Development of bovine herpesvirus 4 as an expression vector using bacterial artificial chromosome cloning

L. Gillet1,{dagger}, V. Daix1,{dagger}, G. Donofrio2, M. Wagner3, U. H. Koszinowski4, B. China5, M. Ackermann6, N. Markine-Goriaynoff1 and A. Vanderplasschen1

1 Department of Infectious and Parasitic Diseases (B43b), Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium
2 Dipartimento di Salute Animale, Facoltà di Medicina Veterinaria, Sezione di Malattie Infettive degli Animali, Università degli Studi di Parma, I-43100 Parma, Italy
3 Department of Pathology, Harvard Medical School, Boston, MA 02115, USA
4 Department of Virology, Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität München, D-81377 Munich, Germany
5 Food Sciences Department (B43b), Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium
6 Institute for Virology, University of Zurich, CH-8057 Zurich, Switzerland

Correspondence
A. Vanderplasschen
A.vdplasschen{at}ulg.ac.be


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Several features make bovine herpesvirus 4 (BoHV-4) attractive as a backbone for use as a viral expression vector and/or as a model to study gammaherpesvirus biology. However, these developments have been impeded by the difficulty in manipulating its large genome using classical homologous recombination in eukaryotic cells. In the present study, the feasibility of exploiting bacterial artificial chromosome (BAC) cloning and prokaryotic recombination technology for production of BoHV-4 recombinants was explored. Firstly, the BoHV-4 genome was BAC cloned using two potential insertion sites. Both sites of insertion gave rise to BoHV-4 BAC clones stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. Reconstituted virus replicated comparably to wild-type parental virus and the loxP-flanked BAC cassette was excised by growing them on permissive cells stably expressing Cre recombinase. Secondly, BoHV-4 recombinants expressing Ixodes ricinus anti-complement protein I or II (IRAC I/II) were produced using a two-step mutagenesis procedure in Escherichia coli. Both recombinants induced expression of high levels of functional IRAC molecules in the supernatant of infected cells. This study demonstrates that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.

{dagger}These authors contributed equally to this work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that has been isolated throughout the world from healthy cattle as well as those exhibiting a variety of diseases (Thiry et al., 1992). Its genome has a B-type structure consisting of a long unique region (L-DNA) flanked by a total of 15 (on average, as determined for the 66-p-347 strain) polyrepetitive DNA (prDNA) elements distributed randomly at both ends of the genome (Fig. 1a). The recent sequencing of BoHV-4 confirmed that it is a member of the genus Rhadinovirus encoding a relatively reduced set of open reading frames (ORFs) homologous to cellular genes (Zimmermann et al., 2001).



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Fig. 1. Schematic representation of the strategies followed to produce two infectious BoHV-4 BAC plasmids. (a, b) The EcoRI restriction map of the entire BoHV-4 V. test strain is shown at the top. A loxP-flanked BAC cassette was inserted into the XhoI (a) or BstEII (b) sites located, respectively, at the left and the right ends of BoHV-4 V. test strain L-DNA. These regions do not contain any ORFs and are located, respectively, in the EcoRI G and EcoRI I restriction fragments of the BoHV-4 V. test strain genome. (c) Flowchart of stages performed to produce BoHV-4 BAC plasmids, to control their infectivity and to demonstrate the possibility of removing the loxP-flanked BAC cassette from the genome of reconstituted virus.

 
Several features make BoHV-4 attractive as a backbone for a viral vector and/or as a model to study gammaherpesvirus biology: (i) its genome is less complex than those of several other herpesviruses (Zimmermann et al., 2001); (ii) it allows the stable insertion of additional genetic material up to at least 10·5 kb (see below); (iii) in contrast to several other rhadinoviruses, it is easy to propagate in cell culture (Thiry et al., 1992); (iv) wild-type virus exhibits limited or no pathogenicity in natural and experimental hosts (Thiry et al., 1992); (v) in contrast to most gammaherpesviruses, BoHV-4 is able to replicate in a broad range of host species both in vitro and in vivo (Thiry et al., 1992); (vi) in some cells, BoHV-4 has been shown to establish non-replicative persistent infection, allowing expression of transgenes for weeks without affecting the viability of the expressing cells (Gillet et al., 2004); and (vii) a small animal model (rabbit) is available (Thiry et al., 1992).

