Analysis of a bovine herpesvirus 1 recombinant virus that does not express the bICP0 protein

V. Geiser, Y. Zhang and C. Jones

Department of Veterinary and Biomedical Sciences, Nebraska Center for Virology and School of Biological Sciences, University of Nebraska, Lincoln, NE 68583, USA

Correspondence
C. Jones
cjones{at}unlnotes.unl.edu


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bovine herpesvirus 1 (BHV-1) infected-cell protein 0 (bICP0) stimulates productive infection by activating viral gene expression. In this study, an attempt was made to construct a recombinant virus with point mutations in the C3HC4 zinc RING finger of bICP0, as this domain is necessary for activating viral transcription and productive infection. A virus was identified in bovine cells that induced small clusters of infected cells resembling a small plaque. Instead of the expected mutations within the zinc RING finger, this virus contained a point mutation within the initiating ATG of bICP0, a point mutation two bases downstream from the ATG mutation and deletion of flanking plasmid sequences used for homologous recombination. The bICP0 mutant was rescued with wild-type (wt) bICP0 sequences and the bICP0-rescued virus produced wt plaques. The bICP0-rescued virus and wt BHV-1, but not the mutant, expressed the bICP0 protein during productive infection of bovine cells, suggesting that the mutant virus was a null mutant. Consequently, the mutant was designated the bICP0 null mutant. Infection of bovine cells with the bICP0 null mutant resulted in at least 100-fold lower virus titres, indicating that bICP0 protein expression is important, but not required, for virus production. When bovine cells infected with the bICP0 null mutant virus were subcultured, the cells continued to divide, but viral DNA could be detected after more than 35 passages, suggesting that the bICP0 null mutant induced a persistent-like infection in bovine cells and that it may be useful for generating additional bICP0 mutants.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Infection of cattle with bovine herpesvirus 1 (BHV-1), a member of the subfamily Alphaherpesvirinae, leads to respiratory disorders, conjunctivitis, genital infections, encephalitis, abortions and a multi-systemic fatal disease in neonates. Following acute infection, BHV-1 establishes latency in sensory neurons within trigeminal or dorsal root ganglia (Jones, 1998, 2003). Latent virus can be reactivated consistently from latency by dexamethasone treatment, which results in virus shedding and spread to susceptible hosts (Rock et al., 1992; Winkler et al., 2000, 2002). Although the main site of BHV-1 latency is sensory neurons, BHV-1 DNA is detected consistently in tonsils (Winkler et al., 2000), peripheral blood cells (Fuchs et al., 1999), lymph nodes and spleen (Mweene et al., 1996) of latently infected calves.

Calves infected acutely with BHV-1 contain high levels of apoptotic CD4+ T cells compared with mock-infected calves (Winkler et al., 1999, 2000). Viral antigens and apoptotic cells localize to germinal centres of pharyngeal tonsil in acutely or latently infected calves. BHV-1 induces apoptosis when cultured T cells, B lymphocytes or monocytes are infected (Hanon et al., 1996, 1998). Glycoprotein D (Hanon et al., 1999), c-myc induction (Hanon et al., 1997), p53 expression and caspase activation (Devireddy & Jones, 1999) play a role in BHV-1-induced apoptosis. As BHV-1 infects lymphoid cells and induces apoptosis, peripheral blood mononuclear cells prepared from calves infected acutely with BHV-1 produce less interleukin 2, have a reduced mitogenic response and exhibit lower natural cytotoxic activities (Carter et al., 1989). BHV-1 also interferes with major histocompatibility complex class I antigen presentation and cytotoxic lymphocyte-mediated killing of virus-infected cells (Hariharan et al., 1993; Nataraj et al., 1997; reviewed by Favoreel et al., 2000). Collectively, these studies indicate that BHV-1 suppresses the immune system of acutely infected calves by several different mechanisms.

Viral gene expression is regulated temporally in three distinct phases: immediate-early (IE), early (E) and late (L) (Jones, 2003). The bICP0 protein is encoded by IE transcription unit 1 (IEtu1) (Wirth et al., 1992), is expressed constitutively during productive infection because it has an IE and E promoter, and activates its own promoters (Fig. 1a) (Fraefel et al., 1994). When BHV-1 DNA is transfected into permissive cells, plaque formation is inefficient. Co-transfection of BHV-1 DNA with bICP0 significantly increases the number of plaques and decreases the time taken for plaques to appear (Geiser & Jones, 2003; Inman et al., 2001b), indicating that bICP0 stimulates productive infection. bICP0 associates with histone deacetylase 1 (HDAC1) and, in quiescent cells, bICP0 relieves HDAC1-induced transcriptional repression (Zhang & Jones, 2001), suggesting that this activity promotes viral transcription.



