Evaluation of the T-REx™ transcription switch for conditional expression and regulation of HSV-1 vectors
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- Knopf, C.W., Zavidij, O., Rezuchova, I. et al. Virus Genes (2008) 36: 55. doi:10.1007/s11262-007-0178-9
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Herpes simplex virus 1 (HSV-1) strain ANG and ANGpath were cloned as bacterial artificial chromosome (BAC). Two different types of BAC genomes were obtained. BAC genomes of type I contained the BAC replicon at the intended target region between the genes of UL48 and UL49. In BAC genomes of type II, the BAC sequences were found to be aberrantly fused between the termini of the HSV-1 genome. Both the BAC types were used to establish a conditional gene expression system for HSV-1 by Flp recombinase-mediated insertion of expression vectors that were modified to respond to the T-REx™ tetracycline (Tet)-inducible transcription switch. During BAC cloning and mutagenesis in E. coli, not only deletions but also defined mutations of the HSV-1 genome were observed. Successful virus reconstitution from BACs with large inserts demonstrated that HSV-1 has a packaging capacity for foreign sequences of at least 8.1% of its genome size. Targets for Tet-regulated gene expression were the viral DNA polymerase gene (pol) and a reporter gene of glycoprotein B fused to enhanced green fluorescent protein (gBGFP). Results with the pol gene as target showed that virus plaque production could not be significantly controlled by the T-REx™ gene switch using vectors encoding one copy of the tetR gene. In contrast, an efficient Tet-response was achieved with the gBGFP reporter, which was optimal in a Tet repressor (TetR)-producing cell line, demonstrating that the TetR concentration provided by the virus was not sufficient for a tight control of Tet-regulated gene expression.
KeywordsHSV-1 ANGANGpathBAC cloningRed/ET recombinationTetracyclineT-REx™ transcription switchGene expression
Herpes simplex virus 1 (HSV-1) has number of properties that make it an attractive platform for new vector developments with the central nervous system as target . The advantage is its pronounced neurotropism, leading to the establishment of latent infections. Its large genome persists as an episome in the neuronal cells, and has a great carrier capacity for therapeutic genes. As a basis for oncolytic vector design and study of viral pathogenicity, our labs have cloned the HSV-1 genomes of strain ANG and its pathogenic derivative (ANGpath) as bacterial artificial chromosome (BAC) by homologous recombination in eukaryotic cells, using the technique exemplified for the human cytomegalovirus (hCMV) genome . In order to construct replication-competent vectors without sequence deletions, the gene junction between UL48 and UL49 was selected for the integration of the BAC replicon. In addition, it was mandatory for the therapeutic application of the recombinant vectors to remove the BAC replicon from the vector genome. Therefore, we applied a BAC replicon that was flanked by loxP sequences, and could be excised by Cre recombinase . The established HSV-1 BACs were employed for the construction of therapeutic vectors with suicide phenotype, in which both expression as well as replication should be controlled by a tetracycline (Tet)-controlled transcription switch, following the strategy exemplified for CMV . The Tet-regulatable expression vectors contained (i) the Tet repressor gene (tetR) under the control of the hCMV major immediate-early enhancer–promoter (PCMV), and (ii) the gene of interest under the control of the Tet-inducible hybrid hCMV promoter (PCMV2tetO) bearing two minimal tet operator sequences of type 2 (2tetO) inserted 10 bp downstream of the TATA element . The regulation vectors were recombined with HSV-1 BAC using Flp recombinase-mediated insertion . In the present study, we have chosen the viral DNA polymerase gene (pol) as target for controlling virus replication by applying a recently introduced Tet-inducible transcription switch (T-REx™; Invitrogen, Karlsruhe, Germany) , which was successfully tested in a replication-defective HSV-1 vector . Using Red/ET and Flp recombinase-mediated recombination for targeted mutagenesis [6, 8], we generated several pol (UL30) knockout (K.O.) mutants, in which PCMV2tetO or only 2tetO elements were inserted at the 5′-control region of the gene. In addition, for in situ visualization and optimization of conditional gene expression, we have constructed a reporter gene that encoded a fusion protein (gBGFP) of HSV-1 glycoprotein B (gB) and the enhanced green fluorescent protein (EGFP). The gBGFP fusion gene under the control of PCMV2tetO was inserted together with the tetR gene at two different locations on the HSV-1 genome: (i) into the ribonucleotide reductase (RR) gene locus (UL39, UL40) of wildtype ANG BAC, and (ii) in the terminal repeat sequences of the large unique region (TRL) of a ANGpath BAC clone, in which the BAC replicon was aberrantly fused between the genome termini. Our results show that the applied T-REx™ transcription switch in a replication-competent HSV-1 vector, encoding only one copy of the tetR gene and with the pol gene under the control of a 2tetO-bearing hCMV promoter, is inappropriate for controlling virus replication as determined by virus plaque formation. In contrast, an apparently tight control of gene expression was achieved with virus vectors, in which the gBGFP reporter was set under the control of T-REx™ Tet-regulation, but only in cells constitutively expressing TetR, manifesting that the TetR concentration is the critical factor for an optimal functioning of the T-REx™ transcriptional switch.
