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Research Article

Bovine herpesvirus-1 US3 protein kinase: critical residues and involvement in the phosphorylation of VP22

Shaunivan L. Labiuk, Vladislav Lobanov, Zoe Lawman, Marlene Snider, Lorne A. Babiuk, Sylvia van Drunen Littel-van den Hurk

  • 1Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada
  • 2University of Alberta, 3-7 University Hall, Edmonton, AB T6G 2J9, Canada
  • 3Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada
  • Correspondence
    Sylvia van Drunen Littel-van den Hurk
    sylvia.vandenhurk{at}usask.ca

    • Journal of General Virology 2010; 91(5):1117–1126 · https://doi.org/10.1099/vir.0.016600-0

    View at publisher PubMed

    Abstract

    The US3 gene product of bovine herpesvirus-1 (BoHV-1) is a protein kinase that is expressed early during infection and capable of autophosphorylation. By examining differentially labelled US3 moieties by co-immunoprecipitation, we demonstrated that the protein kinase interacts with itself in vitro, which supports autophosphorylation by US3. Based on its homology to other serine/threonine protein kinases, we defined two highly conserved lysines in US3, at position 195 within the ATP-binding pocket and at position 282 within the catalytic loop; altering either residue resulted in kinase-dead mutants, demonstrating that these two residues are critical for the catalytic activity of BoHV-1 US3. During immunoprecipitation experiments, US3 interacted weakly with VP22, another tegument protein of BoHV-1. Furthermore, VP22 co-localized with US3 inside the nucleus in BoHV-1-infected cells. In vitro kinase assays demonstrated that VP22 is phosphorylated not only by US3, but also by the cellular casein kinase 2 (CK2) protein. The selective CK2 protein kinase inhibitor, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and the less specific CK2 inhibitor Kenpaullone reduced VP22 phosphorylation, while CK1, protein kinase C or protein kinase A inhibitors did not affect phosphorylation. When US3 was included with VP22 in the kinase assay in the presence of DMAT, a low level of VP22 phosphorylation was observed. These data demonstrate that BoHV-1 VP22 interacts with both CK2 and US3, and that CK2 is the major kinase phosphorylating VP22, with US3 playing a minor role.

    • A supplementary table is available with the online version of this paper.

    INTRODUCTION

    Bovine herpesvirus-1 (BoHV-1) is an alphaherpesvirus that can cause a number of diseases in cattle, including infectious bovine rhinotracheitis, conjunctivitis, reproductive tract lesions, encephalitis and fetal infections (Jones & Chowdhury, 2007; Turin et al., 1999). Major economic losses occur when affected cattle suffer from milk drop, shipping fever, decreased body weights, abortions or death. The BoHV-1 virion consists of a double-stranded DNA genome that is encapsulated by a protein capsid and an envelope, with a complex of about 20 virus-encoded proteins, termed the tegument, between the latter two.

    US3 is a serine/threonine protein kinase expressed in cells upon infection with alphaherpesviruses, including BoHV-1 (Takashima et al., 1999). In BoHV-1, US3 is a 58 kDa protein with kinase activity, but the specific functions remain unknown. According to Takashima et al. (1999), BoHV-1 US3 deletion mutants grow well in cell culture. US3 does not appear to be a critical component in blocking apoptosis, making BoHV-1 US3 different from herpes simplex virus (HSV) US3 in this respect.

