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1 Department of Primary Industries and Fisheries, St Lucia, Brisbane, QLD, Australia
2 School of Veterinary Science, University of Queensland, St Lucia, Brisbane, QLD, Australia
Correspondence
Timothy J. Mahony
Timothy.Mahony{at}dpi.qld.gov.au
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ABSTRACT |
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Present address: Department of Basic Sciences and Institute for Digital Biology, College of Veterinary Medicine, Mississippi State University, PO Box 1600, MS 39762, USA. 
Two supplementary tables detailing primer sequences are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The BoHV-1 genome encodes 73 recognized open reading frames (ORFs) within a 135 301 bp double-stranded DNA genome, which has been completely sequenced. Three subtype variants of BoHV-1, designated BoHV-1.1a, BoHV-1.2a and BoHV-1.2b, are recognized based on genomic DNA restriction endonuclease profiles and clinical observations (Metzler et al., 1985, 1986).
Only a limited number of genes has been identified as being essential or non-essential for the in vitro viability of BoHV-1. These genes have largely been identified from the development of attenuated and recombinant vaccine strains or by direct comparison of homologous genes encoded by human herpesvirus 1 (HHV-1). However, due to the differences in host tropism and clinical signs of BoHV-1, not all gene requirements can be considered identical. Furthermore, the BoHV-1 genome encodes several genes for which there are no HHV-1 homologues. Genes that have been identified as non-essential for in vitro viability of BoHV-1 include the genes encoding glycoproteins C, E, I, G and M (Baranowski et al., 1996; Denis et al., 1996; Schwyzer & Ackermann, 1996; Tikoo et al., 1995), nucleotide metabolism genes encoding the thymidine kinase (Bello et al., 1992; Smith et al., 1994) and deoxyuridine triphosphatase proteins (Bello et al., 1992; Liang et al., 1993; Smith et al., 1994), as well as genes that encode tegument (UL49 and US9), membranous (UL49.5) (Liang et al., 1995, 1993) and regulatory proteins such as the virion serine/threonine protein kinase (US3) (Furth et al., 1997), immediate-early transactivator protein (bICP0) (Geiser et al., 2005), immediate-early and late phase nuclear transrepressor protein (bICP22) and proteins putatively involved in immune evasion (Circ) (Fraefel et al., 1994; Furth et al., 1997; Schmitt & Keil, 1996). Genes of unknown function that are also identified as non-essential for BoHV-1 in vitro viability include those in US2, UL7 and UL24 ORFs (Furth et al., 1997; Schmitt & Keil, 1996; Whitbeck et al., 1994). Although there are some data regarding the essential and non-essential genes required for the in vitro viability of BoHV-1, a comprehensive map detailing this information for all genes encoded by BoHV-1 has not been completed.
In this study, a transposon insertion mutagenesis system (Mahony et al., 2004) and homologous recombination (using the GETrec system; Narayanan et al., 1999) have been utilized to examine the requirement of individual genes for the in vitro viability of an infectious clone of BoHV-1 (Mahony et al., 2002). A map has been established that identifies BoHV-1 genes that are essential or non-essential for in vitro viability. In addition, this assessment provides the basis for further development of BoHV-1 as a vaccine vector.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), was used for this study. pBACBHV37 has a thymidine kinase deficient (TK–) phenotype. TK– BoHV-1 have been extensively investigated and utilized as vaccine candidates due to their reduced virulence in vivo (Bello et al., 1992; Chowdhury, 1996; Kaashoek et al., 1996). pBACBHV37 shows no apparent attenuation and displays indistinguishable replication kinetics to that of a wild-type BoHV-1 in vitro (Mahony et al., 2002).
The mammalian cell line CRIB-1 (ATCC CRL-11883) (Flores & Donis, 1995) was used for the transfection of transposed (pBACBHV37 Tn5
) and gene-deleted (pBACBHV37GET) pBACBHV37 clones. CRIB-1 cells were cultured as described previously (Mahony et al., 2002). Standard liquid and solid preparations of Luria–Bertani (LB) media containing 25 µg chloramphenicol (CAP) ml–1 and/or 30 µg kanamycin (KAN) ml–1 and/or 100 µg ampicillin (AMP) ml–1 were used where appropriate to propagate BAC clones.
Gene disruption library.
A gene disruption library using random Tn5 insertion into pBACBHV37 in DH10B cells was previously constructed by Mahony et al. (2004) and stored at –80 °C until required.
BAC DNA extraction.