Materials and methods
Lipofectamin™, Plus™ reagent, doxycycline, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and Opti-MEM I were purchased from Gibco/Invitrogen (Karlsruhe, Germany). Luria broth (LB) without additions was used for bacterial growth throughout. l-arabinose was purchased from ICN Biomedicals (Meckenheim, Germany). Restriction enzymes and reaction buffers were routinely used from New England BioLabs (Frankfurt a.M., Germany), Roche (Mannheim, Germany), and Fermentas (St. Leon-Rot, Germany). HyperLadder I (Bioline, Luckenwalde, Germany) was used as DNA size standard. DNA oligonucleotide synthesis and DNA sequencing were kindly performed by Wolfgang Weinig and Andreas Hunzicker, respectively (DKFZ Core Facility, Heidelberg, Germany).
Cells and viruses
The baby hamster kidney cell line BHK-21 (clone 13) was a gift of Prof. J. Subak-Sharpe (Medical Research Council, Glasgow), and was cultured in DMEM supplemented with 5% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) (supplemented DMEM/5% FBS). Standard HSV-1 strain ANG and ANGpath virus were propagated as described previously . E. coli strains DH5α, DH10B, and PIR1 were obtained from Invitrogen (Karlsruhe, Germany). E. coli K12 DH5α strain BT340 containing pCP20 was a kind gift of Wilfried Wackernagel (Carl-von-Ossietzky University, Oldenburg, Germany). Mary K. B. Berlyn (E. coli Genetic Stock Center, Yale University, New Haven, CT 06520-8103) kindly provided E.coli strain MG1655 containing pKD46.
The nucleotide positions of the HSV-1 genome provided here refer to McGeoch et al. . Design of plasmids and primers, and sequence handling were performed with Clone Manager 7, version 7.11, Professional Suite (Scientific & Educational Software Durham, NC 2772). Plasmid constructions were routinely confirmed by DNA restriction as well as sequence analysis. All plasmids containing the R6Kγ origin (ori) were propagated in E. coli strain PIR1, which provides the phage lambda Π protein. Plasmid pKD46 (Apr, repA101ts) carries the phage lambda recombination genes red α, β, and γ under the control of the araB (arabinose) promoter . Plasmid pCP20 (Apr, Cmr, ts repA101ts) carries the yeast Flp recombinase gene . The following plasmids were a kind gift of Eva Borst and Martin Messerle (Hannover Medical School, Hannover, Germany): pSLFRTKn , containing the kanamycin resistance (kanr) gene from transposon Tn903, and flanked by two identically orientated Flp recombinase recognition targets (FRT); pHA1, a derivative of pK18, containing a 1.5-kbp fragment from murine gammaherpesvirus 68 and a PacI fragment including the BAC replicon and a gpt (guanosine phosphoribosyl transferase) gene ; pOri6K-Kan1 (1,568 bp) , carrying the kanr gene with one flanking FRT signal site, and the Π protein-dependent R6Kγ ori . Plasmid p6KFRT was constructed from pSLFRTKn by inserting between its NcoI and EcoRV sites the 450-bp EcoRI blunt-end/BspHI R6Kγ ori fragment of pOri6K-Kan1. Plasmid p11 derived from pUC19 containing a 11-kbp EcoRI joint fragment of ANGpath ΔBAC with sequences from nt 5,732 to 12,591 of the HSV-1 genome. The vectors pcDNA3.1, pcDNA4/TO as source for PCMV2tetO, and pcDNA6/TR as source for the tetR