    HSV US3 protein kinase was identified earlier and investigated more thoroughly (Daikoku et al., 1993; Frame et al., 1987; Purves et al., 1987). It is known to be one of at least three protein kinases expressed by HSV and to block cellular apoptosis, as well as facilitate egress of nucleocapsids from the nucleus in HSV-1-infected cells (Leopardi et al., 1997; Reynolds et al., 2002). Although knowledge of the function of this kinase remains limited, certain interactions have come to light in recent years. Emerin, a nuclear membrane protein that interacts with lamin A/C, transcription regulators, actin, nesprins and BAF, is critical for maintaining membrane integrity and is hyperphosphorylated during infection in a manner that is modulated by the US3 and UL34 gene products (Leach et al., 2007; Leopardi et al., 1997; Morris et al., 2007). HSV ICP22, UL31, UL34, UL46, US9 and the host cell protein Bad have been identified as substrates of US3, while UL12 and the cellular proteins cytokeratin 17 and Bid are regarded as putative substrates (Kato et al., 2005; Matsuzaki et al., 2005). While HSV-2 US3 was shown to be distributed throughout the cell with more expression in the nucleus outside the context of infection, in infected cells US3 moved from a predominantly cytoplasmic localization at 4 h after infection to a predominantly nuclear localization at 8 h (Goshima et al., 1998). US3 was again observed in the cytoplasm at 12 h, presumably due to capsid association. Taken together, these results are consistent with the potential of US3 to affect a variety of proteins inside both the nucleus and the cytoplasm and demonstrate the possibility that different proteins may be affected at various time points during the course of HSV infection.

    VP22, the product of the UL49 gene, is one of the most abundant tegument proteins in herpesviruses. Although its function is not entirely clear, VP22 is able to traffic from infected or transfected cells to surrounding cells (Elliott & O'Hare, 1997; Harms et al., 2000; Lemken et al., 2007; Zheng et al., 2006). This property has been found useful for increasing the efficacy of DNA vaccines in mice (Hung et al., 2001; Kim et al., 2004; Zheng et al., 2006) and in cattle (Zheng et al., 2005). VP22 can also interact with HSV VP16, another tegument protein (Elliott et al., 1995), and in BoHV-1 is critical for virulence and the development of disease symptoms in cattle (Liang et al., 1997). VP22 has been implicated in the stabilization and acetylation of cellular microtubules (Elliott & O'Hare, 1998; Harms et al., 2000).

    The phosphorylation states of HSV-1 VP22 found at different stages of infection correspond to the protein's localization. VP22 from HSV-1 is primarily cytoplasmic early during infection but accumulates in the nucleus at later stages, the nuclear form being associated with the highest phosphorylation state (Elliott & O'Hare, 1999; Pomeranz & Blaho, 1999). When expressed outside the context of infection, HSV-1 VP22 is localized primarily to the cytoplasm but associates with chromatin during mitosis (Blouin & Blaho, 2001; Elliott & O'Hare, 2000). In this case, after incorporation in the nucleus it remains localized there. A punctate appearance in the cytoplasm is sometimes observed, which may be due to the association with acidic compartments derived from the Golgi apparatus (Brignati et al., 2003). In BoHV-1, the localization patterns are similar to those of HSV-1 (Lobanov et al., 2009), although qualitative differences in chromatin, nuclear and microtubule association have been noted (Harms et al., 2000). Initial experiments involving the UL13 protein kinase of HSV-1 implicated it as a putative viral factor involved in the phosphorylation of VP22 (Coulter et al., 1993). Subsequent studies demonstrated that cellular casein kinase 2 (CK2) is the major kinase responsible for VP22 phosphorylation (Elliott et al., 1996, 1999), but that UL13 is also involved, both in HSV-1 and HSV-2 (Geiss et al., 2001; Morrison et al., 1998). Recently, UL13 was confirmed to directly phosphorylate VP22 in HSV-1 (Asai et al., 2007). However, in co-transfected Vero cells, US3 from HSV-2 did not appear to be responsible for phosphorylating VP22 (Geiss et al., 2001). This, however, was not confirmed with kinase assays.

    In order to characterize the functions of US3 in BoHV-1 infection, we examined its autophosphorylation and the effects of various protein kinase inhibitors. We defined regions critical to the kinase activity of this protein and identified two lysine residues which, when altered, resulted in kinase-dead US3 mutants. Furthermore, we observed interactions between US3 and VP22, which suggested that VP22, one of the most abundant tegument proteins, might be phosphorylated by US3. Although BoHV-1 VP22 was phosphorylated to some extent by US3, the kinase predominantly phosphorylating VP22 was found to be CK2.