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Fig. 1. Schematic of the bICP0 gene and the targeted site for mutagenesis. (a) Position of IE and LR transcripts. One IE promoter activates expression of IE/4.2 and IE/2.9 and this transcription unit is designated IEtu1. IE/4.2 encodes bICP4 and IE/2.9 encodes bICP0. E/2.6 is the early transcript that encodes bICP0 and its expression is controlled by an E promoter located at the 5' terminus of exon 2 (e2) (Wirth et al., 1991, 1992). All bICP0 protein-coding sequences are contained within e2. The origin of replication (ORI) separates IEtu1 from IEtu2. IEtu2 encodes the protein bICP22. The solid lines in the transcript position map represent exons (e1, e2, e3) and arrows indicate the direction of the respective transcripts. (b) Partial restriction map of plasmid pbICP0x showing the location of bICP0 and LR ORF2. A region of the HindIII L fragment was cloned into pUC19 as described in Methods and this plasmid was designated phd/k. The region surrounding the mutations in bICP0 was then subcloned from plasmid mbICP0 into phd/K (pbICP0x) as described in Methods to facilitate homologous recombination. The positions of primers bICP0a and bICP0b used to amplify the bICP0 gene are denoted by filled rectangles. The HindIII restriction-enzyme site (AAGCTT) was created by linker insertion and was included to facilitate screening of the mutant. Arrows denote the location of zinc RING finger mutations (aa 13 C->G, aa 51 C->A). The procedures for site-directed mutagenesis have been described previously (Inman et al., 2001b). (c) Nucleotide sequence of the zinc RING finger mutations. The MscI restriction-enzyme site (TGGCCA) was incorporated into the mutant oligonucleotide to facilitate screening.

 
The ICP0 homologues encoded by BHV-1 and herpes simplex virus type 1 (HSV-1) contain well-conserved C3HC4 zinc RING fingers near their respective N termini. Mutational analysis has demonstrated the importance of the C3HC4 zinc RING finger domain of bICP0 and ICP0 (Everett, 1987, 1988; Everett et al., 1993; Inman et al., 2001b). ICP0 (Everett et al., 1997, 1999a, b; Maul & Everett, 1994; Maul et al., 1993) and bICP0 (Inman et al., 2001b; Parkinson & Everett, 2000) co-localize with and disrupt the promyelocytic leukaemia protein-containing nuclear domains (ND10 or PODS). ICP0 regulates steady-state levels of cellular and viral proteins because it interacts with protein-degradation machinery (Everett et al., 1997, 1999a) and has E3 ubiquitin-ligase activity (Boutell et al., 2002; Van Sant et al., 2001). The E3 ubiquitin-ligase activity of ICP0 disrupts the cell cycle and alters expression of certain proteins (Hobbs & DeLuca, 1999; Lomonte & Everett, 1999).

A bICP0 mutant was constructed previously by inserting the {beta}-galactosidase gene into bICP0-coding sequences (Koppel et al., 1996). This mutant was not stable because viral sequences were not deleted and, thus, wild-type (wt) virus could be regenerated when the virus was passaged in cultured cells. HSV-1 ICP0 mutants have impaired growth in culture, but the defect in virus production is reduced when infection is conducted at a high m.o.i. (Sacks & Schaffer, 1987; Stow & Stow, 1986). In a human tumour-cell line, U2-OS, ICP0 mutants have wt growth properties, suggesting that cellular factors can substitute for ICP0 functions (Yao & Schaffer, 1995).

In this study, we generated a bICP0 null mutant that did not express detectable levels of the bICP0 protein. Although bICP0 was not required for growth of BHV-1 in cultured bovine cells, expression was necessary for efficient growth and plaque formation. These studies also suggested that the bICP0 null mutant virus established a persistent-like infection in bovine kidney cells.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of plasmids.
Construction of the bICP0 plasmid containing point mutations at aa 13 and 51 of the zinc RING finger (mbICP0) has been described previously (Inman et al., 2001b). The HSV-1 ICP0 plasmid was obtained from S. Silverstein (Columbia University, NY, USA). pUC19 and pcDNA3.1(–) were purchased from New England Biolabs and Invitrogen, respectively. The plasmid phd/k contained the BHV-1 HindIII–KpnI region of the HindIII C fragment (Wirth et al., 1989, 1991, 1992) cloned into pUC19 (Fig. 1).