Dual resistant bacterial colonies harbouring pBACBHV37 Tn5 or pBACBHV37GET DNA were inoculated into 200 ml LB CAP/KAN broth and incubated at 37 °C with moderate shaking for 18–24 h. BAC DNA was extracted from bacterial pellets by alkaline lysis using the Nucleobond BAC 100 extraction kit (Macherey-Nagel) according to the manufacturer's instructions. Extracted BAC DNA was resuspended in 100 µl 10 mM Tris/HCl (pH 8.5) and stored at 4 °C.
Viral genomic DNA extraction.
BoHV-1 genomic DNA of cell culture reconstituted pBACBHV37 virus (RV pBACBHV37) was extracted as follows. CRIB-1 cells were infected at an m.o.i. of 5 and infection was allowed to proceed to completion. Virus culture supernatant was freeze–thaw (–80 °C) and clarified by centrifugation at 5000 g for 15 min at 4 °C. Clarified virus culture supernatant was decanted into open-topped ultracentrifuge tubes (Seaton) and pelleted at 75 000 g for 120 min at 4 °C. Viral DNA was extracted by anion exchange using the Genomic 20G tip (Qiagen) as described in the manufacturer's instructions. Extracted genomic viral DNA was resuspended in 100 µl TE buffer and stored at 4 °C.
Sequencing of pBACBHV37 modified clones.
Direct sequencing was used to determine the site of transposition and to confirm the deletion of targeted genes. Each sequencing reaction (40 µl) contained, 16 µl Big Dye Terminator v3.1 sequencing mix, 5 % (v/v) DMSO, 0.4 µM primer KanME (see Supplementary Table S1, available in JGV Online) and 2 µg purified BAC DNA. Subsequent confirmatory sequencing of selected clones was conducted as described above with the primers IN-UL7, IN-UL16, IN-UL20, IN-UL35, IN-UL49, IN-UL49.5, IN-UL53 and IN-UL54 (Supplementary Table S1). Sequencing cycle conditions were as follows: initial denaturation 95 °C for 5 min followed by 60 cycles of 95 °C for 30 s, 50 °C for 20 s and 60 °C for 4 min, and a completion hold of 4 °C. Sequence products were recovered by precipitation with ethanol/sodium acetate (pH 4.6) and washed with 500 µl 70 % ethanol and air-dried. Dried sequence products were submitted to the Australian Genome Research Facility (Brisbane, Australia) for capillary separation. Sequence data were viewed using the 4Peaks programme by A. Griekspoor and T. Groothuis (www.mekentosj.com) and similarities were searched for using the BLASTN programme (Altschul et al., 1997).
Generation of linear transgenes.
Linear transgenes (LT) were amplified by PCR using the relevant primers (Supplementary Table S2). PCR mixtures (20 µl) contained 10x Platinum Pfx reaction buffer, 1.25 mM each of dATP, dCTP, dGTP and dTTP, 2.5 mM MgSO4, 0.4 µM each primer, 1 U Taq DNA polymerase (Invitrogen) and 1 fmol µl–1 EZ-Tn5 <KAN-2> template (Epicentre Technologies). PCR cycling conditions were as follows: initial denaturation at 94 °C for 2 min, followed by 35 cycles of 94 °C for 15 s, 60 °C for 30 s and 68 °C for 90 s, final extension of 68 °C for 3 min and a completion hold at 4 °C. Reaction products were resolved on a 1 % agarose gel in TBE and products corresponding to the expected size (1300 bp) were recovered using QIAquick gel extraction kit (Qiagen). Gel-purified products were stored at –20 °C until required.
Targeted gene deletion.
Targeted deletion of selected BoHV-1 genes was catalysed by homologous recombination using the GETrec system (Narayanan et al., 1999) and analysed as described previously (Mahony et al., 2002). Modified pBACBHV37GET clones were identified by colony formation on dual selective media and confirmed by sequencing.
Transfection analysis.
Transfection of CRIB-1 cells with pBACBHV37 Tn5 or pBACBHV37GET DNA was conducted as described previously (Mahony et al., 2002). Monolayers transfected with pBACBHV37 Tn5 and pBACBHV37GET were observed daily for the formation of cytopathic effect (CPE) and for enhanced green fluorescent protein (eGFP) expression in pBACBHV37 Tn5 only. Freeze–thaw and clarified (by centrifugaton at 5000 g at 4 °C for 15 min) supernatants from transfected monolayers were passaged on fresh CRIB-1 monolayers three times, irrespective of initial CPE or eGFP observations.
