Major tegument protein VP8 of bovine herpesvirus 1 is phosphorylated by viral US3 and cellular CK2 protein kinases

Introduction

Bovine herpesvirus 1 (BoHV-1) is a member of the family Herpesviridae and is associated with several disease manifestations in cattle (Jones & Chowdhury, 2007; Turin et al., 1999). Infectious bovine rhinotracheitis is a common respiratory form, which may predispose cattle to secondary bacterial infections resulting in shipping fever. BoHV-1 is also associated with conjunctivitis, reproductive tract lesions, encephalitis and fetal infections. Most of the economic losses result from shipping fever, milk drop, decreased body weight, abortions and death. A major feature complicating the control of herpesviruses including BoHV-1 is their ability to establish latency within the host.

The BoHV-1 virion is composed of a double-stranded DNA genome surrounded by a protein capsid, a tegument and an envelope. The tegument consists of a complex of about 20 virus-encoded proteins, the structures and interactions of which are poorly understood. The tegument proteins are among the first proteins to be released and interact with the intracellular environment during the infection process. The 97 kDa UL47 gene product, VP8, is one of the most abundant proteins in the tegument of BoHV-1. Like its herpes simplex virus-1 (HSV-1) homologue, VP13/14 (LaBoissiere et al., 1992; Meredith et al., 1991), VP8 is phosphorylated (Carpenter & Misra, 1991) and glycosylated (van Drunen Littel-van den Hurk et al., 1995) and contains both nuclear localization and nuclear export signals (Verhagen et al., 2006; Zheng et al., 2004). These signals are thought to give VP13/14 the capacity to shuttle mRNA between the nucleus and cytoplasm of infected cells (Donnelly et al., 2007). Early during infection, both VP8 and VP13/14 are localized in the nucleus (Donnelly & Elliott, 2001a, b; van Drunen Littel-van den Hurk et al., 1995). De novo-synthesized VP8 is also observed in the cytoplasm later during infection and in dense inclusions within the Golgi. VP8 has been shown to be an important tegument protein during the induction of humoral and cellular immune responses (van Drunen Littel-van den Hurk et al., 1995).

Studies of HSV-1 have demonstrated the importance of phosphorylation of tegument proteins for dissociation of the tegument structural components (Morrison et al., 1998). Phosphorylation of VP13/14 plays an important role in this respect, and is thought to be mediated by both cellular and virion-associated kinases (Lemaster & Roizman, 1980; Meredith et al., 1991). VP13/14 appears to be phosphorylated by the cellular casein kinase 2 (CK2) and, to a lesser extent, by protein kinase A and protein kinase C (PKC), whereas the virion-associated kinase activity is unclear (Morrison et al., 1998). Phosphorylation may also play a role in primary infection, subcellular trafficking and tegumentation. For example, in varicella-zoster virus, a major transcriptional regulatory protein, IE62, is profoundly influenced in its cellular localization as a result of phosphorylation by the open reading frame (ORF) 66 protein kinase (Eisfeld et al., 2006). In this case, the cytoplasmic form of IE62 is critical if it is to be included in the packaging of the virus as an abundant tegument protein.

The BoHV-1 US3 gene product (Takashima et al., 1999) is a 58 kDa serine/threonine protein kinase, the function of which remains largely unknown, both in vitro and in vivo. Contrary to the HSV homologue (Daikoku et al., 1993; Frame et al., 1987; Leopardi et al., 1997; Purves et al., 1987), BoHV-1 US3 does not appear to be a critical component for blocking apoptosis in infected cells (Takashima et al., 1999). Both HSV-1 and BoHV-1 US3 deletion mutants grow well in cell culture (Purves et al., 1987; Takashima et al., 1999).

Although VP8 of BoHV-1 is clearly phosphorylated, to our knowledge the kinases responsible have not been established. Identifying the kinases involved is a critical step towards understanding the importance and role of VP8 during the infection process, including release of this major tegument protein from the virion, trafficking within the cell and tegumentation. Our approach to identifying kinases interacting with VP8 from BoHV-1 was to immunoprecipitate FLAG-tagged VP8 from BoHV-1-infected cells and to identify co-immunoprecipitating proteins by mass spectroscopy. With this strategy, the viral kinase US3 and the well-known cellular protein kinase CK2 were identified, both of which were found to be capable of phosphorylating VP8.

Methods

Cells and viruses.. Madin–Darby bovine kidney (MDBK) cells, fetal bovine testicular (FBT) cells and Cos-7 cells were cultured in Dulbecco's modified Eagle medium (DMEM; Gibco-BRL) supplemented with 10 mM HEPES, 50 µg gentamicin (Gibco) ml^–1 and 10 % gamma-irradiated fetal bovine serum (FBS; SeraCare Life Sciences), unless otherwise stated. BoHV-1 strain 108 and BoHV-1–GFP–UL47, which expresses VP8 as a green fluorescent protein (GFP) fusion, were propagated in MDBK cells (Misra et al., 1983). Virus infections were accomplished by rocking 150 cm² 85–90 % confluent cell monolayers with virus in 5 ml DMEM supplemented with 5 % FBS for 1 h at 37 °C, followed by incubation with an additional 10 ml DMEM supplemented with 5 % FBS.