The pbICP0x construct was constructed by cloning the XbaI–AscI fragment (genome nt 100236–103953) of plasmid phd/k into pUC19. Plasmid phd/k was digested with HindIII plus XbaI, blunt-ended, ligated, digested with AscI and then religated. The resulting plasmid was digested with BstXI, blunt-ended and HindIII linkers were added to form phd/K+. Plasmids phd/K+ and mbICP0 were digested with HindIII and SalI. The HindIII–SalI fragment obtained from plasmid mbICP0 was gel-purified and ligated into phd/K+ to generate pbICP0x (Fig. 1b). All plasmids were transformed and maintained in the DH5{alpha} strain of Escherichia coli (Life Technologies).

Cells.
Madin–Darby bovine kidney (MDBK) cells were plated at a density of 5x105 cells per 100 mm2 plastic dish in Earle's modified Eagle's medium supplemented with 5 % fetal bovine serum (FBS), penicillin (10 U ml–1) and streptomycin (100 µl ml–1). MDBK cells were split in a 1 : 8 ratio every 3 days. The bovine cell line 9.1.3 was supplemented with medium containing 10 % FBS and split in a 1 : 6 ratio every 3 days, as described previously (Inman et al., 2001b).

Virus.
The Cooper strain of BHV-1 (wt virus) was obtained from the National Veterinary Services Laboratory, Animal and Plant Health Inspection Services, Ames, IA, USA. Viral stocks were prepared by infecting MDBK cells at an m.o.i. of 0·01 from plaque-purified virus and subsequently titrating on MDBK cells.

Extraction of viral genomic DNA.
MDBK cells were infected with wt BHV-1 Cooper strain, the rescued virus or the bICP0 null mutant virus (m.o.i., 0·01) and viral genomic DNA was extracted as described previously (Inman et al., 2001a, b). At 36–72 h after infection [cytopathic effect (CPE) of approximately 80 %], the supernatant was collected and clarified by centrifugation (7000 r.p.m., 4 °C, 20 min in a Beckman J2-21 using a JA-20 rotor). Virus in the supernatant was pelleted through a 30 % sucrose/TE cushion (25 ml virus per 5 ml sucrose solution) by centrifugation (25 000 r.p.m., 4 °C, 2 h in a Beckman L7-65 ultracentrifuge using an SW28 rotor). The pellet was suspended in 623 µl RSB buffer [10 mM Tris/HCl (pH 7·4), 10 mM KCl, 1·5 mM MgCl] and then treated with 6·3 µl DNase I (105 U ml–1) at 37 °C for 2 h. The reaction was stopped by adding 90 µl 20 mM EDTA and incubating at 65 °C for 15 min. The suspension was diluted with 24·3 ml calcium- and magnesium-free PBS (CMF-PBS) and the virus was pelleted as described above. The pellet was suspended in 1·8 ml DNase I-free TE. Virions were disrupted by adding 100 µl 20 % SDS, 15 µg RNase and incubating the solution at 37 °C for 30 min. Proteinase K (10 mg) was then added and the solution was incubated at 65 °C for 30 min. Three phenol/chloroform/isoamyl alcohol (50 : 48 : 2) extractions were performed, followed by one extraction with chloroform/isoamyl alcohol (48 : 2). The aqueous phase was extracted with ether three times. Following ethanol precipitation, viral DNA was electrophoresed on an agarose gel to examine its quality and quantity. Known concentrations of DNA standards were used to estimate the amounts of viral DNA.

Transfections and identification of the bICP0 mutant.
Bovine 9.1.3 cells were co-transfected with 2 µg viral genomic DNA, 6 µg pbICP0x (or phd/k) plasmid and increasing amounts of an HSV-1 ICP0 expression plasmid (0, 0·25, 0·5, 1 or 2 µg) by using Superfect (Qiagen) as described previously (Inman et al., 2001a).

The sequence of the region of the bICP0 mutant virus, the rescued virus and pbICP0x was determined by PCR amplification using primers bICP0a and bICP0b as described below and shown in Fig. 1(b). The PCR products were cloned into the TOPO vector and the insert was sequenced.