    RESULTS

    BoHV-1 US3 autophosphorylation and kinase inhibitors

    When the kinase activity of BoHV-1 US3 was previously examined, autophosphorylation of the US3 kinase itself was observed (Takashima et al., 1999). In order to demonstrate that US3 proteins are interacting to cause this phosphorylation, we performed a co-immunoprecipitation assay using differentially labelled US3 moieties. US3-enhanced cyan fluorescent protein (ECFP) co-immunoprecipitated with US3–haemagglutinin (HA) from lysates of Cos-7 cells transfected with both pUS3–HA and pUS3–ECFP (Fig. 1a⇓). Similarly, US3–HA was co-immunoprecipitated by US3–ECFP. No interaction between US3–ECFP and the anti-HA resin was observed, and only low background interaction was observed between US3–HA and anti-green fluorescent protein (GFP)-protein A–Sepharose. Furthermore, when the assay was repeated using pECFP instead of pUS3–ECFP, no interaction was detected between US3–HA and ECFP (Fig. 1b⇓). The interaction between differentially labelled US3 proteins therefore appears to be specific.

    Figure image not available in archive
    Fig. 1.

    Self-interaction and autophosphorylation of US3. (a) Lysates of Cos-7 cells transfected with pUS3–HA, pUS3–ECFP or both were incubated with anti-HA agarose or anti-GFP polyclonal antibody and protein A–Sepharose, washed identically and analysed by Western blotting. (b) Lysates of Cos-7 cells transfected with pUS3–HA, pECFP or both were incubated with anti-HA agarose or anti-GFP polyclonal antibody and protein A–Sepharose, washed identically and analysed by Western blotting. US3–ECFP and ECFP were identified by using GFP-specific polyclonal antibody and US3–HA was identified with monoclonal antibody specific for the HA tag. (c) A kinase assay was performed with US3–HA from lysates of Cos-7 cells transfected with pUS3–HA in the absence of inhibitor or in the presence of 10 μM DMAT, 20 μM D4476, 10 μM Ro-32-0432 or 50 μM Kenpaullone. (d) A kinase assay was performed with US3–HA from lysates of Cos-7 cells transfected with pUS3–HA in the absence of inhibitor or in the presence of Kenpaullone at 50, 100 or 150 μM. Proteins were separated on a 10 % SDS-PAGE gel before the detection of γ-32P by exposure to an imaging screen for 30 min.

    US3–HA purified from lysates of Cos-7 cells transfected with pUS3–HA was used in the kinase assay to show that it maintains its kinase activity and to assess the effects of various kinase inhibitors (Fig. 1c⇑). Upon examining the autoradiograph, we observed a single band corresponding to phosphorylated US3–HA, similar to that reported by others (Takashima et al., 1999). Autophosphorylation did not appear to be inhibited by 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), a selective protein kinase CK2 inhibitor (Pagano et al., 2004), nor by D4476, a CK1 inhibitor (Rena et al., 2004) nor Ro-32-0432, a PKC inhibitor (Birchall et al., 1994). There was a low degree of inhibition by Kenpaullone, a less specific kinase inhibitor which also inhibits CK2 (Zaharevitz et al., 1999). Kenpaullone, however, did not eliminate autophosphorylation of US3 even at concentrations of 150 μM (Fig. 1d⇑).

    K195 and K282 are critical for the kinase activity of BoHV-1 US3

    In order to determine which residues within BoHV-1 US3 are critical for its kinase activity, we compared the sequence to those of other serine/threonine protein kinases. By submitting the BoHV-1 US3 sequence to the NCBI Conserved Domain Search Tool (Marchler-Bauer & Bryant, 2004), we obtained alignments showing that the catalytic loop and ATP-binding pocket regions of other serine/threonine protein kinases are conserved (Fig. 2a⇓). The positively charged residues K282 in the catalytic loop and K195 in the ATP-binding pocket appeared to be highly conserved within these critical regions. The protein sequence for US3 from BoHV-1 was subsequently compared to that of HSV-1 using blastp 2.2.18. There was significant correlation between the C-terminal two-thirds of the protein sequences (Fig. 2b⇓). Residue K195 of BoHV-1 US3 appeared to be equivalent to residue K220 of HSV-1, which has been mutated to create kinase-dead mutants by others (Kato et al., 2008), while K282 of BoHV-1 appeared equivalent to K307 of HSV-1. Site-directed mutagenesis on the pUS3–HA plasmid allowed us to obtain four mutants: K195E, K282A, K282E and K282M. Each of these mutants was verified by sequence analysis.