gene, controlled by PCMV, were obtained from Invitrogen (Karlsruhe, Germany). pcDKpn-nR derived from pcDNA3.1 clone containing the KpnI n fragment of wildtype (wt) HSV-1 ANG from nt 52,732 to 57,433. pcD4gB (5,690 bp) derived from pcDNA4/TO containing between EcoRV and XbaI sites the NruI/XbaI fragment from pcDKpn-nR comprising the UL27 gene from nt 52,732 to 56,097. pgBP77K79 derived from pUC19, containing an EcoRI/HindIII fragment generated by PCR with the gB gene of wt HSV-1 ANG spanning nt 53,020–56,086, in which the SanDI site was changed into a BamHI site (G- > A) at nt 55,597. Plasmid pEGFP derived from pIRES-EGFP (Clontech-Takara, Saint-Germain-en-Laye, France) by BamHI collapse. pgBGFP (6,181 bp) originated from pgBP77K79 containing the blunt-ended 1,757-bp NruI/XhoI fragment from pEGFP between the blunt-ended BamHI and SnaBI sites. Plasmid pgBGFP0 (5,442 bp) derived from pgBGFP by BamHI collapse. pgBGFP2 (4,607 bp) was constructed from pgBGFP0 by BsrGI collapse. pcD4gBG2 (7,351 bp) was obtained by inserting the 1,662-bp BsiWI/BstEII fragment of pgBGFP2 into pcD4gB. pcD4gBG05 (7,173 bp) derived from pcD4gBG2 by HindIII/BsiWI collapse. pR6KTR (3,738 bp) was constructed by inserting the blunt-ended SpeI/NotI fragment, encoding the tetR gene, from pcDNA6/TR into the EcoRV site of pOri6K-Kan.
Construction of Tet-regulated expression vector pRTRgBG
Plasmid pRTRgBG (6,658 bp) was constructed by inserting the 2,915-bp NruI/EcoRV fragment of pcDgBG05, containing the gBGFP gene, into the blunt-ended BstEII site of pR6KTR.
Construction of Tet-regulated expression vectors pRTRPOL and pRTRPOLi
Plasmid pPOLKZ served as source for the HSV-1 ANG pol gene with a Kozak initiation codon , and was constructed from pSKIILORF  by exchanging the HindIII/SnaBI fragment against a 204-bp HindIII/SnaBI-cleaved PCR fragment, generated with primer PATG 5′-ACC AAC CTT GCC ACC ATG TTT TCC GGT GGC GGC GGC CC, and primer PSNAB 5′-TCA TCG CAT TCG CTA TAG TAC G. pRTRPOL (8,735 bp) was constructed by inserting the 3,874-bp ClaI/blunt-ended XbaI pol gene fragment from pPOLKZ between the FspI and AccI sites of pRTRgBG (configuration a). pRTRPOLi (8,735 bp) was identically constructed as described for pRTRPOLi using pRTRgBG (configuration b).
Construction of Tet-regulated expression vector pRTRPOLII
Plasmid pRTRPOLII (7,609 bp) with a promoterless pol gene was constructed by inserting the 3,867-bp blunt-ended HindIII/XbaI pol fragment from pPOLKZ into the XmnI site of pR6KTR.
Construction of HSV-1 ANG BAC and ΔBAC
HSV-1 ANG BACs were constructed following the protocol of Borst et al. . For homologous recombination the PacI fragment of pHA1 were flanked by subcloning in pUC19 with UL48 sequences, from nt 104,155 to 105,345 and with UL49 sequences, from nt 105,346 to 106,754. Gel purified 9,974-bp HindIII/SacI BAC replicon fragments were cotransfected with CsCl density gradient-purified wt or ANGpath DNA into BHK-21 cells, that were cultured in the presence of 100 μM mycophenolic acid and 25 μM xanthine. After four rounds of selection, Hirt DNA  was prepared as described previously  and electroporated into DH10B cells. Positive clones were characterized by restriction enzyme digestion.