Applied and fundamental research on herpesviruses requires genomic modifications such as insertion of transgenes and/or deletion of viral ORFs. Both types of modification have been applied to BoHV-4 by homologous recombination in eukaryotic cells (Gillet et al., 2004; Markine-Goriaynoff et al., 2004). However, this approach is extremely slow and laborious. Further studies on BoHV-4 require a faster approach to modify its genome. Manipulation of large herpesvirus genomes has recently been facilitated by using bacterial artificial chromosome (BAC) vectors (Messerle et al., 1997; Wagner et al., 2002). These vectors allow the maintenance and mutagenesis of the viral genome in Escherichia coli followed by the reconstitution of progeny virions by transfection of the BAC plasmid into permissive eukaryotic cells.

In the present study, we addressed the feasibility of exploiting BAC cloning and prokaryotic recombination technology for further applied and fundamental research on BoHV-4. Firstly, in order to BAC clone the BoHV-4 genome, two non-coding regions located at both extremities of the L-DNA were selected for insertion of a loxP-flanked BAC cassette. Both sites of insertion led to the production of BoHV-4 BAC clones stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. Reconstituted viruses replicated comparably to wild-type parental virus and the loxP-flanked BAC cassette was excised by growing them on permissive cells stably expressing Cre recombinase. Secondly, prokaryotic recombination technology and one of the BoHV-4 BAC clones produced were used to generate BoHV-4 recombinants expressing functional Ixodes ricinus anti-complement protein I or II (IRAC I/II) (Daix et al., 2001). IRACs are inhibitors of the alternative pathway of the complement system and are secreted in the saliva of I. ricinus ticks.

Taken together, the data of the present study demonstrated that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cell lines and virus strains.
Madin–Darby bovine kidney (MDBK; ATCC CCL-22), embryonic bovine lung (EBL; DSMZ ACC 192) and 293T cells (ATCC CRL-11268) were cultured in minimum essential medium (MEM; Invitrogen) containing 10 % fetal calf serum (FCS; BioWhittaker). The BoHV-4 V. test (Thiry et al., 1981) strain was used throughout this study.

Production of a stable cell line expressing Cre recombinase.
EBL cells stably expressing Cre recombinase fused to a nuclear localization signal (EBL NLS–Cre) were produced as follows. Briefly, NLS–Cre was excised by NheI and SpeI digestion from the pGBF-NLS-Cre vector (provided by Dr D. Pirottin, University of Liège, Belgium) and cloned into the XbaI site of the pEFIN3 bicistronic expression vector (provided by Dr M. Parmentier, Free University of Brussels, Belgium) resulting in pEFIN3-NLS-Cre. After linearization by ScaI digestion, the plasmid was transfected into EBL cells. After 2 weeks of selection under G418 (final concentration 200 µg ml–1; Invitrogen), all resistant cells expressed NLS–Cre (data not shown) and were used without further cell cloning.