PCR.
PCR was performed with DNA extracted from infected MDBK cells at 48 h after infection. PCR was conducted with 5 µl 10x commercial PCR buffer, 2 µl 25 mM MgCl2, 10 µl GC-Melt (BD), 1 µl 40 mM dNTPs, 1 µM each primer and 1 U Taq polymerase for each 50 µl reaction. To detect the bICP0 null mutant, PCR was performed on extracted DNA by using the bICP0a (nt 102828–102845, 5'-GCCTTTCGCCGCCCGCCC-3') and bICP0b (nt 102469–102452, 5'-CAACGCGCCGTCCGCCCC-3') primers. The bICP0 region of the mutant virus was also amplified by using primers bICP0a and bICP0b-EcoRI (5'-CGGAATTCAACGCGCCGTCCGCCCC-3') and then cloned into pUC19.

The bICP22 primers were 5'-GCGCTGGTCCTCCGGCTCC-3' (upstream primer) and 5'-CTCGCTGGCGGCGCTTGG-3' (downstream primer) (Schang & Jones, 1997). The latency-related (LR) gene and bICP0 primers were 5'-TTCTCTGGGCTCGGGGCTGC-3' and 5'-AGAGGTCGACAAACACCCGCGGT-3' (L3B primers) (Hossain et al., 1995). The glycoprotein C (gC) primers were 5'-GAGCAAAGCCCCGCCGAAGGA-3' and 5'-TACGAACAGCAGCACGGGCGG-3' (Schang & Jones, 1997).

After a hot start for 3 min, each PCR cycle consisted of 95 °C for 30 s, incubation at the annealing temperature for the indicated time and 72 °C for 1 min. The annealing temperatures and times were as follows: bICP0, 66·6 °C, 45 s; bICP22, 65 °C, 45 s; L3B, 65 °C, 1 min; gC, 63 °C, 45 s. PCR was carried out for 35 cycles (bICP0, bICP22, gC) or 40 cycles (L3B). To ensure complete elongation of the amplified products, the reaction mixture was incubated at 72 °C for an additional 10 min. PCR products were electrophoresed on a 2 % agarose gel and the DNA was visualized by ethidium bromide staining.

Measurement of virus titres in bovine cells.
MDBK cells were infected at an m.o.i. of 0·01 for the designated viruses (1 h at 37 °C). Cultures were then rinsed twice with CMF-PBS and complete medium was added. Supernatant from infected cultures or cell lysate was subjected to three freeze–thaw cycles. After cell debris had been pelleted, the supernatant was titrated on MDBK cells. The final dilution eliciting a CPE was determined for each sample.

At 48 h post-infection (p.i.), total cell lysate was collected and subjected to three freeze–thaw cycles. Virions were pelleted by using a sucrose cushion and treated with DNase I to remove cellular and non-encapsidated DNA. Total viral genomic DNA was purified as described above for extraction of viral genomic DNA and the DNA was analysed by gel electrophoresis. The equivalent of a 100 mm plate was loaded for each virus, along with 10-fold dilutions of the bICP0 rescued virus to facilitate quantification. Total virion genomic DNA was quantified by using a ChemiDoc XRS imaging system and analysed with the Quantity One program (Bio-Rad).

Protein analysis.
To determine protein levels of bICP0 and {beta}-actin, whole-cell lysate was prepared by using a high/low-salt lysis procedure as described previously (Zhang & Jones, 2001). Proteins in the gel were transferred to Immobilon P membranes (Millipore) and Western blotting was performed as described previously (Hossain et al., 1995; Inman et al., 2001b).

To estimate viral protein levels, virions were collected at 48 h p.i. and purified virions suspended in 60 µl per 100 mm2 plate of 2x lysis buffer [7·84 mM Tris/HCl (pH 8·0), 39·2 mM NaCl, 0·784 mM EDTA, 2 mg iodoacetamide ml–1, 0·05 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium o-vanadate, 5 µg leupeptin ml–1, 5 µg pepstatin ml–1, 5 µg antipain ml–1, 10 mM PMSF]. Boiling 10 % SDS (60 µl) was added to the cell pellet from a 100 mm dish, the sample was vortexed and the solution was boiled for 5 min. Samples were centrifuged for 5 min at 4 °C (15 000 r.p.m.) in a Beckman Avanti 30 centrifuge. The supernatant was transferred to a new tube along with 120 µl per 100 mm2 plate of 2x sample buffer [62·5 mM Tris/HCl (pH 6·8), 2 % SDS, 50 mM dithiothreitol, 0·1 % bromophenol blue, 10 % glycerol]. The cell lysate was boiled for 5 min and the proteins separated by 10 % SDS-PAGE. BHV-1-specific antibodies were detected by Western blotting with a primary anti-BHV-1 serum (VMRD) diluted 1 : 500. Detection of proteins was conducted by using a secondary donkey anti-goat antibody (Santa Cruz Biotechnology) diluted 1 : 2000.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of a bICP0 mutant virus
The C3HC4 zinc RING finger located near the N terminus of bICP0 is required for transactivation of viral promoters and activation of productive infection (Inman et al., 2001b; Zhang & Jones, 2001). To examine the role that it plays during productive infection, we attempted to construct a BHV-1 recombinant virus with mutations in the C3HC4 zinc RING finger.