Primers for gene knockouts used in this study
5′ TGC ACA TGC CGG CCC GGG CGA GCC TGG GGG TCC GGT AAT TTT GCC ATC CCT CCC ATG TGC AGG TGC TGA ATT CG
5′ CTT GAA TGT CAC GCA CGC CAC CCC CAA CAG GTG GGA GAA GTA ATA GTC CGG GTG ACC ACG TCG TGG AAT GCC TT
5′ GCC CAC CGG CTA CGT CAC GCT CCT GTC GGC CGC CGG CGG TCC ATA AGC CCT CCC TAT CAG TGA TAG AGA TCT CCC TAT CAG TGA TAG AGA TCC CAT GTG CAG GTG CTG AAT TCG
5′ TGC ACA TGC CGG CCC GGG CGA GCC TGG GGG TCC GGT AAT TTT GCC ATC CCT ATC AGT GAT AGA GAT CTC CCT ATC AGT GAT AGA GAT CCC ATG TGC AGG TGC TGAA TTC G
5′ CAT GGA AGG AAC ACA CCC CCG TGA CTC AGG ACA TCG GTG TGT CCT TTT GGT CCC ATG TGC AGG TGC TGA ATT CG
5′ CCC GCG TCC CTG ACA AGA ATC ACA ATG AGA CCC AAA GTT TGG TTC AGA GGG GTG ACC ACG TCG TGG AAT GCC TT
Transformation of E. coli DH10B
Transformation of E. coli with plasmids was routinely performed with the ROTI® Transform Kit and protocol (Roth, Karlsruhe, Germany). Electroporation was used for BAC DNA and DNA fragments. E. coli DH10B cells were made competent for electroporation as follows. Microfuge tubes (2 ml) containing 1.4 ml LB and proper antibiotics were inoculated with 30 μl of a fresh overnight culture, and grown at the required temperature by shaking at 1,000 rpm using a thermomixer. After 3 h, bacteria were collected by brief centrifugation (10,000 ×g, 30 s, 4°C) and resuspended in 2 ml ice-cold and sterile distilled water. After two further rounds of centrifugation and resuspension, cell pellets were resuspended in 45 μl ice-cold and sterile distilled water, 5 μl (0.5 μg) of DNA was added, and the cell/DNA mixture was transferred into electroporation cuvettes (0.2 cm Ø) and kept on ice until used. Electroporation was performed with a Micropulser (Biorad, München, Germany) at 1.35 kV and 5 ms pulses for DNA fragments, and at 2.5 kV for BAC DNA. Electroporated cells were taken up in 1-ml prewarmed LB and incubated at the selected temperature with 1,000 rpm for 1 h. Aliquots were plated on selection agar.
Generation of recombinant viral BACs for conditional gene expression
Escherichia coli DH10B cells, containing the recombinant KFRT BACs, were transformed with the temperature-sensitive Flp recombinase expression plasmid pCP20, and selected by overnight growth at 30°C on agar plates containing 17 μg Cm/ml and 100 μg Ap/ml. Kns/Apr/Cmr transformants, containing both pCP20 and FRT BAC, were obtained by replica plating on agar plates with 100 μg Ap/ml at 30°C, 30 μg Kn/ml at 30°C, and 17 μg Cm/ml at 37°C, respectively. Individual colonies were grown up, made competent with ROTI® Transform, and transformed with the Tet-regulated expression vectors. After 70-min incubation at 30°C, selection was carried by overnight growth on agar plates containing 17 μg Cm/ml and 30 μg Kn/ml at 42°C. Three to five colonies were grown up on a small scale for DNA analysis.
Analysis of BAC DNA
For restriction analysis, BAC DNA was isolated from 2-ml overnight E. coli cultures by the alkaline lysis procedure , and the BAC DNA suspended into 50-μl 10 mM Tris-HCl/1 mM EDTA pH 7.5 (TE). For large preparations, BAC DNA was isolated from 12× 2-ml bacterial cultures as follows. After neutralization and centrifugation (step 3 of the alkaline lysis procedure), supernatants were combined in a 15-ml centrifuge tube, and incubated for 60 min at 37°C in the presence of 25 mM EDTA and 50 μg/ml RNAse A (DNase free) (Roche, Mannheim, Germany). BAC DNA was extracted with one volume of phenol/chloroform/isoamyl alcohol (ratio 25:24:1) and separated by centrifugation (3,000× g/10 min/4°C) using a swing-out rotor. BAC DNA was precipitated by adding one volume of 2-propanol and carefully mixing, and collected by centrifugation (3,000× g/30 min/4°C). The DNA pellet was taken up into 80% ethanol and transferred into 2-ml reaction tubes. After centrifugation at 15,000× g and 4°C for 5 min in a tabletop centrifuge, the pellet was air-dried and resuspended in 300 μl TE.