    Figure image not available in archive
    Fig. 2.

    Mutagenesis of US3 from BoHV-1. (a) The amino acid sequence of US3 (NP_045368) was used as input to the NCBI Conserved Domain Search Tool which compared it to other known serine/threonine protein kinases. Using the default parameters, the catalytic loop region and ATP-binding pocket were identified and the top six alignments reproduced here. The program output uses upper-case letters to represent amino acids that are aligned. Blue and red residues are used to indicate the degree of conservation within the multiple sequence alignment with red designating the most highly conserved residues. The hash marks (#) designate the residues that are involved in the conserved feature. K282 of the catalytic loop and K195 of the ATP-binding pocket are highly conserved. Descriptions: NP_045368, BoHV-1 US3; 1JNK, C-Jun N-Terminal Kinase; 1DAW_A, Protein Kinase CK2 (Alpha subunit); 1F3M_C, Human Serine Threonine Kinase PAK1; 1PME, Human Erk2 Map Kinase; 1TKI_A, Serine Kinase domain of the Giant Muscle protein Titin; 2PHK_A, Phosphorylase Kinase. (b) Based on blastp 2.2.18, the C-terminal two-thirds of US3 are conserved between BoHV-1 and HSV-1 and K195 and K282 correspond to K220 and K307 of US3 from HSV-1, respectively. (c) A kinase assay was performed with US3(wt)–HA or the mutants US3(K195E)–HA, US3(K282A)–HA, US3(K282E)–HA or US3(K282M)–HA in the presence of 10 μM DMAT, which was added to inhibit potential CK2-mediated phosphorylation. Proteins were separated on a 10 % SDS-PAGE gel before transfer to nitrocellulose and detection of γ-32P by overnight exposure to an imaging screen (left panel), or detection with US3-specific monoclonal antibody by Western blot (right panel).

    Subsequently, the US3–HA mutants were tested using the kinase assay in the presence of DMAT to inhibit potential CK2-mediated phosphorylation. The mutants showed almost no incorporation of 32P compared with wild-type (wt) US3–HA, indicating that residues K195 and K282 are both critical to the kinase activity and autophosphorylation of US3 from BoHV-1 (Fig. 2c⇑, left panel). In the absence of DMAT, phosphorylation of the US3 mutants was observed as well, presumably due to phosphorylation by CK2 (data not shown). Detection of wt US3 and mutant US3 by Western blotting confirmed that all mutants had the correct molecular mass and that the mutant proteins were at least equally loaded in comparison to the wt protein (Fig. 2c⇑, right panel).

    BoHV-1 US3 interacts with VP22, the product of UL49

    BoHV-1 VP22, the product of UL49 and one of the major tegument proteins, is known to be highly phosphorylated. Since the kinases responsible have not been identified, we examined whether it might interact with the US3 kinase. We first showed that VP22 is co-immunoprecipitated with US3–HA from the lysate of BoHV-1-infected fetal bovine testicular (FBT) cells that had previously been transfected with the pUS3–HA plasmid (Fig. 3a⇓). No VP22 was observed to co-immunoprecipitate with the anti-HA resin from BoHV-1-infected cells without US3–HA present. When FLAG–VP22 and US3–HA were co-expressed in Cos-7 cells, interactions between the proteins were also observed (Fig. 3b⇓). This experiment was performed using somewhat less stringent wash conditions (100 mM NaCl and 38 mM Tris/HCl, pH 7.4) as the interaction was not apparent under the usual higher stringency conditions (data not shown). There was no interaction between US3–HA and the anti-VP22-protein A–Sepharose or between FLAG–VP22 and the anti-HA resin when US3–HA was not present. Therefore, although the interaction between VP22 and US3 is relatively weak, it appears to be specific.