In vitro replication capacity.
To indirectly assess the in vitro replicative capacity of mutant clones, the cycle threshold (Ct), established by real-time PCR (qPCR) from passage three (P3) viral culture supernatants, was determined. Total DNA from P3 supernatants (200 µl) was extracted using the DNeasy DNA extraction kit (Qiagen). qPCR mixtures (20 µl) contained 10 µl TaqMan Universal PCR master mix (Applied Biosystems), 0.6 µM of primers BHVF and BHVR (Supplementary Table S1), 0.2 µM probe (BHVP) labelled with 6-carboxy-
uorescein (FAM) (Supplementary Table S1) and 1 µl template DNA. Cycling was conducted using the Rotorgene RG-3000 (Corbett) thermocycler with initial denaturation of 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. A Ct of 0.2 was used for data acquisition.
Restriction endonuclease profiling.
To discount the presence of unmodified pBACBHV37 and to show complexity of pBACBHV37 Tn5 and/or pBACBHV37GET DNA comparable to that of pBACBHV37 DNA, REP of selected clones was conducted using SalI or co-digested using HindIII/DraI, according to the manufacturer's insrtructions (NEB). Digests were resolved on a 0.5 % agarose gel in TBE containing 200 ng ethidium bromide ml–1. Restriction endonuclease profiles (REP) were visualized and photographed using the Gel-Doc system and Quantity-One software (Bio-Rad).
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RESULTS |
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clones DNA from DH10B cells, transposon insertion position was determined by sequencing. Sequence reactions were amplified with primer KanME, which binds 330 bp upstream of the Tn5 terminus within the kanamycin resistance (KanR) cassette, generating sequences spanning the junction of the transposon terminus and the flanking BoHV-1 DNA region. The transposon insertion positions harboured by pBACBHV37 Tn5 library clones are schematically represented in Fig. 1. Of the 100 pBACBHV37 Tn5 clones sequenced, transposition of 42 ORFs occurred, with 20 ORFs singularly disrupted and 22 ORFs disrupted two or more times.
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). A number of intergenic insertions were duplicated as they occurred between the dual copy genes, bICP4 and bICP22, within the repeat regions; these insertions were designated IG-5 and IG-6 with respect to their internal repeat sequence (IR) or terminal repeat sequence (TR) position, respectively (Table 4). Similarly, transposition of the dual copy gene, bICP4 (Table 1), was also duplicated with respect to their IR or TR position. Sequence analysis of 100 pBACBHV37 Tn5 clones (data not shown) demonstrated that 43 of the 73 recognized ORFs encoded by BoHV-1 were disrupted at least once. In order to complete the analysis, the remaining 30 ORFs were deleted using GETrec (Narayanan et al., 1999).
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clones DNA was transfected into CRIB-1 cells to determine if the disrupted gene was essential or non-essential to virus viability in vitro. Following transfection and three passages of pBACBHV37 Tn5 DNA, the occurrence of lytic CPE was interpreted as virus reconstitution and demonstration that the ORF was non-essential to virus viability. Conversely, the absence of lytic CPE was interpreted as demonstration that the ORF was essential to virus viability. The essential or non-essential designations of the BoHV-1 ORFs disrupted by transposition are presented in Tables 1
, 2 and 3 according to their transcriptional regulation. In general, the requirement of the majority of ORFs disrupted by transposon mutagenesis conformed to the requirements of the respective HHV-1 homologue (Roizman, 1996). However, transposon disruption of the ORFs UL54, UL42, US3 (Table 2), UL38 and UL16 (Table 3), displayed an opposite requirement when compared with their HHV-1 homologue (Roizman, 1996).
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DNA harbouring intergenic insertions demonstrated the majority of intergenic regions disrupted were not required for BoHV-1 in vitro viability (Table 4). However, transposon insertion of IG-1 and IG-4 indicated the positions were required for reconstitution of viable virus (Table 4). Furthermore, results obtained from the transfection of pBACBHV37 Tn5 DNA harbouring insertions within the dual copy gene bICP4 (Table 1) were inconclusive and the requirement for bICP4 was unable to be determined by transposon mutagenesis.
Several clones were identified as harbouring transposon insertions in the same ORF, though at different positions within the ORF. The majority of BoHV-1 ORFs that were disrupted two or more times in separate transposition events (Tables 1, 2 and 3) yielded the same requirement for replication for that particular ORF. However, several clones with multiple transposition events in the same ORFs showed conflicting requirements when transfected into cells. This included ORFs UL36, UL28, UL19, US4 (Table 3) and UL29 (Table 2). A determination of the requirement of these ORFs could not be made solely on Tn5 transposition.