Plasmid pbICP0x contained the entire coding sequence of bICP0 with at least 1 kb of flanking sequences on both sides of the zinc RING finger mutations to facilitate homologous recombination with BHV-1 (Fig. 1b). The zinc RING finger contained mutations at aa 13 (C->G) and aa 51 (C->A) (Inman et al., 2001b). The mutation at aa 51 had a unique MscI restriction site to allow identification of the mutation (Fig. 1c). A unique HindIII restriction site was also present in pbICP0x to facilitate screening. A plasmid encoding HSV-1 ICP0 was co-transfected with BHV-1 DNA and pbICP0x into bovine cells to stimulate plaque formation. ICP0 or bICP0 stimulates the number of plaques and reduces the time for plaque formation when cells are transfected with BHV-1 DNA (Geiser, 2001; Geiser & Jones, 2003; Inman et al., 2001b). Three days after transfection, cultures were subjected to three freeze–thaw cycles and the virus in the supernatant was allowed to form plaques on MDBK cells. A small area of infected cells resembling a plaque was identified. After three rounds of plaque purification, this virus maintained the small-plaque phenotype (Fig. 2a). Cells comprising the small plaque were distinct from mock-infected cells (Fig. 2c) and did not yield the typical plaques observed following infection of MDBK cells with wt BHV-1 (Fig. 2b). The putative bICP0 mutant was rescued by using a plasmid containing the wt bICP0 gene and designated bICP0-rescued virus. The bICP0-rescued virus yielded plaques that were indistinguishable from those produced by wt BHV-1 (Fig. 2d).



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Fig. 2. Plaque formation in infected MDBK cells. MDBK cells were infected with the bICP0 null mutant (m.o.i., 0·00005) (a) or wt BHV-1 (m.o.i., 0·00003) (b), were mock-infected (c) or were infected with the bICP0-rescued virus (m.o.i., 0·00002) (d). After incubation for 1 h at 37 °C, monolayers were rinsed twice with CMF-PBS and overlaid with 0·6 % SeaPlaque agarose. Cells were incubated for 3 days at 37 °C and plaques were photographed at 10x magnification by using a Leica DM IRB microscope. Images were captured with the Leica DC viewer software (Leica Microsystems).

 
Two primers (bICP0a and bICP0b; Fig. 3a) were used to amplify the region of the bICP0 gene containing the unique HindIII site and mutations in the zinc RING finger. These primers amplified a virus-specific 377 bp fragment from all three viruses (Fig. 3b). The HindIII site was present in the amplified product from the bICP0 mutant virus, but not from cells infected with wt or the bICP0-rescued virus (Fig. 3c). The bICP0 mutant virus did not contain the expected zinc RING finger mutations, as the amplified product was not cleaved with MscI (Fig. 3d). DNA sequencing of the amplified 377 bp fragment revealed that the bICP0 mutant virus contained a mutation in the first in-frame ATG (mutated to GTG), a C->A substitution two bases downstream of the ATG codon and an 11 nt deletion from the multiple cloning site upstream of pbICP0x (Fig. 4). The rest of the bICP0-coding sequences within the PCR product had wt sequences. In addition, promoter-proximal sequences were also wt (data not shown). These results suggested that we had generated a bICP0 null mutant.



