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
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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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.
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METHODS |
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Production of a stable cell line expressing Cre recombinase.
EBL cells stably expressing Cre recombinase fused to a nuclear localization signal (EBL NLSCre) were produced as follows. Briefly, NLSCre 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 ml1; Invitrogen), all resistant cells expressed NLSCre (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 ml1; 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 IEIRAC I/IIBGHpoly(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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Immunoblotting.
Concentrated cell supernatants were diluted four times in NuPAGE LDS sample buffer (Invitrogen) containing a reducing agent. After electrophoresis on NuPAGE Novex 412 % 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 ml1 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 OD412background sample OD412)/(total lysis sample OD412background 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.
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RESULTS |
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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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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 NLSCre 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 NLSCre 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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DISCUSSION |
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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-
-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.
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ACKNOWLEDGEMENTS |
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Received 25 October 2004;
accepted 20 December 2004.