Antibodies.. Polyclonal antibodies specific for US3 were prepared using the synthetic peptide MERAAERLARQRARGLWRSRFACCVAA, which corresponds to the N-terminal 27 aa of US3. Rabbits were injected with 0.5 mg keyhole limpet haemocyanin (KLH)-conjugated peptide in Freund's complete adjuvant (Sigma). Two subsequent 0.3 mg booster immunizations, alternately with ovalbumin–peptide and KLH–peptide conjugates in Freund's incomplete adjuvant (Sigma), were performed at 21-day intervals. Serum was collected and tested 10 days after the last injection. The production of VP8-specific polyclonal antibodies has been described previously (van Drunen Littel-van den Hurk et al., 1995). Monoclonal anti-casein kinase 2β clone 6D5 (anti-CK2) and monoclonal anti-haemagglutinin (HA) clone HA-7 (anti-HA) were obtained from Sigma. The procedure involving animals was performed in accordance with the guidelines of the Canadian Council for Animal Care.

Gene isolation and cloning.. A cDNA library was constructed from BoHV-1-infected MDBK cells. Briefly, a nearly confluent monolayer of MDBK cells was infected with BoHV-1 at an m.o.i. of 10. After 7 h, total RNA was collected using 1 ml TRIzol Reagent (Invitrogen) according to the supplier's instructions. The resulting RNA had a 28S : 18S ratio of 1.7. The RNA was treated with amplification-grade DNase I [1 U (µg RNA)^–1; Invitrogen] in the supplied buffer for 15 min at room temperature, followed by incubation with 2.5 mM EDTA at 65 °C for 10 min. An Omniscript kit (Qiagen) was used for cDNA synthesis. Briefly, 2 µg RNA was added to 0.5 mM each dNTP, 0.5 µg oligo(dT) (Invitrogen), 20 U RNaseOut RNase inhibitor (Invitrogen), 8 U Omniscript reverse transcriptase and Buffer RT in a final volume of 40 µl, incubated at 37 °C for 1 h and heat-inactivated at 93 °C for 5 min. The resulting cDNA was stored at –80 °C.

Based on the GenBank sequence for BoHV-1 (GenBank accession no. NC_001847[GenBank] ), the gene encoding the US3 protein (NP_045368[GenBank] ) was amplified from the cDNA library by PCR (HotStar HiFidelity Polymerase kit; Qiagen). The forward primer incorporated an NheI restriction endonuclease site (5'-TAGCTAGCACGACCCGACGTTCTTG-3') and the reverse primer an EcoRV site (5'-CAGATATCAGAGGCCGCACCGAAGA-3') (restriction sites in italics). The PCR product was cloned into the NheI and EcoRV sites of a pcDNA3.1 vector (Invitrogen) modified to contain a double HA tag (Zhu et al., 2009) in frame with the double HA tag at the 3' end. The resulting plasmid (pUS3-HA) was sequenced to confirm the correct incorporation. We noted a minor difference with the U_S3 sequence in GenBank (a composite of strains K22, Cooper, P8-2, 34 and Jura), as bases 319, 324, 325 and 327 are guanine instead of adenine in strain 108. With the exception of two lysines at positions 107 and 109 that were glutamates, these base substitutions did not alter the amino acid sequence of the gene. The U_L47 gene was derived from a VP8–yellow fluorescent protein plasmid described previously (Zheng et al., 2004) and subcloned into the EcoRV site of pFLAG-CMV-2 (Sigma), in frame with the gene encoding the FLAG peptide at the 5' end. The correct sequence of the resulting plasmid (pFLAG-VP8) was confirmed.

Preparation of cell lysates.. Cos-7 or FBT cells in 150 cm² monolayers at approximately 85–90 % confluence were transfected with 16 µg pFLAG-VP8 or pUS3-HA using Plus Reagent and Lipofectamine in Opti-MEM (all from Invitrogen) according to the manufacturer's instructions and incubated for 48 h in DMEM. Cells were washed twice with PBS (pH 7.3) and harvested in 2 ml lysis buffer [150 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 50 mM Tris/HCl (pH 7.4)] supplemented with Protease Inhibitor Cocktail for use with mammalian cell and tissue extracts (Sigma) at the recommended concentration. After gently rocking for 2–3 min, cell lysates were collected on ice and stored at –20 °C until use. To prepare BoHV-1-infected FBT cells, infection was initiated 10 h prior to cell lysis. Cell lysates were cleared by centrifugation at 1600 g for 10 min at 4 °C prior to use.