    Figure image not available in archive
    Fig. 3.

    Co-immunoprecipitation of VP22 with US3 in BoHV-1-infected and transiently transfected cells. (a) BoHV-1-infected FBT cell lysates, pUS3–HA-transfected FBT cell lysates or pUS3–HA-transfected and infected FBT cell lysates were collected, incubated with anti-HA agarose, washed identically and analysed by Western blotting as indicated. (b) Co-immunoprecipitation of FLAG–VP22 and US3–HA expressed transiently in Cos-7 cells, using anti-HA agarose or anti-VP22-protein A–Sepharose. In this case, the samples were washed with 100 mM NaCl and 38 mM Tris/HCl, pH 7.4 rather than the standard wash buffer. VP22 and FLAG–VP22 were identified with VP22-specific polyclonal antibody and FLAG-specific monoclonal antibody, respectively, and US3–HA was identified with monoclonal antibody specific for the HA tag.

    Subcellular localization of US3 and VP22 from BoHV-1

    In order to examine the localization of US3 in infected cells, a near-confluent monolayer of Madin–Darby bovine kidney (MDBK) cells was infected with BoHV-1 at an m.o.i. of 0.001, and incubated for 41 h in the presence of neutralizing serum. After cell fixation and detection by anti-US3 serum, isolated plaques were observed (Fig. 4a⇓). This allowed us to conclude that US3 localizes predominantly to the nucleus in BoHV-1-infected cells with less fluorescence in the cytoplasm. Furthermore, nuclear localization occurred at all stages of infection from early (at the outer rims of the plaques) to late (near the centre of the plaques) stages. We subsequently determined whether the nuclear localization was dependent on the presence of other viral proteins. A Cos-7 cell monolayer was transfected with pUS3–ECFP, which expresses US3 as an ECF fusion protein. When live cells were examined, expression of US3–ECFP was observed primarily in the nucleus with less fluorescence being observed in the cytoplasm (Fig. 4b⇓). The predominantly nuclear localization was confirmed by 4,6-diamidino-2-phenylindole (DAPI) staining of the nuclei.

    Figure image not available in archive
    Fig. 4.

    Subcellular localization of US3. (a) Localization of US3 in MDBK cells infected with BoHV-1. A nearly confluent MDBK cell monolayer was infected with BoHV-1 at an m.o.i. of 0.001 for 41 h in the presence of neutralizing serum. Cells were fixed with paraformaldehyde and permeabilized with acetone prior to immunostaining with US3-specific rabbit serum and Alexa Fluor 488 goat anti-rabbit IgG. Analysis of plaques was performed with a confocal microscope. (b) Localization of US3 in Cos-7 cells transfected with pUS3–ECFP. Nearly confluent Cos-7 cell monolayers were transfected with 1.6 μg pUS3–ECFP in a six-well plate and live-cell images were taken at 8 h post-transfection. DAPI (2 μl ml−1) was added 15 min prior to microscopy to identify nuclei.

    VP22 is localized to the nucleus at late stages of infection, so it seemed likely that any interaction with US3 would occur during later stages of the infection process. In order to explore the co-localization of these two proteins, we examined plaques from BoHV-1-UL49-enhanced yellow fluorescent protein (EYFP)-infected MDBK cells. After 48 h infection, cells were fixed and examined by immunofluorescence by using rabbit serum specific for US3 (Fig. 5⇓). We observed that around the edges of the plaques, which represent cells early during the infection process, VP22 is localized predominantly to the cytoplasm. Near the centre of the plaques, which represent cells at later stages of infection, VP22 was present in the nucleus. US3 was again mostly observed in the nucleus both at the edges and at the centre of the plaques. Overlap of the chromophores occurred only in the cells near the centre of the plaques. Therefore, it is likely that any interactions between VP22 and US3 occur at late stages of BoHV-1 infection.

    Figure image not available in archive
    Fig. 5.