BAC cloning of BoHV-4.
Two strategies of insertion were used to BAC clone BoHV-4 genome. A BAC cassette was inserted either into the far left XhoI site or into the far right BstEII site of BoHV-4 L-DNA. These regions of insertion do not contain any ORFs (Zimmermann et al., 2001). The BAC cassette was obtained from a modified pBeloBAC vector in which additional restriction sites were inserted upstream and downstream of the loxP sequences (GenBank accession no. AY665170) (Fig. 1a and b). Insertion of the BAC cassette into the BoHV-4 genome was performed by homologous recombination as described previously (Markine-Goriaynoff et al., 2004). The recombination cassettes were produced as follows. For the left insertion (Fig. 1a), the EcoRI restriction fragment G of the BoHV-4 V. test strain genome was first cloned into the pGEM-T Easy vector (Promega) resulting in pGEM-T-EcoRI G. The BAC cassette was then released by PmeI digestion from the modified pBeloBAC vector and introduced into the blunted XhoI site of pGEM-T-EcoRI G resulting in pGEM-T-EcoRI G BAC. Similarly, for the right insertion (Fig. 1b), the EcoRI restriction fragment I of the BoHV-4 V. test strain genome was first cloned into the pGEM-T Easy vector resulting in pGEM-T-EcoRI I. The BAC cassette released from the modified pBeloBAC vector by PmeI digestion was then ligated into the blunted BstEII site of pGEM-T-EcoRI I resulting in pGEM-T-EcoRI I BAC. The vectors pGEM-T-EcoRI G BAC and pGEM-T-EcoRI I BAC were used to produce BoHV-4 V. test BAC G and V. test BAC I recombinant strains, respectively (Fig. 1c). EGFP-expressing recombinant viruses were enriched by six rounds of plaque purification. The viral genomes were then transferred into bacteria as described elsewhere (Smith & Enquist, 2000).

Southern blotting.
Southern blot analysis was performed as described previously (Markine-Goriaynoff et al., 2003).

Antibodies.
The mouse monoclonal antibody (mAb) 35 raised against BoHV-4 early-late glycoprotein complex gp6/gp10/gp17 was used in this study (Dubuisson et al., 1991). Mouse sera raised against IRAC I and II (anti-IRAC I and anti-IRAC II) were also used in this study (Daix et al., 2001).

Indirect immunofluorescent staining.
Cells grown on glass coverslips were fixed in PBS containing 4 % (w/v) paraformaldehyde (Merck) for 10 min on ice and then for 20 min at 20 °C. After washing with PBS, samples were permeabilized in PBS containing 0·1 % (w/v) NP-40 (Fluka) at 37 °C for 10 min. Immunofluorescent staining (incubation and washes) was performed in PBS containing 10 % FCS. Samples were incubated at 37 °C for 45 min with one of the following primary antibodies: mAb 35 (diluted 1 : 1000), anti-IRAC I (diluted 1 : 100) or anti-IRAC II (diluted 1 : 100). After three washes, samples were incubated at 37 °C for 30 min with R-phycoerythrin (PE)-conjugated F(ab')2 goat anti-mouse Ig (5 µg ml–1; Dako) as the secondary conjugate. Samples were mounted as described elsewhere (Vanderplasschen et al., 2000).

Multi-step growth curves.
Triplicate cultures of MDBK cells were infected at an m.o.i. of 0·5 p.f.u. per cell. After an incubation period of 1 h, cells were washed and then overlaid with MEM containing 5 % FCS. Supernatant of infected cultures was harvested at successive intervals after infection and the amount of infectious virus was determined by plaque assay on MDBK cells as described previously (Vanderplasschen et al., 1993).

Production of BoHV-4 recombinants expressing IRAC I or IRAC II by mutagenesis in bacteria.
Mutagenesis of wild-type V. test BAC G plasmid was performed by homologous recombination in bacteria using a modified version of the shuttle plasmid pST76K-SR expressing recA recombinase (Hobom et al., 2000). Briefly, pBluescript SK(+) vector (Stratagene) was digested with NspI and BsrFI to release the lacZ gene containing the multiple cloning site. The latter fragment was then inserted into pST76K-SR digested with AvaI and SphI, resulting in pST76KSR-lacZ. Plasmids to induce homologous recombination were constructed as follows. Firstly, IRAC I and II ORFs with their stop codon deleted were inserted into the pcDNA4-TO His/Myc vector (Invitrogen) digested with BamHI and XhoI, resulting in pcDNA4-TO IRAC I and II. Secondly, DNA fragments containing human cytomegalovirus (HCMV) immediate-early (IE) promoter fused to IRAC (I or II) ORF and bovine growth hormone poly(A) [HCMV IE–IRAC I/II–BGHpoly(A)] were released from the latter plasmids by PvuII and BspHI digestion. After filling in both ends with Klenow, the fragments were inserted into the blunted XhoI site of pGEM-T-EcoRI I resulting in pGEM-T-EcoRI I IRAC I or II. Finally, pGEM-T-EcoRI I IRAC I or II plasmids were digested with NotI and EcoNI. After blunting, the appropriate fragments were inserted into the SmaI site of the pST76KSR-lacZ shuttle vector resulting in pST76KSR-lacZ IRAC I or II. Recombinant BoHV-4 BAC plasmids were produced by a two-step mutagenesis procedure as described elsewhere (Hobom et al., 2000) (see also Fig. 3).