Mass spectroscopy (MS).. Anti-FLAG M2 affinity gel (Sigma) was incubated with cell lysate (40 µl gel per 5 ml lysate) overnight at 4 °C and washed three times with 1 ml wash buffer [150 mM NaCl, 50 mM Tris/HCl (pH 7.4)] according to the manufacturer's instructions. Bound proteins were eluted with 100 µl SDS-PAGE sample buffer and boiled. Subsequently, 50 µl of each sample was analysed by SDS-PAGE (8 % gel). The gels were stained with Coomassie Brilliant Blue R250 in 40 % methanol, 10 % acetic acid. Proteins of interest were prepared for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS using an in-gel protease digestion protocol (method according to the Columbia Proteomics Center at the University of Missouri, ). Briefly, protein bands were excised, washed, destained in wash solution (50 mM ammonium bicarbonate in 50 % acetonitrile), dehydrated in 100 % acetonitrile and dried completely using a centrifugal evaporator. Subsequently, the gel pieces were rehydrated in 150 µl reduction solution (10 mM dithiothreitol, 100 mM ammonium bicarbonate) for 30 min at 56 °C, followed by 100 µl alkylation solution (50 mM iodoacetamide, 100 mM ammonium bicarbonate) for 30 min at room temperature and then washed with wash solution, dehydrated in 100 % acetonitrile and dried in a centrifugal evaporator. The gel pieces were rehydrated in a minimal volume of 50 mM ammonium bicarbonate containing modified proteomics-grade porcine trypsin (20 µg ml^–1; Sigma) and incubated overnight at 37 °C. Two extractions were performed with 50 µl extraction solution (60 % acetonitrile, 1 % trifluoroacetic acid) with agitation by vortexing, for 10 min each. Extracts were combined and dried by centrifugal evaporation to near dryness and resuspended in 5 µl resuspension solution (50 % acetonitrile, 0.1 % trifluoroacetic acid). Samples were submitted to the National Research Council of Canada/Plant Biotechnology Institute (Saskatoon, Canada) for analysis of the tryptic peptide molecular masses by MALDI-TOF MS on a Voyager DE-STR instrument (Applied Biosystems). The data were searched against a comprehensive non-redundant protein sequence (NCBInr) database (February 2007) using Mascot (Perkins et al., 1999).

Immunoprecipitation and Western blot analysis.. Anti-FLAG M2 affinity gel or monoclonal anti-HA–agarose conjugate (clone HA-7; Sigma) was incubated with cell lysate (40 µl resin per 4 ml Cos-7 lysate or 10 ml FBT lysate) overnight at 4 °C and washed three times with 1 ml wash buffer [150 mM NaCl, 50 mM Tris/HCl (pH 7.4)]. Bound proteins were eluted by boiling in 100 µl SDS-PAGE sample buffer for 5 min. In each experiment, 20 µl of each sample was analysed by SDS-PAGE (10 % gel) and the proteins were transferred to nitrocellulose membranes and incubated with the appropriate antibodies at a 1 : 2000 dilution. After washing, the membranes were incubated with 1 : 2000-diluted alkaline phosphatase-labelled goat anti-rabbit IgG or goat anti-mouse IgG (KPL). Western blots were developed with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (Sigma). In each experiment, samples were treated identically, and equal loading was verified by detection of antibody heavy chains by Coomassie blue staining or in the Western blots.

Microscopy.. MDBK or FBT cells were plated on Permanox 2 chamber slides (Lab-Tek) in DMEM supplemented with 5 % FBS. The next day, when the cells were approximately 90 % confluent, they were infected with BoHV-1–GFP–UL47 at an m.o.i. of 0.6 in DMEM supplemented with 1 % FBS. After 7 h incubation at 37 °C, the cells were washed three times with PBS, incubated with 10 % buffered neutral formalin (VWR) for 30 min and then washed again three times with PBS. Subsequently, the cells were permeabilized with ice-cold acetone for 2 min at –20 °C. After washing again with PBS, the fixed cells were incubated with 5 % normal goat serum in PBS overnight at 4 °C. Cells were incubated with anti-US3 rabbit serum at a dilution of 1 : 1000 for 2 h, and then with Alexa Fluor 594-labelled goat anti-rabbit IgG (Invitrogen) at a dilution of 1 : 500 for 1 h at room temperature. Finally, slides were washed with PBS, stained with 4',6-diamidino-2-phenylindole (DAPI; 2 µl ml^–1), washed in deionized water for 10 min, air dried and mounted using Vectashield HardSet Mounting Medium (Vector Laboratories) for fluorescence.

Kinase assays.. Cell lysates were prepared from Cos-7 cells transfected with pFLAG-VP8 or pUS3-HA, or from untransfected Cos-7 cells. Twenty microlitres of anti-FLAG M2 affinity gel or anti-HA–agarose conjugate was washed according to the manufacturer's instructions and incubated overnight at 4 °C with 1 ml of the appropriate lysate. If replicate samples were required, incubations as well as the subsequent washes were all performed as one batch, and the beads were then split equally for the actual assay to ensure that all samples were identical. The beads were washed at least five times with 1 ml wash buffer [50 mM Tris/HCl, 150 mM NaCl (pH 7.4)].