Restriction enzyme digestions were carried out following the protocols of the manufacturers. Incubations using BamHI and EcoRI were routinely performed for 60 s at 650 W in a microwave oven with a turntable.
Reconstitution of recombinant viruses from BACs
For reconstitution of recombinant virus, HSV-1 BAC DNA was transfected into BHK-21 cells. When cytopathic effect (CPE) was complete, cells were subjected to three cycles of freezing and thawing, and virus pools prepared from the supernatants of a low speed centrifugation (500× g for 10 min at room temperature). To enrich for infectious virus, half-confluent BHK-21 cell monolayers grown on six-well plates were infected with aliquots of the supernatant virus, and viral growth continued until CPE was complete. Then virus stocks were prepared as stated above.
Transfection of HSV-1 BACs
HSV-1 BAC DNA was transfected into BHK-21 cells using Lipofectamine™ and Plus™ reagent according to the protocol of the manufacturer (Invitrogen, Karlsruhe, Germany). In brief, for transfection in six-well plates, 1 μg BAC DNA was diluted in 245 μl Opti-MEM and complexed with 5 μl Plus™ reagent, and in a separate reaction tube 245 μl Opti-MEM was mixed with 5 μl Lipofectamine™. After 20-min incubation at room temperature, both mixtures were combined, and kept for another 30 min at room temperature. The DNA-lipofectin complexes were applied dropwise to the BHK-21 monolayers which were kept in serum-free DMEM. After 4 h of incubation at 37°C with 5% CO2, 1.5 ml DMEM with 10% FBS was added; then incubation continued for 2–3 days until the CPE was complete. For fusion protein induction, 5 μg Dox/ml was added at the time of medium replenishment.
Growth kinetics of reconstituted BAC virus
Half-confluent BHK-21 cell monolayers grown in a series of 35-mm dishes were inoculated with 0.05 plaque-forming units (PFU) per cell for 1 h at 37°C. Then virus was removed and 4 ml of supplemented DMEM/5% FBS was added. At the stated times of infection, viral growth was terminated by placing the dishes in a −70°C freezer. At the end of the kinetics, all dishes were subjected to three cycles of freezing and thawing, and then virus pools were prepared from the supernatants by sedimentation at 500× g, and titrated.
From serial dilutions of virus stocks made in serum-free DMEM, 0.333 ml aliquots were inoculated per well of 12-well dishes containing the specified half-confluent cell monolayers. After 1 h of incubation at 37°C, each inoculum was removed, and virus growth continued until plaque formation in supplemented DMEM/5% FBS containing 1.5% carboxymethyl cellulose (low viscosity; Sigma, Taufkirchen, Germany) and the indicated Dox concentrations. Virus plaques were determined by direct microscopic visualization as well as from cell monolayers fixed with 5% formaldehyde, and stained with 1% crystal violet.
Phase contrast and fluorescence microscopy was applied to determine virus plaque formation and for in situ gBGFP expression using a Leica DMIL fluorescence microscope with a C Plan L20×/0.30 objective. Images were collected with a Leica DFC350 FX black-and-white camera using the Leica Software FireCam 1.2.0 (Leitz, Wetzlar, Germany).
BACs with the genomes of the HSV-1 ANG standard virus and the pathogenic variant ANGpath were established using the technique exemplified for hCMV . To achieve conditional Tet-regulated transcription for controlling viral replication and expression, we have adopted the system previously introduced for conditional CMV replication . It consists of the insertion of a Tet-regulated expression vector into the BAC genome via FRT-mediated recombination. Selected gene K.O. mutants with a single FRT signal site were prepared using Red/ET and Flp recombinase-mediated recombination [6, 8]. The latter technique was also used to integrate a T-REx™ regulation vector, encoding the target gene under control of the 2tetO-bearing PCMV2tet0 or the endogenous promoter, and the tetR gene with PCMV. The BAC recombinants were then transfected into eukaryotic cells to generate infectious virus. Supernatant virus was collected and used to produce larger virus stocks. The Tet response of the reconstituted BAC virus was determined by the capability of viral plaque formation or the induction of gBGFP expression. In addition, a TetR-expressing cell line was applied for virus infection, in order to assess the effect of TetR concentration on Tet-regulation.