    Subcellular localization of VP22 and US3 in BoHV-1-infected MDBK cells. A nearly confluent MDBK cell monolayer was infected with BoHV-1-UL49–YFP at an m.o.i. of 0.001 for 48 h in the presence of neutralizing serum. Cells were fixed with paraformaldehyde and permeabilized with acetone prior to immunostaining with anti-peptide serum specific for US3 and Alexa Fluor 633 goat anti-rabbit IgG. Confocal microscopy of a plaque (a) revealed a predominantly cytoplasmic localization for VP22–YFP near the outer edges and a primarily nuclear localization near the centre of the plaque, as well as a mostly nuclear localization of US3 in all cells. Higher magnification of a region near the centre of the plaque (b) demonstrated co-localization of VP22 and US3, once VP22 had translocated to the nucleus.

    BoHV-1 VP22 is phosphorylated by CK2 and US3

    To investigate whether VP22 might be phosphorylated by US3, we analysed VP22 from lysates of Cos-7 cells transfected with pFLAG–VP22 by using a kinase assay. Without kinase inhibitors present, we were unable to draw any conclusions because VP22 appeared to be phosphorylated both in the presence and absence of US3. However, when we examined VP22 without US3 in the presence of 10 μM DMAT, a CK2 kinase inhibitor, the phosphorylation was completely eliminated (Fig. 6a⇓). We interpreted this to mean that the cellular protein kinase CK2 must be heavily phosphorylating VP22 in the kinase assay. Phosphorylation of VP22 by CK2 was also inhibited by the less specific Kenpaullone, but not the CK1 inhibitor D4476, or the PKC inhibitor Ro-32-0432, or the PKA inhibitors KT5720 or 14-22 Amide.

    Figure image not available in archive
    Fig. 6.

    Phosphorylation of VP22 by the cellular protein kinase CK2 and the viral protein kinase US3. (a) Phosphorylation of purified FLAG–VP22 in the absence or presence of kinase inhibitors, 10 μM DMAT, 20 μM D4476, 10 μM Ro-32-0432, 50 μM Kenpaullone, 20 μM KT5720 or 20 μM 14-22 Amide. Proteins were separated on a 10 % SDS-PAGE gel before detection by VP22-specific rabbit serum (bottom panel) or γ-32P by exposure to an imaging screen overnight (top panel). (b) Kinase assays were performed with FLAG–VP22, FLAG–VP22+DMAT, FLAG–VP22+DMAT+US3–HA, US3–HA+DMAT, US3–HA, FLAG–VP22+DMAT+US3(K195E)–HA or FLAG–VP22+DMAT+US3(K282A)–HA, as indicated. Proteins were separated on a 10 % SDS-PAGE gel before detection by US3-specific rabbit serum (top panel), VP22-specific rabbit serum (middle panel) or γ-32P by overnight exposure to an imaging screen (bottom panel). The intensities of the γ-32P FLAG–VP22 bands were measured with a Molecular Imager FX (Bio-Rad). (c) Interaction of VP22 and CK2. Anti-FLAG M2 affinity gel was used for immunoprecipitation from Cos-7 cells (lane 1) or Cos-7 cells transfected with pFLAG–VP22 (lane 2). FLAG–VP22 was identified by using polyclonal antibody specific for VP22, and CK2 was identified by using monoclonal antibody specific for CK2β. Based on apparent molecular mass, CL is likely to be the light chain from the antibody of the affinity gel.

    With this information, we were able to determine the susceptibility of VP22 to phosphorylation by US3 by including the CK2 inhibitor DMAT in the kinase assay (Fig. 6b⇑). DMAT again eliminated phosphorylation of VP22 by CK2. When US3–HA was added together with DMAT, autophosphorylation of US3, as well as increased phosphorylation of VP22, was observed. The intensities of the γ-32P FLAG–VP22 bands were 2302 and 583 counts mm−2, when VP22 was phosphorylated by CK2 (lane 1) or US3 (lane 3), respectively. When the kinase-dead US3(K195E)–HA and US3(K282A)–HA mutants were used in the assay instead of US3–HA, we observed no phosphorylation of VP22. Thus, it appears that the major kinase activity responsible for phosphorylating VP22 is CK2, while US3 plays a minor role. When we examined lysates of Cos-7 cells transfected with pFLAG–VP22, we confirmed that CK2 co-immunoprecipitates with FLAG–VP22 (Fig. 6c⇑). In this case, the CK2β subunit was observed as a band immediately above the light chain (CL) of the anti-FLAG antibody. No interaction between the anti-FLAG resin and CK2 was detected, confirming the specificity of the interaction between VP22 and CK2.