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Fig. 3. Production of BoHV-4 recombinant strains expressing IRAC I or II by mutagenesis of V. test BAC G plasmid in bacteria. (a) The IRAC I or II expression cassette was inserted in the BstEII site of the V. test BAC G plasmid corresponding to the far left BstEII site of BoHV-4 L-DNA. The insertion was performed by homologous recombination between V. test BAC G and pST76KSR-lacZ IRAC I or II plasmids. (b) Flowchart of stages performed to produce BoHV-4 strains expressing IRAC I or II.

 
Concentration of cell supernatant.
Cell supernatants (15 ml per 75 cm2 flask) were collected 2 days post-infection or post-transfection, centrifuged at 100 000 g for 2 h and concentrated to a final volume of 250 µl using Amicon Centricon Plus-20 columns (10 000 nominal molecular weight limit; Millipore).

Immunoblotting.
Concentrated cell supernatants were diluted four times in NuPAGE LDS sample buffer (Invitrogen) containing a reducing agent. After electrophoresis on NuPAGE Novex 4–12 % Pre-cast Bis-Tris gels (Invitrogen), proteins were transferred to nitrocellulose membranes and identified with specific antibodies and a chemiluminescence peroxidase substrate kit (Sigma-Aldrich). Mouse anti-IRAC I and anti-IRAC II sera were used as primary antibodies at a dilution of 1 : 3000.

Anti-complement alternative pathway assay.
The ability of IRAC molecules to inhibit the alternative pathway of the complement system was tested using the AH50 assay (Coligan et al., 1992). Briefly, 50 µl unsensitized rabbit erythrocytes (Erab) [2x108 cells ml–1 in ice cold gelatin/veronal-buffered saline with MgCl2 and EGTA (GVB/Mg EGTA)] were added on ice to 20 µl human serum and increasing amounts of the concentrated cell supernatant to be tested. The final volume of each sample was made up to 150 µl with GVB/Mg EGTA buffer. For background and total lysis samples, 100 µl GVB/Mg EGTA buffer and 100 µl water were added to the 50 µl Erab, respectively. After an incubation period of 60 min at 37 °C, 1·2 ml ice-cold 0·15 M NaCl was added to each sample to stop haemolysis. After centrifugation at 1250 g for 10 min at 4 °C, the supernatant was collected and its optical density measured at 412 nm (OD412). Relative haemolysis of each sample was calculated as: (test sample OD412–background sample OD412)/(total lysis sample OD412–background sample OD412).

Confocal microscopy analysis.
Confocal microscopy analyses were performed with a TCS SP confocal microscope (Leica) as described previously (Vanderplasschen & Smith, 1997).

Statistical analysis.
Statistical comparisons were assessed by analysis of variance. When F ratios were significant (P<0·05), the method of Scheffe's post hoc tests was used.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
As mentioned above, BoHV-4 has several intrinsic features that make it attractive as a backbone for a viral vector and/or as a model to study gammaherpesvirus biology. However, these developments have been impeded by the difficulty of manipulating its large genome. Manipulation of large herpesvirus genomes has recently been facilitated by using BAC vectors and prokaryotic recombination technology. In the present study, we addressed the feasibility of applying these techniques to BoHV-4.

