCV-1 cells; �, no extract added (free probe); �, 10 �g of nuclear extract added;forsk, stimulation of cells with 10 �M forskolin for 1 h; CREB soma, competitionwith 200-fold excess of unlabeled oligonucleotide (1 pmol) containing a CREB sitefrom the somatostatin promoter (the other competing oligonucleotides,HCMV19, AP-1, SV40 and AP-1 Ad5, were used in the same amount of excess).Arrows mark the bandshift signals from the respective transcription factors.

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containing the AP-1 sites from SV40 or adenovirus 5 (Ad5) (16, 31).These findings revealed that in addition to synthetic oligonucleotides,heterologous binding-site-containing DNA fragments from othersources are also able to exert enhancer activity for SV40 transcription.

DISCUSSION

The data presented here demonstrate that short sequence mo-tifs, represented by synthetic double-stranded DNA oligonu-cleotides, can be assembled in vivo to convert an enhancerlessSV40 into a functional virus. Novel enhancers are assembledfrom different nonmatching sequence motifs, most likely viathe pathway of nonhomologous end joining (NHEJ) or micro-homology-mediated end joining (MMEJ). Preligation of stickyends of these oligonucleotides facilitates the assembly of afunctional virus but is not mandatory, thanks to the efficientcellular repair activities. Functional enhancers were readily as-sembled from a mixture of synthetic CMV-derived oligonucleo-tides or metal response elements, representing MREs from a numberof target genes of MTF-1. It is of particular interest that the resultingviruses could display a novel property, like preferential growth, incells loaded with the heavy metals zinc and cadmium. It remains to beseen how such synthetic enhancers with repeated elements mightevolve further (see also reference 32).

As a side note, we observed the occasional integration of bovineDNA fragments from the calf serum in the culture medium intothe enhancerless viral backbone although the DNA in the serumwas hardly detectable. Interestingly, three independent isolates ofrecombinant viruses contained similar but nonidentical frag-ments of the bovine genome.

Taken together, our results show that (i) SV40 harboring syntheticenhancers can readily be generated by transfection of host cells with amix of enhancerless DNA and synthetic oligonucleotides containingtranscription factor binding sites, (ii) the oligonucleotides do noteven have to contain ligatable ends since the cellular repair machineryjoins them to build an enhancer by trimming and/or filling up single-stranded overhangs, and (iii) the recombinant viruses can have novelproperties. With synthetic MREs, viruses could be obtained thatgrew poorly, if at all, in cells kept in normal medium but grewwell in heavy metal-loaded cells. Similarly, viruses containingcytomegalovirus-derived enhancer motifs displayed altered prop-erties in that they acquired the ability of directing early gene ex-pression and viral DNA replication in human HEK293 cells. How-ever, this effect did not result in viruses with a fully expanded hostrange since substantial early gene expression and DNA replicationin human cells were not sufficient to allow for a progressive spreadof infection. Among the four different HCMV repeats (17, 18, 19,and 21 bp), stress conditions of transfection and infection clearlyfavored the 18-bp oligonucleotide with its NF-�B site in CV-1cells.

By using defined binding sites for transcription factors, thissystem of combining synthetic components followed by naturalselection could be used to create made-to-measure enhancers thatfit a particular need, e.g., to obtain viruses with a certain cell typespecificity. This might be useful for various applications, such astargeted gene therapy.

ACKNOWLEDGMENTS

We are indebted to Antonia Manova for technical advice, to Kurt Steinerfor analysis of DNA sequences, and to George Hausmann for critical read-ing of the manuscript.

This work was supported by the Schweizerischer Nationalfonds andthe Kanton Zürich.

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