Kinase assays were performed largely as described elsewhere (Takashima et al., 1999). Briefly, 5 µl [γ-³²P]ATP [0.3 µCi (11.1 kBq) µl^–1; Perkin Elmer] was added to the substrate mixture, which consisted of 20 µl FLAG–VP8 on anti-FLAG M2 affinity gel or 20 µl ddH₂O (Gibco), 5 µl 10x kinase buffer [500 mM MgCl₂, 1 % Triton X-100, 10 mM 2-mercaptoethanol, 500 mM Tris/HCl (pH 8.0)] and 1 µl 3x FLAG peptide (Sigma), with ddH₂O to a total volume of 40 µl. The substrate mixture was then added to the US3–HA bound to 20 µl anti-HA–agarose conjugate and incubated at room temperature for 30 min. The 3x FLAG peptide was included in all samples whether FLAG–VP8 was present or not. In some cases, the following protein kinase inhibitors were included: InSolution Casein Kinase II Inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT; 10 µM), InSolution Casein Kinase I Inhibitor D4476 (20 µM), Ro-32-0432 (10 µM) or kenpaullone (50 µM) (all from Calbiochem). After incubation, the samples were boiled with 70 µl SDS-PAGE sample buffer, loaded equally and separated by SDS-PAGE (10 % gel). The gels were then dried and exposed to Imaging Screen-K for visualization on a Molecular Imager FX (Bio-Rad).

Virus growth properties.. To examine the effect of treatment with CK2 inhibitor on virus replication, MDBK cells were infected with BoHV-1–GFP–UL47 at an m.o.i. of 0.6 or 0.01. After 1 h incubation, the medium was replaced with fresh medium in the absence or presence of 40 µM DMAT or 50 µM kenpaullone. The progression of the infection at an m.o.i. of 0.6 was monitored by phase-contrast and fluorescent microscopy. Furthermore, cells were collected every 4 h for 16 h and at 24 and 48 h from the cells infected at an m.o.i. of 0.01. The virus was released from the cells by two freeze–thaw cycles. All samples were clarified by low-speed centrifugation and stored at –80 °C prior to determination of the infectious titre by plaque assay on MDBK cells.

Results

Identification of proteins co-precipitated with VP8 by MS
In order to establish whether protein kinases interact with VP8, FLAG–VP8 was immunoprecipitated from FBT cells transfected with pFLAG-VP8 and then infected with BoHV-1. FBT cells were chosen as they can both efficiently be transfected and infected with BoHV-1. After transfection with FLAG–VP8 and infection with BoHV-1, FBT cell lysates were generated and incubated with anti-FLAG M2 affinity gel. Bound proteins were separated by SDS-PAGE (Fig. 1). Several well-resolved differences were observed in the Coomassie blue-stained gels between uninfected and infected lysates, presumably corresponding to BoHV-1 proteins that interact with VP8. In total, 49 bands of interest were excised. The gel pieces were treated according to an in-gel trypsin digestion protocol, and the peptides were extracted and analysed by MALDI-TOF MS. As controls, the most intense band corresponding to BoHV-1 VP8 (Mascot score=219), as well as the heavy chain from the anti-FLAG mouse antibody (C_H; Mascot score=84), were positively identified. The database queries also identified two of the processed bands as containing tryptic peptides corresponding to two protein kinases: the BoHV-1 US3 serine/threonine protein kinase (Mascot score=178) and the cellular CK2 (Mascot score=189). In both cases, the Mascot scores were very high, indicating a positive match.

(56K): Fig. 1. Proteins co-immunoprecipitated with VP8. Lysates from FBT cells, BoHV-1-infected FBT cells, FBT cells transfected with pFLAG-VP8 or FBT cells transfected with pFLAG-VP8 and subsequently infected with BoHV-1 were incubated with anti-FLAG M2 affinity gel, and bound proteins were separated by SDS-PAGE (8 % gel) to detect co-immunoprecipitated proteins. The most prominent band was positively identified as VP8, as well as CH, the heavy chain from the affinity gel. The two arrows indicate the bands that were identified as CK2+β-actin (190 kDa) and US3 (58 kDa). Based on apparent molecular mass, the lower band is probably the light chain (CL) from the antibody of the affinity gel.

The masses of the tryptic peptides and sequences of matching peptides from the database queries for US3 and CK2 are shown in Supplementary Tables S1 and S2 (available in JGV Online). Several matches to CK2 from various species were observed, including mouse (Mascot score=114), rat (Mascot score=114) and human (Mascot score=121), which is not surprising as CK2 is highly conserved among mammals (Russo et al., 2004). The highest score (189) was obtained with a combination of CK2 and β-actin. This is also reflective of the apparent 190 kDa molecular mass of the analysed band. CK2 is a multimeric enzyme with a total molecular mass of approximately 150 kDa, whilst β-actin is a highly conserved protein with an apparent molecular mass of approximately 42 kDa. Additional putative binding partners identified by MALDI-TOF were BoHV-1 ICP4 and gL, as well as β-actin, eTIF3 and damage-specific DNA-binding protein 1.