Cloning of BoHV-4 genome in E. coli
The approach depicted in Fig. 1(c) was used to BAC clone BoHV-4. Two sites located in non-coding regions of BoHV-4 L-DNA were selected for insertion of the BAC cassette (Fig. 1a and b). Simultaneous transfection of BoHV-4 V. test strain genome and pGEM-T-EcoRI G BAC or pGEM-T-EcoRI I BAC plasmids into MDBK cells generated the BoHV-4 V. test BAC G and V. test BAC I recombinant strains, respectively. The molecular structures of these recombinant strains were confirmed by a combined HindIII restriction endonuclease/Southern blot approach (Fig. 2a) and by sequencing of the regions used to target recombination (data not shown).



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Fig. 2. Characterization of BoHV-4 BAC plasmids and derived strains. (a) Characterization of BoHV-4 BAC plasmids and derived BoHV-4 strains by a combined restriction endonuclease/Southern blot approach. V. test BAC G and V. test BAC I plasmids and the genome of BoHV-4 V. test BAC G, V. test BAC I, V. test BAC G excised and V. test BAC I excised strains were analysed by HindIII restriction (left panels) and further tested by Southern blotting using a probe corresponding to nt 1430–9940 of the modified pBeloBAC plasmid (right panels). Arrows indicate restriction fragments containing the BAC cassette. Marker sizes (MS) in kb are indicated on the left. (b) Characterization of BoHV-4 strains derived from BoHV-4 BAC plasmids by confocal analysis of viral plaques. MDBK cells grown on glass coverslips were infected with BoHV-4 wild-type V. test (i–iii), V. test BAC G (iv–vi) and V. test BAC G excised (vii–ix) strains and then overlaid with MEM containing 5 % FCS and 0·6 % (w/v) carboxymethylcellulose (Sigma-Aldrich) to obtain isolated plaques. Three days after infection, plaques were revealed by indirect immunofluorescent staining using mAb 35 and PE-conjugated goat anti-mouse Ig as the primary and secondary antibodies, respectively. Each set of three horizontal panels (i–iii, iv–vi and vii–ix) represents analysis of the same plaques. EGFP fluorescence is shown in (i), (iv) and (vii), and PE fluorescence in (ii), (v) and (viii). The merged EGFP and PE signals are shown in (iii), (vi) and (ix). The side of each panel corresponds to 75 µm. Similar results were obtained with the strains derived from V. test BAC I plasmid (data not shown). (c) Replication kinetics of BoHV-4 strains derived from BoHV-4 BAC plasmids compared with the parental BoHV-4 V. test strain, determined as described in Methods. The data presented are the means of triplicate measurements.

 
Circular intermediates of BoHV-4 V. test BAC G and V. test BAC I genomes were isolated from infected cells and electroporated into E. coli DH10B to generate BoHV-4 V. test BAC G and I plasmids, respectively. These plasmids were characterized as described above by a combined HindIII restriction endonuclease/Southern blot approach (Fig. 2a). This method confirmed that BoHV-4 V. test BAC G and I plasmids were two BAC clones of BoHV-4 genome.

Stability of the BoHV-4 genome in E. coli
To assess the stability of BoHV-4 genome as a BAC plasmid, E. coli DH10B containing BoHV-4 V. test BAC G or I plasmids was serially cultured for 20 consecutive days, each day representing approximately 36 generations. After various periods of culture, the BAC plasmids were isolated and characterized by HindIII endonuclease digestion. No difference was observed among plasmids grown for various periods of time, demonstrating the stability of V. test BAC G and I plasmids in E. coli (data not shown).

Reconstitution of infectious virus from BoHV-4 BAC plasmids and excision of the BAC cassette from reconstituted virus
The usefulness of BAC cloning technology for manipulation of large DNA viruses requires the ability to reconstitute infectious virus from the BAC plasmid. Consequently, we tested whether infectious particles could be produced by electroporation of BoHV-4 V. test BAC plasmids (Fig. 2b). HindIII restriction analysis of the DNA of reconstituted viruses revealed restriction profiles identical to the patterns observed for BoHV-4 V. test BAC plasmids (Fig. 2a).