BAC cloning of HSV-1 ANG genomes resulted in two distinct BAC types
BAC mutants for Tet-inducible expression of DNA polymerase
Tet-regulated expression vector pRTRPOLII, containing a promoterless pol gene (Fig. 2), was inserted into TETO1FRT and TETO2FRT, yielding the recombinant BACs TETO1POL and TETO2POL (Fig. 3C). These constructions were performed to test the effect of the 2tetO positioning in the context of the endogenous promoter. In all the three vector constructs, the 5′-upstream sequence of the translation initiation codon of the pol gene was modified according to the Kozak consensus (AAGCTT GCCACC ATG) .
POL and POLi were calculated to have a size of 164.7 kbp, and with the size increase of 8.1%, these BAC recombinants carried the largest DNA insertion. For TETO1POL and TETO2POL theoretical sizes of 163.4 kbp and 163.6 kbp were calculated.
It should be noted that during the initial cloning of HSV-1 BACs, we consistently observed variations in the BamHI pattern, affecting predominantly the size of DNA fragments with known palindromic or repetitive sequence content, such as BamHI fragments u and b. Variations of fragment b that embraces internal repeat sequences of the large unique region, were also noted in the BamHI profile of TETO2POL clones (Fig. 3D). In two independent settings of the pol vector integration experiments, affecting one out of three progeny clones, and three out of three progeny clones, a larger BamHI fragment a was identified (Fig. 3D) that could arise only by the fusion of fragments a and c’ (Fig. 1, BamHI map).
BAC mutants for Tet-inducible expression of gBGFP
As a consequence of the insertion of pRTRgBG in RRgBG the original BamHI fragments h, h’, and o were altered, and four new fragments were identified in the BamHI profile of a representative clone, as depicted in Fig. 4C. The theoretical size of RRgBG was calculated to be 161.3 kbp.
Reconstitution of infectious virus from Pol BAC
For generation of infectious virus, BHK-21 cell monolayers were transfected with Pol BAC and cultured in the presence of Dox as described in “Materials and methods”. Microscopic analyses, performed 2 days after transfection, clearly showed in all BAC-transfected monolayers the formation of more or less extended syncytial plaques, a plaque type typical for strain ANG. According to the progress of infection, culture supernatants were collected between 3 and 5 days after transfection, and used to prepare virus stocks, as described in “Materials and methods”. Aliquots of virus stocks were used for another round of infection in the presence of Dox, and analyzed for plaque formation. Results showed that supernatant virus, rescued from transfections with POL, TETO1POL, and/or TETO2POL, produced syncytial plaques. In contrast, no plaques were detected in POLi-infected monolayers. The latter finding was repeatedly obtained with POLi constructs from wt ANG and ANGpath BAC genomes (data not shown), and is currently under study.
Titer of reconstituted BAC virus in the presence and absence of doxycycline on BHK-21 and LMTK tetR cells
2.3 × 105
2.4 × 105
1.4 × 105
1.0 × 105
1.1 × 104
1.1 × 104
3.4 × 103
4.0 × 103
1.6 × 106
1.2 × 106
1.2 × 105
1.0 × 105
Since the reconstituted POL virus carried the largest DNA insert, and was the first engineered herpesvirus that contained a pol gene under the control of PCMV2tetO, the replication capability of this virus was examined by performing a growth kinetic as described in “Materials and methods”. While wt virus titers plateaued at 24 h after infection with 108 PFU/ml, the POL virus titer also leveled off at 24 h but remained three log units lower at 2 days after infection, indicating that POL virus replication was by far less efficient than that of the wt virus.
Dox response of gBGFP expression in cells infected with reconstituted virus of RRgBG and ΔgBG
Generation of BAC mutants for conditional gene expression
In this report, we analyzed whether or not a previously published conditional viral gene expression system is applicable for HSV-1 . For this purpose, the genomes of wt HSV-1 strain ANG and of its pathogenic variant, ANGpath, were cloned as BACs following the protocol of Borst et al. . Using Red/ET and Flp recombinase-mediated recombination [6, 8, 11], we could establish very quickly the described BAC mutants for Tet-controlled pol and gBGFP gene expression starting from FRT-bearing knockout mutants of HSV-1 BACs of type I, and of ΔBAC. With the exception of POLi BAC, infectious virus was successfully reconstituted from all BAC constructs, exhibiting calculated sizes between 157.9 kbp and 164.7 kbp. From POL BAC with the largest DNA insert, and its successful reconstitution and titration can be derived that mature virions of HSV-1 ANG have a packaging capacity for foreign sequences that extends the standard genome by at least 8.1%. Wade-Martins et al.  reported for HSV-1 amplicons a maximal packaging size of 153 kbp. This size discrepancy may be related to the different genomes. It should be mentioned that, in the context of this article, we have not examined whether the virus yield can be improved by removing the floxed BAC replicon from POL BAC.