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Fig. 3. Analysis of the bICP0 mutant virus by PCR analysis. (a) Schematic of the bICP0 region and strategy used for PCR amplification. PCR amplification of BHV-1 DNA using the bICP0a and bICP0b primers yielded a virus-specific 377 bp band. If homologous recombination between pbICP0x and the viral genome occurred, two bands (124 and 253 bp) would be observed following digestion with HindIII. If the mutation at aa 51 from C->A was present in the zinc RING finger, digestion with MscI would yield two bands (99 and 278 bp). Locations of zinc RING finger mutations are denoted by asterisks. (b) PCR amplification of viral DNA. DNA samples prepared from MDBK cells were amplified with primers bICP0a and bICP0b. (c, d) Digestion of the amplified products with (c) HindIII or (d) MscI. The bICP0 null mutant virus was subjected to four rounds of plaque purification prior to PCR amplification. PCR was performed on DNA extracted from the bICP0 null mutant virus (N), wt virus (W), bICP0-rescued virus (R), mock-infected cells (M), a no-template control (NT) and the bICP0 mutant plasmid (pbICP0x) using bICP0a and bICP0b primers.

 


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Fig. 4. DNA sequence analysis of the bICP0 null mutant virus. The 377 bp amplified product was gel-purified, cloned into the TOPO vector and sequenced. The bICP0 null mutant contained the HindIII restriction-endonuclease site (AAGCTT), but had a wt zinc RING finger that was consistent with the absence of the MscI restriction-endonuclease site (TGGCCA). Additional mutations were present in the 5' region of the gene, including the initiating methionine (ATG) and absence of cloning sequences in bICP0x. To confirm the sequencing results, the bICP0 region was also cloned directionally into pUC19. The bICP0 region was PCR-amplified by using primers bICP0a and bICP0b-EcoRI. The PCR product was digested with HindIII and the gel-purified product was cloned into the EcoRI and HindIII sites of pUC19. The pbICP0x plasmid was sequenced by using the TOPO vector and contained the expected mutations in the zinc RING finger. The bICP0-rescued virus was sequenced by using the TOPO vector and contained the expected wt sequence.

 
Western blot analysis of the bICP0 mutant virus
To test for bICP0 protein expression, MDBK cells were infected with the bICP0 null mutant, wt BHV-1 or the bICP0-rescued virus. bICP0 protein expression was detected readily in MDBK cells infected with wt BHV-1 and the bICP0-rescued virus, but not with the bICP0 null mutant (Fig. 5a). As expected, similar levels of {beta}-actin protein were detected in all of the samples (Fig. 5b). RT-PCR analysis revealed that the bICP0 null mutant expressed bICP0 mRNA at a reduced level relative to wt BHV-1 and the bICP0-rescued virus (data not shown). The results in Fig. 5 confirmed that the mutant was a bICP0 null mutant.



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Fig. 5. Western blot detection of bICP0 expression of the bICP0 null mutant, bICP0-rescued virus and wt virus in MDBK cells. MDBK cells were mock-infected (M) or infected with the bICP0 null mutant virus (N), bICP0-rescued virus (R) or wt virus (W) at an m.o.i. of 0·1. At 48 h p.i., whole-cell lysate (3x106 cells, left-hand lanes; 1x106 cells, right-hand lanes) was electrophoresed by SDS-PAGE (10 % gel) and electroblotted on to Immobilon P membranes. (a) The membrane was probed with a polyclonal antibody generated against the N-terminal 361 aa of bICP0 (1 : 500 dilution). Detection of bound primary antibody was performed by using the ECL detection system with donkey anti-rabbit antibody diluted 1 : 2000. The predicted molecular mass of bICP0 is 97 kDa (Fraefel et al., 1994). (b) The membrane was probed with an antibody directed against {beta}-actin (Santa Cruz Biotechnology).

 
Analysis of the BHV-1 bICP0 mutant virus in MDBK cells
Infection of MDBK cells with wt BHV-1 led to an obvious CPE, destruction of the cell monolayer and efficient plaque formation (Fig. 2b and d). In contrast, the impaired cytotoxicity of the bICP0 null mutant made it difficult to quantify virus titres accurately by standard plaque assays. To estimate the levels of infectious virus in bovine cells following infection with the bICP0 null mutant, fresh medium was added to wells and the final dilution that elicited a CPE was used to estimate the virus titre. Infection of MDBK cells with the bICP0 null mutant virus resulted in at least 100-fold lower virus titres of cell-associated (Fig. 6a) and cell-free (Fig. 6b) virus. The wt and bICP0-rescued viruses had maximal titres at 36 h p.i., whereas the bICP0 null mutant increased up to 48 h p.i. (Fig. 6a and b).