To excise the BAC cassette, BoHV-4 V. test BAC G and I reconstituted viruses were propagated in EBL NLS–Cre cells to generate BoHV-4 V. test BAC G and I excised strains, respectively. Deletion of the BAC cassette was confirmed by a combined restriction endonuclease/Southern blot approach (Fig. 2a) and by monitoring the expression of EGFP [Fig. 2b, compare (iv) and (vii)].

Finally, in order to investigate the putative effect of the recombination processes described above (insertion/excision of the BAC cassette) on BoHV-4 growth in vitro, BoHV-4 wild-type V. test, V. test BAC G or I and V. test BAC G or I excised strains were compared using the growth assay described in Methods (Fig. 2c). All viruses tested exhibited similar growth curves (P<=0·05).

Production of BoHV-4 recombinant strains expressing IRAC molecules by mutagenesis in bacteria
In the second part of this study, we tested whether the V. test BAC G plasmid described above could be modified by mutagenesis in bacteria in order to produce BoHV-4 recombinants to be used as in vitro expression vectors. To challenge this approach, we decided to produce BoHV-4 recombinants expressing IRAC I or II proteins under the control of the HCMV IE promoter. To this end, IRAC expression cassettes were inserted into the far right BstEII site of BoHV-4 L-DNA using the V. test BAC G plasmid and a two-step replacement procedure (Fig. 3a). The resulting modified plasmids, called V. test BAC G IRAC I or II, were analysed by a combined HindIII restriction endonuclease/Southern blot approach (Fig. 4a). IRAC I ORF probe, which only cross-reacts very weakly with the IRAC II sequence, hybridized to the expected 4·25 kb band in the HindIII profile of the V. test BAC G IRAC I plasmid. The IRAC II ORF probe, which cross-reacts with the IRAC I sequence, hybridized to the expected 3·95 kb and 232 bp bands in the HindIII profile of the V. test BAC G IRAC II plasmid. V. test BAC G IRAC I and II plasmid structures were further confirmed by sequencing of the regions used to target recombination (data not shown). Next, V. test BAC G IRAC I and II plasmids were transfected into permissive EBL cells to reconstitute BoHV-4 V. test BAC G IRAC I and II recombinant viruses, respectively. These viruses were grown on EBL NLS–Cre to generate BoHV-4 V. test BAC G IRAC I and II excised strains in which the BAC cassette had been deleted. The molecular structure of the four recombinant strains produced was analysed using the combined HindIII restriction endonuclease/Southern blot approach described above (Fig. 4a). Once it had been determined that the recombinants had the correct molecular structure, IRAC expression was tested (Fig. 4b). Immunostaining of BoHV-4 V. test BAC G IRAC I or II and V. test BAC G IRAC I or II excised plaques revealed that both transgenes were expressed during the virus replication cycle [Fig. 4b, (ii), (v), (viii) and (xi)].



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Fig. 4. Characterization of BoHV-4 recombinant strains expressing IRAC I or II. (a) Characterization of BoHV-4 recombinant strains expressing IRACs using a combined restriction endonuclease/Southern blot approach. The DNA of the various intermediates described in Fig. 3(b) was analysed by HindIII restriction (left panel) and further tested by Southern blotting using IRAC I or II ORF probes (middle and right panels). Arrows indicate the restriction fragments containing IRAC ORFs. Marker sizes (MS) in kb are indicated on the left. (b) Immunodetection of IRACs expressed by BoHV-4 recombinant strains. MDBK cells were infected at an m.o.i. of 0·01 with BoHV-4 V. test BAC G IRAC I (i–iii), V. test BAC G IRAC I excised (iv–vi), V. test BAC G IRAC II (vii–ix) and V. test BAC G IRAC II excised (x–xii) strain. After an incubation period of 48 h at 37 °C, cells were analysed by indirect immunofluorescent staining as described in Methods. Anti-IRAC I (i–vi) and anti-IRAC II (vii–xii) mouse sera were used as primary antibodies and revealed using PE-conjugated goat anti-mouse secondary antibodies. Cells were then examined by confocal microscopy for EGFP (i, iv, vii and x) and R-PE (ii, v, viii and xi) signals. The merged EGFP and R-PE signals are shown in (iii), (vi), (ix) and (xii). The side of each panel corresponds to 150 µm.