To identify the essential or non-essential requirement of ORFs that show ambiguous requirements with respect to multiple Tn5 insertions (UL36, UL29, UL28, UL19 and US4), an opposing requirement when compared to the HHV-1 homologue (UL54, UL42, UL38, UL16 and US3) and to determine the requirement of 30 ORFs where no transposon insertions were identified, the complete deletion of these ORFs by GETrec was conducted.
Sequence analysis of pBACBHV37GET clones
Specific gene deletion through replacement with the relevant LT was confirmed by sequencing. pBACBHV37GET clones were amplified with KanME, generating products incorporating 330 bp of the KanR cassette, 50 bp of the homologous recombination region and part of the flanking BoHV-1 DNA region. Sequence analysis of all pBACBHV37GET clones demonstrated that all of the ORFs targeted had been replaced with the LT (data not shown).
Transfection analysis of pBACBHV37GET clones
The results of the transfection analysis of pBACBHV37GET clones are shown in Tables 1, 2 and 3 and mapped on Fig. 1. The majority of deleted pBACBHV37 ORFs, including those that were initially determined to be ambiguous (UL36, UL29, UL28, UL19 and US4) or discordant (UL54, UL42, UL38 and US3) by transposon disruption, conformed to the reported requirement of the respective HHV-1 homologue (Roizman, 1996). However, the UL53, UL49.5, UL49, UL35, UL20, UL16 (Table 3), UL54 and UL7 (Table 2) ORFs, predominantly encoding viral structural proteins, displayed differing requirements when compared with the respective HHV-1 homologue (Roizman, 1996). The ORFs UL54, UL49.5, UL49, UL35 and UL7 were determined to be non-essential to BoHV-1 viability and the UL53, UL20 and UL16 ORFs were determined to be essential to virus viability in vitro.
In vitro replication capacity
The determined Ct values of transposed and gene-deleted pBACBHV37 clones are shown in Tables 1, 2, 3 and 4. Ct values of >36 were considered indicative of an essential gene with values >45 representing non-detection of viral DNA. Ct values <20 were considered indicative of mild attenuation and showed a comparable replicative capacity to that of unmodified pBACBHV37 (Ct =18). Clones with Ct values between 20 and 36 demonstrated attenuation, with those clones approaching Ct 36 displaying severe attenuation. In general, the determined Ct values for transposed or gene-deleted clones concurred with the essential and non-essential designation of the respective ORF.
Restriction Enzyme Profiles
The REPs of HindIII/DraI-digested pBACBHV37 DNA harbouring either complete deletion and/or transposition of UL53, UL20 and UL16 are shown in Fig. 2. The REPs display a complexity comparable to that of pBACBHV37 (Fig. 2, lane 1), suggesting that there are no large deletions or rearrangements within the modified clones. The profiles confirm that the alterations in the REPs are a result of specific modifications made to the respective ORF (Fig. 2).
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UL53 (Fig. 2, lane 3) contains an additional fragment at 9 kb with concurrent loss of a fragment between 6.4 and 7.2 kb compared with pBACBHV37. The UL53 ORF is 999 bp and contains no DraI, but three HindIII restriction sites at positions 24, 570 and 867. By replacing the UL53 ORF with the LT, which encodes no DraI or HindIII restriction sites, the latter two sites, but not the restriction site at position 24 would have been removed, since this site is present within the 5' recombination arm of the LT.
The REP generated by pBACBHV37GET UL20 (Fig. 2, lane 4) is comparable to the profile of pBACBHV37. However, the loss of a fragment of approximately 9 kb is most likely a result of expansion of the target ORF by replacement with the LT. The UL20 ORF is 716 bp and contains no DraI or HindIII sites. Replacement of the ORF with the LT has expanded the fragment by approximately 500 bp, resulting in the increase in fragment size to 9.5 kb (Fig. 2).
The REPs for pBACBHV37GET UL16 (Fig. 2, lane 5) and pBACBHV37 Tn5UL16 digested DNA (Fig. 2, lane 6) represent the contrast between the two mutagenesis methods utilized. The UL16 ORF is 1020 bp and harbours no HindIII or DraI restriction sites. Deletion of the UL16 ORF with LT does not introduce HindIII or DraI restriction sites; however, disruption of the ORF with the Tn5 transposition cassette introduces two HindIII sites and one DraI restriction site and is the likely cause of the loss of a fragment at 15 kb and the generation of additional fragments at 5.7 and 13 kb.