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Fig. 6. Growth properties of the bICP0 null mutant, bICP0-rescued virus and wt virus. (a, b) The final dilution eliciting a CPE was determined at various times after infection of MDBK cells with cell-associated virus (a) or cell-free virus (b). MDBK cells were infected by using an m.o.i. of 0·01. The bICP0 null mutant virus (N) is denoted by open bars, the bICP0-rescued virus (R) by shaded bars and wt Cooper virus (W) by filled bars. (c) The total amount of virion DNA at 48 h p.i. was determined for each virus. Total cell lysate was collected and subjected to three freeze–thaw cycles. Virions were pelleted by using a sucrose cushion and treated with DNase I to remove non-encapsidated DNA. After DNase I inactivation, total viral genomic DNA was extracted and analysed by 0·75 % gel electrophoresis. Virions from a 100 mm2 plate were loaded for wt (W), bICP0 null mutant virus (N) and bICP0-rescued virus (R), along with 10-fold dilutions of the bICP0-rescued virus to facilitate quantification. Total viral DNA from virions was quantified by using the ChemiDoc XRS imaging system and analysed with the Quantity One program. (d) Western blot analysis of virion proteins prepared from infected cells. MDBK cells were infected with the bICP0 null mutant virus at an m.o.i. of 0·1 (N), bICP0-rescued virus at an m.o.i. of 0·01 (R) or wt BHV-1 at an m.o.i. of 0·01 (W). As controls, mock-infected cells were included (M). At 48 h p.i., cells and media were collected from infected cultures and subjected to three freeze–thaw cycles. Virions in the cell-free lysate were pelleted through a 30 % sucrose/TE cushion. Proteins were purified from pelleted virions by using the 2x SDS lysis method as described in Methods. Protein from 1x107 cells was separated by SDS-PAGE (10 % gel) and electroblotted on to Immobilon P membranes. Virus-specific antibodies were detected by Western blotting using the ECL detection system as described for bICP0. The primary anti-BHV-1 antiserum was diluted 1 : 500 and the secondary donkey anti-goat antibody was diluted 1 : 2000.

 
To confirm the differences in virus titres, we estimated the quantity of viral DNA that was present in viral particles at 48 h p.i. Viral DNA was extracted from virions as described in Methods and analysed by agarose-gel electrophoresis. The bICP0 null mutant consistently contained at least 10-fold less virion genomic DNA from the same number of cells at 48 h p.i. (Fig. 6c).

Total virion proteins were also detected by Western blot analysis using antiserum directed against BHV-1. Lower levels of viral proteins were detected in MDBK cells infected with the bICP0 null mutant relative to cells infected with wt or the bICP0-rescued virus (Fig. 6d).

Survival of MDBK cells infected with the bICP0 null mutant virus
As the bICP0 null mutant virus was not as cytotoxic as wt BHV-1 or the bICP0-rescued virus, we tested whether MDBK cultures infected with the bICP0 null mutant were capable of growing. When MDBK cells were infected with the bICP0 null mutant, we were able to subculture the infected cells at least 35 times. Cultures infected with the bICP0 null mutant contained numerous cells with rounded nuclei and/or elongated fusiform morphology (Fig. 7a) relative to mock-infected cells (Fig. 7b). Viral DNA was detected by PCR using primers that detected several different viral genes after 12 passages (Fig. 7c) and following 35 passages (data not shown). After 14 passages, low levels of virion DNA and infectious virus were detected in the supernatant of MDBK cells infected with the bICP0 null mutant (data not shown). Collectively, these studies suggested that the bICP0 null mutant induced a persistent infection in MDBK cells because the infected cells survived, the cultures contained viral DNA and low levels of infectious virus were released from cultures.



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Fig. 7. Persistence of the bICP0 null mutant virus in MDBK cells. (a, b) MDBK cells infected with the bICP0 null mutant virus (a) or mock-infected (b) were subcultured at 48 h p.i. Representative cells were photographed 24 h after passaging and imaged at 20x magnification. Images were captured with the Leica DC viewer software (Leica Microsystems). (c) Detection of BHV-1 DNA in MDBK cells. DNA was extracted from MDBK cells infected with the bICP0 null mutant virus (N), bICP0-rescued virus (R), wt BHV-1 (W) or from mock-infected cells (M) at 48 h p.i. MDBK cells infected with the bICP0 null mutant virus that were subcultured for 12 passages are designated S. PCR was performed on extracted DNA by using the designated primers. Amplified products were electrophoresed on a 2 % agarose gel and DNA was visualized by staining with ethidium bromide.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we constructed a stable bICP0 null mutant. Although we intended to construct a recombinant virus with mutations in the zinc RING finger, this virus was not identified. The bICP0 null mutant was identified because it developed small clusters of infected cells that appeared to form a plaque. Further purification yielded a virus that retained this phenotype. Similar growth properties of the bICP0 null mutant were observed in another bovine cell line (9.1.3 cells) and rabbit skin cells (data not shown), suggesting that this virus, in general, has poor growth properties in cell types that are permissive for BHV-1 productive infection. PCR amplification and DNA sequencing revealed that this virus contained a mutation in the first in-frame ATG, but contained a wt zinc RING finger. Furthermore, the remainder of the bICP0 gene appeared to possess wt sequences. The bICP0 null mutant was probably a product of homologous recombination between wt BHV-1 and the pbICP0x plasmid, as it contained a diagnostic HindIII restriction site that was present in the pbICP0x plasmid, but not in wt BHV-1. The bICP0 null mutant appeared to be a stable null mutant because it did not express detectable levels of the bICP0 protein and it lacked the first in-frame ATG.