Expression of US3–HA
To study further the interactions between VP8 and US3, we constructed the vector pUS3-HA and raised US3-specific rabbit serum. A protein band at approximately 60 kDa was detected by Western blotting in lysate from Cos-7 cells transfected with pUS3-HA (Fig. 2). The apparent molecular mass of 60 kDa corresponded to the expected molecular mass of HA-tagged US3. Furthermore, a protein at approximately 58 kDa was detected in lysate from BoHV-1-infected FBT cells, corresponding to the expected molecular mass of US3. The US3-specific serum used for detection did not react with any other cellular or viral proteins, confirming the specificity of this antiserum. These results demonstrated expression and detection of US3 and US3–HA in BoHV-1 infected FBT cells and pUS3-HA-transfected Cos-7 cells, respectively.

(12K): Fig. 2. Expression of US3 and US3–HA. Equal amounts (20 µl) of cell lysate from uninfected FBT cells (lane 1), FBT cells infected with BoHV-1 (lane 2) or Cos-7 cells transfected with pUS3-HA (lane 3) were analysed by Western blotting to reveal bands of the expected molecular masses. The protein band in lane 2 corresponds to US3 and the protein band in lane 3 to US3–HA. Molecular mass markers are shown on the left.

Confirmation of the interactions of VP8 with US3 and CK2 by co-immunoprecipitation
To verify the interaction of VP8 with US3, we first showed that VP8 is co-immunoprecipitated with US3–HA from the lysate of FBT cells that were transfected with pUS3-HA followed by infection with BoHV-1 (Fig. 3a). No VP8 was observed to co-immunoprecipitate with the anti-HA resin from BoHV-1-infected cells without US3–HA present. When FLAG–VP8 and US3–HA were co-expressed in Cos-7 cells, an interaction between the proteins was also clearly observed (Fig. 3b). No interactions were detected between US3–HA and the anti-FLAG resin, nor between FLAG–VP8 and the anti-HA resin. The interaction between VP8 and US3 therefore appeared to be specific. The interaction between VP8 and protein kinase CK2 was confirmed by co-immunoprecipitation of lysates from Cos-7 cells transfected with pFLAG-VP8 (Fig. 3c). In this case, the CK2 β subunit was observed as a band immediately above the light chain of the anti-FLAG antibody (C_L). No interaction between the anti-FLAG resin and CK2 was detected.

(26K): Fig. 3. Interaction of VP8 with US3 and CK2 in BoHV-1-infected and transiently transfected cells. (a) Lysates of BoHV-1-infected FBT cells, pUS3-HA-transfected FBT cells, or pUS3-HA-transfected and BoHV-1-infected FBT cells were incubated with anti-HA agarose conjugate, washed identically and analysed by Western blotting. (b) Lysates of Cos-7 cells transfected with FLAG–VP8 and/or US3–HA were incubated with anti-FLAG M2 affinity gel or anti-HA agarose conjugate, washed identically and analysed by Western blotting. (c) Anti-FLAG M2 affinity gel was used for immunoprecipitation of proteins from Cos-7 cells (lane 1) or Cos-7 cells transfected with pFLAG-VP8 (lane 2). VP8 and FLAG–VP8 were identified with a polyclonal antibody specific for VP8. US3–HA was identified with a monoclonal antibody specific for the HA tag. CK2 was identified with monoclonal antibody against CK2β. Based on apparent molecular mass, the lower band is probably the light chain (CL) from the antibody of the affinity gel.

Subcellular localization of US3 and co-localization with VP8
It is known that CK2 is highly conserved across species and is expressed in the cell nucleus (Krek et al., 1992), whilst VP8 from BoHV-1 is also localized in the nucleus at early time points after infection (van Drunen Littel-van den Hurk et al., 1995), which would allow interactions between these proteins to occur. To support the evidence for US3 and VP8 interactions, we verified that US3 from BoHV-1 is also present in the nucleus during infection. MDBK or FBT cells were infected with BoHV-1–GFP–UL47. After 7 h, the cells were fixed and examined by immunofluorescence using US3-specific rabbit serum (Fig. 4). Both US3 and VP8 were observed predominantly within the nuclei, and this was confirmed by DAPI staining. The nuclear localization of BoHV-1 US3 was generally consistent with that of HSV-2 US3, both after transient expression and during viral infection (Goshima et al., 1998).

(51K): Fig. 4. Co-localization of VP8 and US3 in MDBK (a) or FBT (b) cells infected with BoHV-1–GFP–UL47. Cells were infected with BoHV-1–GFP–UL47 at an m.o.i. of 0.6 for 7 h, fixed with formalin and permeabilized with acetone prior to immunostaining with US3-specific rabbit serum. GFP–VP8 (green), US3 (red) and the overlay (merge, yellow) are shown. Nuclei were counterstained with DAPI.