The REP of SalI-digested pBACBHV37 DNA harbouring complete deletion of the ORFs UL54, UL49.5, UL49, UL35 and UL7 are shown in Fig. 3. The REPs demonstrate complexity comparable to that of pBACBHV37 with no observable deletions occurring. Fragment shifts most likely result from either recombination of the LT into a small ORF or removal of SalI restriction sites.
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UL54 is comparable to the profile generated by SalI digestion of pBACBHV37. The UL54 ORF is 1203 bp and does not encode any SalI restriction sites. Replacement of the ORF with the LT (1300 bp including the recombination arms) expands the ORF by 37 bp (Table 2). This small addition was not resolved in the REP, resulting in a largely unchanged profile when compared to pBACBHV37 (Fig. 3, lane 2). However, replacement of the ORF was confirmed by sequencing of the 3' recombination region.
pBACBHV37GET UL49.5 (Fig. 3, lane 3) shows an increase in fragment size from 8.4 to 9.4 kb. The UL49.5 ORF is 291 bp and does not contain any SalI restriction sites. Replacement of the ORF with a 1300 bp LT that does not contain any SalI sites resulted in an approximately 1 kb increase in fragment size (Fig. 3, lane 3). A smaller increase in fragment size from 8.4 to 9 kb was also seen in the profile generated by pBACBHV37GET UL49 DNA (Fig. 3, lane 4). The UL49 ORF is 777 bp and adjacent to the UL49.5 ORF. Replacement of the ORF with the 1300 bp LT has increased the fragment size by approximately 500 bp (Fig. 3, lane 5).
The REP of pBACBHV37GET UL35 (Fig. 3, lane 5) shows the loss of a 4.9 kb fragment and the addition of a 14.5 kb fragment. The UL35 ORF is 375 bp and encodes one SalI restriction site at 190 bp within the ORF. Replacement of the ORF with the LT has deleted the SalI site, resulting in the generation of the high molecular mass fragment observed at approximately 14.5 kb (Fig. 3).
The profile of pBACBHV37GET UL7 DNA (Fig. 3, lane 6) shows the loss of a 7 kb fragment. The UL7 ORF is 900 bp and does not encode any SalI sites. Replacement of the ORF with the LT expanded the ORF by 300 bp, most likely resulting in co-migration with the adjacent higher molecular mass fragment.
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DISCUSSION |
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). A summary of those clones displaying a discordant phenotype is presented in Table 5. The significance of the differences (or similarities) observed between BoHV-1 and HHV-1 ORF requirement and those ORFs of BoHV-1 subtypes is unknown and further work is required. However, this study represents the most comprehensive investigation to date concerning the in vitro requirement of genes encoded by the prototype ruminant alphaherpesvirus, BoHV-1.
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), BoHV-1 encodes several ORFs with no HHV-1 homologues. These unique ORFs are designated Circ, UL0.5, UL0.7, US1.5 (Table 1) and UL3.5 (Table 3) (Schwyzer & Ackermann, 1996; Schwyzer et al., 1996). The requirement of the Circ ORF for BoHV-1 replication was previously shown to be non-essential for the in vitro replication of the BoHV-1.1a strain Jura (Fraefel et al., 1994). However, complete deletion of the Circ ORF from the BoHV-1.2b subtype in this study did not result in the production of infectious BoHV-1 (Table 1). No CPE was observed upon initial transfection or subsequent passages of culture supernatant. The reason for this is unclear; however, replacement of the Circ ORF with the KanR cassette may have disrupted immediate-early transcription across the covalently joined ends of the circularized BoHV-1 genome, resulting in the observed phenotype (Fraefel et al., 1993).
The requirement of the ORFs UL3.5 and US1.5 have also been previously reported for BoHV-1.1a Cooper strain and the findings of the current study were consistent with the essential (Table 3) and non-essential (Table 1) determinations for the ORFs (Furth et al., 1997; Lam & Letchworth, 2004). The agreement of our findings with those of previous studies suggests that the requirement of these ORFs may be consistent for all subtypes of BoHV-1. The requirements of ORFs UL0.5 and UL0.7 have not been previously reported. This study has demonstrated that both ORFs are non-essential for the in vitro viability of BoHV-1 (Table 1).