 
The results presented above suggested that infection of cells by BoHV-4 V. test BAC G IRAC I or II excised strains could represent an alternative to cell transfection for the expression of secreted functional IRAC molecules. To test this hypothesis, MDBK and 293T cells were either transfected with pcDNA4-TO-IRAC I or II, or infected with BoHV-4 V. test BAC G IRAC I or II excised strains (Fig. 5). The pEGFP-N1 vector and the BoHV-4 V. test strain were used as negative controls, respectively. Western blot analysis of concentrated cell supernatants revealed that infection and transfection of 293T cells led to comparable high level of IRAC I and II protein expression. It is important to note that transfection of 293T cells by IRAC-encoding vectors represented the most efficient expression system among many tested (data not shown). In MDBK cells, expression of IRACs induced by transfection was barely detectable, while expression induced by infection was comparable to the level observed in 293T cells. Finally, anti-complement assays performed with concentrated supernatants suggested that IRAC molecules resulting from transfection or infection are comparably active.



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Fig. 5. Ability of IRAC molecules expressed by BoHV-4 recombinants to inhibit the alternative pathway of the complement system. MDBK and 293T cells grown in 75 cm2 flask were transfected with pEGFP-N1 or pcDNA4-TO IRAC I or II plasmids, or infected at an m.o.i. of 1 p.f.u. per cell with BoHV-4 V. test, V. test BAC G IRAC I excised or V. test BAC G IRAC II excised strain. Forty-eight hours after transfection or infection, cell-culture supernatants were collected and concentrated. Proteins contained in 5 µl of concentrated supernatants were resolved by SDS-PAGE, blotted and detected with anti-IRAC I or anti-IRAC II mouse sera as described in Methods. The ability of concentrated cell supernatants to inhibit the alternative complement pathway was determined by the addition of various amounts of concentrated cell supernatant to the test. Data are expressed as the percentage of lysis observed compared with the addition of mock-transfected or mock-infected concentrated cell supernatant and represent the means of triplicate measures. SDs were lower than 0·2.

 
Taken together, the results presented above demonstrate that the BoHV-4 BAC clones produced in the present study can be manipulated in bacteria for production of efficient BoHV-4 expression vectors.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
BAC cloning has speeded up fundamental and applied research on several herpesviruses. In the present study, we BAC cloned the BoHV-4 genome and demonstrated the quality of the plasmids obtained for production of BoHV-4 recombinants using prokaryotic recombination technology. The recombinants produced were used to illustrate the potential of BoHV-4 as an in vitro expression vector.

The BoHV-4 BAC plasmids produced in this study exhibited several interesting features for the production of BoHV-4 recombinant vectors. Firstly, it was possible to propagate them stably in bacteria for at least 720 generations. Despite this relative high number, recombination events were not detected, as described, for example, for murid herpesvirus 4 BAC (Adler et al., 2000). Secondly, infectious BoHV-4 recombinant viruses could be efficiently reconstituted by transfection of the BoHV-4 BAC plasmid into permissive cells. This feature contrasts with HCMV (Borst et al., 1999) BAC plasmids, which required the expression of transactivating factor for efficient production of virions, and with BoHV-1 (Mahony et al., 2002) and herpes simplex virus 1 (Stavropoulos & Strathdee, 1998) BAC plasmids requiring drug treatment to promote IE gene expression. Thirdly, a major advantage of herpesvirus vectors is their very large cloning capacity when compared with other viral vectors (Kootstra & Verma, 2003; Pfeifer & Verma, 2001; Verma & Somia, 1997). The data presented in this study demonstrated that at least 10·5 kb of foreign DNA could be inserted into the BoHV-4 wild-type genome without affecting its replication properties (Fig. 4b).