Phosphorylation of VP8 by CK2 and US3
As VP8, US3 and CK2 are all present in the nucleus, and VP8 interacts with both US3 and CK2, we considered it likely that the kinases might phosphorylate VP8. When VP8 from lysates of Cos-7 cells transfected with pFLAG-VP8 was analysed using a kinase assay, obvious phosphorylation was observed (Fig. 5a). This phosphorylation was almost completely eliminated when 10 µM DMAT, a selective inhibitor of protein kinase CK2 (Pagano et al., 2004), was included in the assay. This confirmed that CK2 remains bound to FLAG–VP8 after washing the anti-FLAG M2 affinity gel using standard conditions. Upon increasing the salt concentration of the buffer, phosphorylation of VP8 by CK2 was reduced but not prevented, even at 100 mM Tris/HCl and 300 mM NaCl (Fig. 5b). Phosphorylation of VP8 was also inhibited by kenpaullone, a less specific CK2 inhibitor (Zaharevitz et al., 1999), whilst VP8 phosphorylation was not inhibited by D4476, a CK1 inhibitor (Rena et al., 2004), nor by Ro-32-0432, a PKC inhibitor (Birchall et al., 1994) (Fig. 5a). These results were highly supportive of our hypothesis that CK2 interacts strongly with VP8 and is capable of phosphorylating this protein.

(26K): Fig. 5. Phosphorylation of VP8 by cellular CK2 and viral US3 protein kinases. (a) A kinase assay was performed with FLAG–VP8, in the absence or presence of 10 µM DMAT, 20 µM D4476, 10 µM Ro-32-0432 or 50 µM kenpaullone. (b) FLAG–VP8 phosphorylation without kinase inhibitors with increasing stringency of the wash conditions: 50 mM Tris/HCl and 150 mM NaCl, 75 mM Tris/HCl and 225 mM NaCl, and 100 mM Tris/HCl and 300 mM NaCl (left to right). (c) A kinase assay was performed with FLAG–VP8, FLAG–VP8+DMAT, FLAG–VP8+DMAT+US3–HA, US3–HA+DMAT or US3–HA, as indicated. Proteins were separated by SDS-PAGE (10 % gel) before detection of γ-32P by exposure to an imaging screen for 30 min.

Subsequently, we included US3 in the assay to assess its ability to phosphorylate VP8 (Fig. 5c). Again, DMAT prevented phosphorylation of VP8 by CK2. Furthermore, when US3–HA was added together with the CK2 inhibitor DMAT, strong phosphorylation of VP8 was again observed. In addition, US3 demonstrated the same autophosphorylation reported by others (Takashima et al., 1999), confirming that the US3–HA construct retains its kinase activity. The autophosphorylation was not inhibited by the presence of DMAT in the assay, further suggesting that DMAT does not inhibit US3 kinase activity.

Consensus sequences for CK2 and US3 in VP8
The minimal consensus sequence required by US3 for recognition and phosphorylation of substrates is (R)_n-X-(S/T)-Y-Y, where n must be at least two residues and X may be absent or any residue, with a preference for arginine, alanine, valine, proline or serine. Y is similar to X but cannot be absent and cannot be aspartate, glutamate or proline. The optimal sequence has n equal to at least three and X present (Leader et al., 1991; Purves et al., 1986). Upon examination of the VP8 sequence (GenBank accession no. AY530215[GenBank] ), we detected four of these basic regions where the consensus holds (Table 1). They all have n=2 and the site beginning at residue 629 is especially favourable as it has X as valine as well as the Y-Y segment consisting of the favourable residues valine and arginine. Thus, several possible US3 phosphorylation sites exist on VP8, further supporting our results. To our knowledge, the viral kinase responsible for phosphorylation of VP13/14 has not been identified. However, we also note that several such consensus sites exist on VP13/14 from HSV-1 (GenBank accession no. NP_044649[GenBank] ), with an especially strong one beginning at residue 9 (RRRRASTR) (Table 1), which suggests that US3 may play a role in phosphorylation of VP13/14 as well.

Table 1. Prediction of CK2 and US3 phosphorylation sites on VP8 from BoHV-1 and on VP13/14 from HSV-1 The GenBank accession numbers of the sequences examined are AY530215 (VP8 of BoHV-1) and NP_044649 (VP13/14 of HSV-1).

The recognition site for CK2 phosphorylation is more complex (Meggio & Pinna, 2003; Pinna, 2002). It can be summarized as (E/D/X)-(S/T)-(D/E/X')-(E/D/X)-(E/D)-(E/D/X), where X can be any residue except basic residues and X' is similar to X but also cannot be proline. In general, the phospho-acceptor is serine or threonine and the third residue after the phospho-acceptor must be the acidic aspartate or glutamate. There is also a strong preference for more than one acidic residue in the site. We note that, in the VP8 sequence, there are two such strong motifs present. The first, starting at residue 64, contains four acidic residues and the second, starting at residue 81, contains three, making them the most probable locations of phosphorylation by CK2. Similarly, we identified two strong motifs in HSV-1 VP13/14, starting at residues 46 and 311 (Table 1).