Sequence deletions as well as mutations occur during BAC cloning in E. coli
During BAC cloning in E. coli, we have noted that the oriL sequences were deleted in all established HSV-1 BACs (Figs. 1 and 3). Deletions of the oriL sequences were previously reported to occur during cloning via plasmids in E. coli [25–27], but could be prevented by using a different vector or another E. coli strain [28–30]. Mutations in oriL that abolish the initiation of viral DNA synthesis have little effect on viral replication in cultured cells, but reduce pathogenesis during acute infection of mice and impair reactivation from latency . It remains to be shown whether virus reconstituted from BAC genomes with deleted oriL shares the same characteristics, that could have a considerable drawback of its in vivo application as oncolytic vectors.
For BAC propagation in E. coli, it is known that tandemly repeated and repetitive sequences cannot be stably maintained [13, 32]. So far, there is no report that mutations are occurring during BAC cloning in E. coli. In the course of our cloning experiments, we found subclones with a larger BamHI fragment a (Fig. 3D). Since sequence analysis reveals that the location of the deleted BamHI site between fragment c’ and a in strain ANG (BamHI map in Fig. 1) is not embedded in a palindromic or repetitive sequence structure, the observed alteration can be explained only as a result of a sequence mutation affecting the respective BamHI site. In a more recent study, during cloning of gB K.O. BAC mutants, one out of five examined clones from a recombination experiment carried the same mutation (N. Strempel, unpublished observation). Presently, we have no clue how this mutation was generated and how frequently mutations in general occur during BAC cloning. Nevertheless, one should bear in mind that the E. coli DH10B cell might represent an error prone environment for the maintenance of viral genomes.
Characterization of BAC mutants for conditional gene expression
The choice of the viral Pol as target for Tet-controlled HSV-1 replication and its monitoring by virus plaque formation has turned out to be a very sensitive measurement. With several alternative constructions, in which the T-REx™ transciptional switch with PCMV2tetO  and the endogenous pol promoter with differently placed 2tetO elements were applied, no convincing Tet-controlled Pol expression was achieved as assessed by virus titration (Table 2). No improvement was gained with a host cell that constitutively produced TetR. Recent experiments were also unsuccessful, in which the tetR gene was replaced by the gene of the Tet-controlled transcriptional silencer  suggesting that the latter is not functioning in an HSV-1 environment (N. Strempel and C.W. Knopf, unpublished data). It may be argued that the positioning of the 2tetO elements in TETO1 and TETO2 was not optimal, while this should not play a role in POL, in which pol gene expression is controlled by PCMV2tetO. The finding that Tet regulation of the pol gene with the applied system was negative with the Pol mutants even by using a TetR producing cell line may be explained by the fact that the escape of only a few Pol copies is sufficient to replicate the viral genome.
More successful were the results for Tet-inducible gBGFP expression by RRgBG and ΔBAC (Fig. 6). The reason for this could be that, in contrast to Pol, the gBGFP fusion protein represents a rather insensitive probe for the in situ visualization, because in order to detect EGFP by conventional fluorescence microscopy about 10,000 molecules in single living cells are required . In the same context belong recent positive results of the regulation of galactosidase expression with the T-REx™ transcription switch system using replication-defective HSV-1 vectors . Whereas a less tight Tet-regulated gBGFP expression was achieved with BHK-21 cells that were infected with reconstituted virus from RRgBG and ΔgBG, a considerable improvement of the Tet-regulated transcription switch was gained when LMTK tetR cells, constitutively expressing TetR, were used as host for the infection. This result suggested that the TetR concentration supplied from the vector, encoding one copy of the tetR gene, was apparently not sufficient to warrant a stringent Tet-regulation. It remains to be shown, whether the introduction of two copies of the tetR gene into the vector as suggested  could overcome the TetR shortage, and possibly enable the construction of HSV-1 vectors for conditional expression and replication.
This research was supported by a BMBF grant for the scientific and technical collaboration with the Slovakian Republic, Project SVK No. 00/006.