Nine of the transposed clones were shown to harbour insertions within six intergenic (IG) regions (IG-1–IG-6) of the BoHV-1 genome (Table 4). Interestingly, clones harbouring transposon insertions within IG-1 and IG-4 produced no CPE following transfection into cells and were designated to be essential for virus viability. The remaining IG insertions did not prevent the reconstitution of BoHV-1, though four of the nine IG clones were clustered within IG-5 and IG-6, a 291 bp region of the BoHV-1 genome (Fig. 1).
It is not readily apparent why IG insertions would prevent in vitro replication of BoHV-1. IG insertions at IG-1, located 12 bp downstream of the essential ORF UL53 (Table 3, Fig. 1), and IG-4, located adjacent to the essential gene UL19 (Table 4, Fig. 1), probably reflect the phenotype of the associated ORF. The insertions may have affected the transcription of these ORFs, thus resulting in the observed phenotypes.
While the exact reasons for the essential nature of these IG insertions can not be explained fully without further experimental studies, the identification of these sites illustrates the power of transposon-based mutagenesis studies which are not reliant on existing sequence knowledge. Furthermore, four other IG sites were identified (IG-2, IG-3, IG-5 and IG-6) that did not prevent in vitro replication of BoHV-1 (Table 4, Fig. 1).
The replicative capacity of mutant viruses was assessed by qPCR as an indirect measure of viral attenuation. The Ct values reflected the essential or non-essential designation of the majority of clones; however, exceptions were identified. Surprisingly, viral DNA was detected in the supernatants for some deletion mutants for which no CPE was observed. In some cases, the Ct values were very high and most likely reflect carry-over of residual BAC DNA from the initial transfection. For example, the deletion mutant for UL5 (Table 2) had a Ct value lower than some recovered viruses with visible CPE. This clone may have produced a viable virus that did not result in visible CPE. Though beyond the scope of the current study, examination of these clones for the presence of intracellular virus and the presence of virus antigen using immunohistochemistry would help to resolve this inconsistency.
In contrast, some mutants with visible CPE appeared to yield low amounts of extracellular virus, for example UL4 and UL11 (Tables 1 and 3, respectively). It is possible that these clones could produce virus that is highly cell-associated and this requires further investigation. Use of the techniques described above could aid in resolving the functions and phenotypic characteristics of these mutants.
BoHV-1 bICP4 is a homologue of HHV-1 ICP4 which is essential to HHV-1 viability in vitro (Roizman, 1996) and is reported to mediate the circularization of the linear HHV-1 genome in infected cells (Su et al., 2006). This study could not determine the requirement for this ORF for BoHV-1.2b in vitro viability (Table 1), as the presence of the duplicated ORF ensures that a complementary functional copy remains irrespective of any random or specific mutagenesis event occurring in either copy (Fig. 1). Furthermore, due to the dual nature of the bICP4 ORF, mutagenesis of either copy of bICP4 may result in two outcomes. Firstly, the transposed or gene-deleted bICP4 may be duplicated upon replication, resulting in two non-functional bICP4 ORFs. Secondly, the complementary wild-type bICP4 ORF may be positively selected, resulting in reconstitution of a wild-type, revertant virus harbouring two functional bICP4 ORF copies. In fact, both virus species may have arisen. Sequencing data (not shown) of both transposon-disrupted and gene-deleted clones putatively harbouring these mutations was inconclusive to the specific location of either the IR or TR bICP4 ORF; however, these data did show that clones harbouring the transposon or LT were present. The presence of revertant virus could not be confirmed.
In this study, transposon insertion and homologous recombination have been utilized to determine the requirement of the ORF complement of BoHV-1 for in vitro viability (Fig. 1). The complementary use of both techniques and subsequent transfection analysis has identified that the ORFs UL54, UL53, UL49.5, UL49, UL35, UL20, UL16 and UL7 differ in requirement for the in vitro viability of BoHV-1 when compared with HHV-1 homologues (Roizman, 1996). Overall, of the 73 recognized ORFs encoded by BoHV-1, 33 were determined to be essential and 36 to be non-essential (the requirement of two dual copy genes remains un-determined). Clearly, the role of each ORF requires further investigation, such as gene complementation and the construction of revertant viruses, which are beyond the scope of the current study. However, the construction of this restriction endonuclease map will contribute to the identification of appropriate targets for antiviral chemotherapy, the development of BoHV-1-derived vectors and to the general understanding of the highly successful alphaherpesvirus subfamily.
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ACKNOWLEDGEMENTS |
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Received 27 March 2008;
accepted 21 June 2008.
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