In general, the phenotype of the bICP0 null mutant was similar to that of HSV-1 ICP0 null mutants, as ICP0 is not required for growth in cultured cells (Sacks & Schaffer, 1997; Stow & Stow, 1986). Although bICP0 protein expression was not absolutely required for productive infection in bovine cells, there was a marked reduction in infectious virus and the mutant did not form clear-cut plaques. One reason why the bICP0 null mutant virus did not grow efficiently is that bICP0 activates viral transcription (Fraefel et al., 1994; Inman et al., 2001b; Wirth et al., 1992; Zhang & Jones, 2001). We also predicted that other bICP0 functions enhance productive infection and virus yield. For example, we have recently discovered that bICP0 inhibits beta interferon (IFN-{beta}) promoter activity in transient-transfection assays (Y. Zhang & C. Jones, unpublished data). HSV-1 genes encoding ICP0, 34.5 and US11 inhibit the IFN response and do not grow efficiently (Katze et al., 2002; Mossman & Smiley, 2002; Mossman et al., 2000, 2001). BHV-1 apparently does not encode a 34.5 homologue, suggesting that bICP0 is the major viral protein that inhibits the IFN response. Mice lacking type I and type II IFN receptors in combination with RAG-2 gene deletions die within a few days of infection with BHV-1 (Abril et al., 2004). BHV-1 infection of wt mice does not lead to clinical symptoms, underscoring the importance of the IFN-signalling pathways and BHV-1 replication. In summary, activation of transcription and inhibition of IFN-dependent transcription are two bICP0 functions that are likely to be necessary for efficient productive infection.

When the HSV-1 ICP0 gene is deleted, the viral genome is maintained persistently, but cytotoxicity is reduced (Samaniego et al., 1998). A similar situation apparently occurred following infection of MDBK cells with the bICP0 null mutant (Fig. 7). To date, MDBK cells infected with the bICP0 null mutant have been subcultured more than 35 times and the viral genome is still detected. We recently demonstrated that bICP0 indirectly induces caspase 3, which leads to apoptosis (Henderson et al., 2004). In the absence of bICP0, BHV-1 infection may not induce a strong apoptotic response, which is indicative of infection with wt virus (Devireddy & Jones, 1999). Our studies suggested that cells infected with the bICP0 null mutant survived for numerous passages and produced low levels of infectious virus. Although we were able to detect infectious virus in the ‘persistently infected’ cultures, we do not know whether the genome was maintained stably in all infected cells and what percentage of infected cells produced virus. Additional studies will be necessary to understand these complex virus–host interactions fully.

We recently generated a panel of bICP0 transposon mutants (Zhang et al., 2005). This study revealed that bICP0 contains several important functional domains: (i) the zinc RING finger; (ii) two separate domains that activate transcription; and (iii) a C-terminal nuclear-localization sequence that is necessary for efficient transactivation. We believe that these mutations can be recombined readily into the bICP0 null mutant, because the fitness of the bICP0 null mutant should be improved. Consequently, it should be possible to generate additional bICP0 mutants with specific mutations in the bICP0 protein-coding sequences. These mutants will be useful to help us understand the function of bICP0 in the context of productive infection and the latency-reactivation cycle in cattle.


   ACKNOWLEDGEMENTS
 
This work was supported by two USDA grants (2002-35204 and 2003-02213) and a Public Health Service grant 1P20RR15635. V. G. received support from the National Institutes of Health under a Ruth L. Kirschstein National Research Service Award 1 T32 AIO60547(National Institute of Allergy and Infectious Diseases).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 25 January 2005; accepted 17 March 2005.