Inhibition of CK2 phosphorylation during transient expression of VP8 and BoHV-1 infection
There is evidence for some tegument proteins that phosphorylation may influence their nuclear localization and/or functional properties. To determine the effect of CK2 phosphorylation on the subcellular localization of VP8, Cos-7 cells were transfected with a plasmid encoding GFP–VP8 (pGFP-VP8). The pGFP-VP8-transfected cells were either left untreated or treated with DMAT or kenpaullone immediately after transfection. As shown in Fig. 6, even at high concentrations of the CK2 inhibitors where toxic effects on the cells started to be observed, VP8 was localized in the nucleus, suggesting that CK2 phosphorylation is not a requirement for nuclear transport of VP8. The results were identical at lower concentrations of inhibitors (<50 µM DMAT or ≤50 µM kenpaullone; data not shown). In order to test a potential effect of CK2 phosphorylation of VP8 on virus replication, MDBK cells were infected with BoHV-1–GFP–UL47, and 1 h later DMAT or kenpaullone was added. In the absence of inhibitors the infection progressed as expected leading to rounding of the cells by 12 h; however, in the presence of the CK2 inhibitor DMAT and even more so with the less specific kinase inhibitor kenpaullone, the infection progressed significantly more slowly (Fig. 7a). Furthermore, the amounts of GFP–VP8 were notably reduced (Fig. 7b) and virus titres were significantly lower in inhibitor-treated BoHV-1-infected cells (Fig. 7c).

(69K): Fig. 6. Effect of CK2-mediated phosphorylation on subcellular localization of VP8. Cos-7 cells were transfected with pGFP-VP8 and either left untreated or treated immediately with 100 µM DMAT or 100 µM kenpaullone. Cells were observed live by fluorescent (left panels) and light (right panels) microscopy, and images were captured 24 h after transfection.

(73K): Fig. 7. Effect of CK2 inhibition on BoHV-1 infection. MDBK cells were infected with BoHV-1–GFP–UL47 at an m.o.i. of 0.6 (a, b) or 0.01 (c), and 1 h later 40 µM DMAT or 50 µM kenpaullone was added. BoHV-1–GFP–UL47-infected cells were harvested every 4 h up to 16 h and at 24 and 48 h p.i. (a, b) Phase-contrast (a) and fluorescent (b) images captured at different time points after infection. (c) Viral titres. The titre of infectious virus progeny in each sample was determined by plaque assay on MDBK cells.

Discussion

The results described here suggest that BoHV-1 VP8 interacts with both the viral protein kinase US3 and the cellular protein kinase CK2. This was first demonstrated by MALDI-TOF MS of protein bands co-immunoprecipitating with FLAG–VP8 from BoHV-1-infected cells, and then confirmed in co-immunoprecipitation experiments involving BoHV-1-infected cells and cells transfected with plasmids encoding VP8 or US3 and detection by Western blotting. Furthermore, VP8, US3 and CK2 are all present in the nucleus. Finally, both US3 and CK2 were capable of phosphorylating VP8 in in vitro kinase assays. We speculate that US3, being associated with the virions, may be more important during the initial release of the tegument into the cytoplasm of the cell, whereas CK2 may play a more prominent role in phosphorylating VP8 after release of VP8 from virions.

We believe it is the phosphorylation of VP8 by US3 that accounts for the band of approximately 95 kDa observed by others in lysates of BoHV-1-infected MDBK cells using a similar kinase assay with non-tagged US3 (Takashima et al., 1999). Our results are also consistent with those reported by Morrison et al. (1998) who demonstrated that HSV-1 VP13/14 is phosphorylated by CK2. Although in this study a viral kinase was also shown to be involved in VP13/14 phosphorylation and necessary for release of VP13/14 from the virus particle, UL13, another HSV-1 protein kinase, was not responsible (Morrison et al., 1998), whilst the impact of US3 was not investigated. However, as US3 is present in both BoHV-1 and HSV-1 (Takashima et al., 1999; Zhang et al., 1990), it seems plausible that this viral kinase could play a role.