The present study illustrates the potential of BoHV-4 as an in vitro expression vector. However, several properties of BoHV-4 make it a good candidate for the development of recombinant vaccines for cattle. Firstly, in contrast to BoHV-1, there is no eradication scheme against BoHV-4. Secondly, bovine monocytes and macrophages support BoHV-4 replication (M. Lambot, unpublished data) suggesting that expression of the transgene in those cells should induce a strong and complete immune response. Thirdly, the immunity developed against the vector should confer cross-protection against alcelaphine herpesvirus 1, the causative agent of malignant catarrhal fever (Rossiter et al., 1989), which has an important economic impact in Africa. Lastly, development of BoHV-4 as a recombinant vaccine will benefit from the rabbit model (Naeem et al., 1990). The technology applied in the present study for BoHV-4 mutagenesis could contribute to its development as a recombinant vaccine in the following ways: (i) a major disadvantage of herpesviruses in the context of vaccine development is their ability to establish latent infection and to reactivate. Several strategies could be adopted to prevent BoHV-4 latent infection such as overexpression of ORF50 (encoding IE2) or deletion of ORF73 (encoding LANA) (Fowler & Efstathiou, 2004). Strategies based on ORF73 deletion could be compatible with the use of the ORF73 regulatory region to provide long-term transgene expression, as described recently for herpesvirus saimiri (Giles et al., 2003). (ii) BAC technology could allow the production of non-replicative vector with no dissemination potential. (iii) The safety of BoHV-4 could be improved by deletion of non-essential genes responsible for its deleterious effects. For example, our recent study showed that non-replicative infection by BoHV-4 protects infected cells against TNF-{alpha}-induced apoptosis (Gillet et al., 2004), an effect mainly due to ORF71 encoding BoHV-4 v-FLIP (F. Minner, unpublished data). Deletion of the latter gene among others could improve further the safety and the cloning capacity of the vector. (iv) In the context of viral vaccine development, BACs represent a new form of vaccine that combines the advantages of DNA vaccines and modified live viruses, since virus can be reconstituted in vivo after administration of infectious DNA (Meseda et al., 2004; Petherbridge et al., 2003; Suter et al., 1999; Tischer et al., 2002). Moreover, recent work (Cicin-Sain et al., 2003) showed that infectious viruses could be reconstituted in vivo after direct transfer of the viral genome from bacteria to the vaccinated animal. These authors postulated that a bacterial vaccination vector delivering an attenuated, yet infectious virus might present the basis for efficient vaccines that are easy to store, distribute and administer. (v) Finally, the maximum cloning capacity of BoHV-4 could be reached by deletion of all non-essential ORFs. BAC-based technology could be used both to identify non-essential genes and to delete them. The former goal could be reached by random transposon mutagenesis of the BoHV-4 genome in E. coli (Brune et al., 1999).

BAC cloning has speeded up fundamental and applied research on several herpesviruses. In the present study, we have BAC cloned the BoHV-4 genome and demonstrated the potential of prokaryotic recombination technology for the production of BoHV-4 recombinants. This step forward will greatly facilitate further research on this gammaherpesvirus.


   ACKNOWLEDGEMENTS
 
A. V., N. M.-G. and L. G. are a Senior Research Associate, Postdoctoral Researcher and Research Fellow of the ‘Fonds National Belge de la Recherche Scientifique’ (FNRS), respectively. Thanks are due to Dr D. Pirottin (University of Liège, Belgium) and Dr M. Parmentier (Free University of Brussels, Belgium) for providing the pGBF-NLS-Cre and pEFIN3 plasmids, respectively. This work was supported by the following grants: ‘Recherche d'initiative du Ministère de la Région Wallonne’ programme no. 14628 ‘convention 315543 Région Wallonne’ and ‘Service public et Fédéral santé publique, sécurité de la chaîne alimentaire et environnement’ (Belgium) programme no. S-6146. M. W. and U. H. K. were supported by the Deutsche Forschungsgemeinschaft through SBF 455. M. W. is supported by a Human Frontiers Science Program long-term fellowship.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 25 October 2004; accepted 20 December 2004.