Given the critical role of protein phosphorylation at specific sites in processes within the cell, it is not surprising that VP8 would be phosphorylated by at least two kinases, each modifying its own unique site or set of sites. Of particular interest in this regard are the effects of phosphorylation on nuclear localization. VP8 localizes to the nucleus and is capable of shuttling between the nucleus and cytoplasm (Verhagen et al., 2006; Zheng et al., 2004). Four nuclear localization signals have been predicted for VP8 (NLS1–4), and NLS1 and NLS2 have been implicated in nuclear localization (Verhagen et al., 2006; Zheng et al., 2004). It is interesting that all four of the sequences proposed as potential US3 phosphorylation sites on VP8 fall within or upstream of these NLSs (¹¹RRPRR, ⁴⁸PRVRRPR, ¹⁶⁸PAQRARR and ⁶⁸¹PLAGKRR). In the case of NLS2, not only is there a potential US3 phosphorylation site immediately upstream, but a potential CK2 phosphorylation site is also located immediately downstream. It is conceivable that phosphorylation of one or perhaps different combinations of these regions could modulate the rate and level of nuclear localization. Phosphorylation-dependent enhancement or inhibition of nuclear localization mediated by NLSs has been demonstrated for a number of viral proteins (Alvisi et al., 2008). In some cases, the effect of phosphorylation can be so strong that modification at a single site can eliminate nuclear localization (Shen et al., 2008). However, treatment of pGFP-VP8-transfected cells with CK2 inhibitors did not influence the subcellular localization of VP8, which suggests that phosphorylation by CK2 is not critical. This was also observed for HSV-2 VP22, as phosphorylation by endogenous cellular kinases did not affect its subcellular localization (Geiss et al., 2004). In contrast, the CK2 inhibitor DMAT, and even more so the less specific inhibitor kenpaullone, reduced BoHV-1 replication, suggesting that CK2-mediated phosphorylation of VP8 and/or additional viral proteins is critical for progression of BoHV-1 infection. Although cellular proteins are also phosphorylated by CK2, the inhibitor concentrations used were not toxic to the cells. As putative consensus sites for phosphorylation of VP8 by CK2 or US3 were identified (Table 1), future studies will involve mutagenesis of these sequences in order to confirm that these sites are functional and to determine more precisely the effects of phosphorylation at these sites on the subcellular localization and functions of VP8, as well as BoHV-1 replication.

Phosphorylation by CK2 plays a role in the functional properties of a number of herpesvirus proteins. For example, HSV-1 ICP27 (Wadd et al., 1999), human herpesvirus 8 ORF57 (Malik & Clements, 2004) and Epstein–Barr virus EB2 (Cook et al., 1994) are phosphorylated by CK2. Phosphorylation of ORF57 by CK2 enhances its interaction with heterogeneous nuclear ribonucleoprotein K; this may then modulate ORF57-mediated regulation of viral gene expression (Malik & Clements, 2004). Recently, phosphorylation of Epstein–Barr virus EB2 by CK2 was shown to be needed for its ability to regulate production of infectious virus particles; this was correlated with reduced cytoplasmic accumulation of several late viral mRNAs (Medina-Palazon et al., 2007). US3 has a number of substrates in HSV-1, including ICP22, UL31, UL34, UL46, US9, gB and the host-cell proteins Bad, Emeri and lamin A/C; UL12 and the cellular proteins cytokeratin 17 and Bid are regarded as putative substrates (Kato et al., 2005, 2009; Leach et al., 2007; Matsuzaki et al., 2005; Mou et al., 2007; Wisner et al., 2009). Several functional studies on the role of US3 in phosphorylation of herpesvirus proteins have been performed. HSV-1 UL31 and UL34 form a complex at the inner nuclear membrane of infected cells, with US3 also present. Phosphorylation of UL31 by US3 plays an important regulatory role during primary envelopment of nucleocapsids (Mou et al., 2009), whereas phosphorylation of UL34 by US3 is not critical (Ryckman & Roller, 2004). Similarly, although US3 affects the intracellular localization of UL34, as well as pseudorabies virus morphogenesis, this appeared to be not correlated with phosphorylation of UL34 by US3 (Klupp et al., 2001). Furthermore, the stability of UL46 is dependent on US3-mediated phosphorylation (Matsuzaki et al., 2005). Recently, phosphorylation of gB was found to be required for fusion of gB with the outer nuclear membrane during viral egress (Wisner et al., 2009), and phosphorylation of gB by US3 downregulated gB cell-surface expression (Kato et al., 2009). Although BoHV-1 US3 was shown to phosphorylate several viral proteins (Takashima et al., 1999), the identities of these proteins and the functional significance have thus far not been studied.

In conclusion, our results indicate that VP8 from BoHV-1 associates with and is phosphorylated by the cellular protein kinase CK2. Furthermore, the viral protein kinase US3 also interacts with and has a profound effect on the phosphorylation state of VP8. This should prove to be a critical step in understanding the role of these kinases during the BoHV-1 infection process, as well as the ultimate effects of phosphorylation on VP8.

Acknowledgements

MS data were collected and analysed at the National Research Council of Canada/Plant Biotechnology Institute (NRC/PBI), Saskatoon. The authors wish to acknowledge the technical assistance and advice of Doug Olson (NRC/PBI), Marlene Snider, Vladislav Lobanov, Jennifer McIntosh, Scott Napper, Palok Aich, Sam Attah-Poku, Natalia Vasilenko and the animal support staff at VIDO. Funding was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR). S. L. L. is the recipient of a Saskatchewan Health Research Foundation post-doctoral fellowship. L. A. B. is the recipient of a Canada Research Chair in Vaccinology. Published as VIDO journal series no. 533.