    DISCUSSION

    The results of this study suggest that at least some of the autophosphorylation observed for BoHV-1 US3 results from self-interaction of US3 moieties and is not inhibited by CK1, CK2 or PKC inhibitors. Furthermore, residues K195 and K282 correspond to highly conserved positively charged residues within the ATP-binding pocket and catalytic loop, respectively, and are critical for the catalytic activity of US3 from BoHV-1. In co-immunoprecipitation assays, US3 appeared to interact with the product of the viral UL49 gene, VP22, with co-localization occurring predominantly at later time points during infection. VP22 was susceptible to phosphorylation, to a certain extent by US3, but primarily by CK2, in in vitro kinase assays, and finally, interaction between VP22 and CK2 was confirmed by a pull-down assay.

    The minimal consensus target sequence of US3 has been determined to be (R)nX-(S/T)-Y-Y, where n is at least two residues, X may be absent or any residue with a preference for arginine, alanine, valine, proline or serine, and Y is similar to X but cannot be absent nor the residues aspartate, glutamate or proline (Leader et al., 1991; Purves et al., 1986). Optimally, the sequence has three or more residues for n and X is present. Examining the sequence of VP22 from BoHV-1 (NP_045310), we found support for our results in that there are two regions with a strong correlation to this consensus. Beginning at residue 53, the sequence RRASVR is present, while at residue 92, RRSSSR is present. Both have n=2 and favourable residues at the X and Y positions. The recognition site for CK2 phosphorylation can be summarized as (E/D/x)-(S/T)-(D/E/x’)-(E/D/x)-(E/D)-(E/D/x), where x is any residue except a basic residue and x’ is similar to x but also cannot be proline (Meggio & Pinna, 2003; Pinna, 2002). Serine or threonine is generally the phospho-acceptor and the third residue from the phospho-acceptor must be aspartate or glutamate. Further, there is a strong preference for more than one acidic residue within the site. In the BoHV-1 VP22 sequence, there are two such sites present: PSEDED starting at residue seven and ESGSDD beginning at residue 30.

    Cellular CK2 appears to be the major kinase responsible for phosphorylation of the major tegument protein VP22 of both BoHV-1 and HSV-1 (Elliott et al., 1996, 1999; Morrison et al., 1998). However, there are differences in the characteristics of the US3 protein kinase from HSV-1 and BoHV-1. In HSV-1-infected cells US3 is a critical component for blocking apoptosis, while this is not the case in BoHV-1 (Takashima et al., 1999). By examining the phosphorylation of BoHV-1 VP22 from a qualitative perspective we noticed that VP22 was not only phosphorylated by CK2, but also to a lower extent by US3. In contrast, it has been noted that, in HSV-2, US3 does not appear to be responsible for VP22 phosphorylation in Vero cells expressing US3 and VP22 transiently, although this was not confirmed in kinase assays (Geiss et al., 2001). Although according to the rules outlined above there are potential recognition sites for US3 in VP22 from HSV-1 (ACM62272: 4RRSVK) and HSV-2 (NP_044519: 4RRSVK and 84RRSAS), these are not as favourable as those found on VP22 from BoHV-1 (NP_045310: 53RRASVR and 92RRSSSR). In particular, they are lacking residue ‘X’. Since, conversely, VP22 from BoHV-1 has two very strong recognition sequences, we believe that this may explain why US3 is capable of phosphorylating BoHV-1 VP22. Thus, this suggests that, as in HSV-1, in BoHV-1 CK2 is the major kinase responsible for phosphorylation of VP22, but that US3 may also play a role.

    METHODS

    Cells and virus.

    Cos-7, FBT and MDBK cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL) supplemented with 10 % gamma-irradiated fetal bovine serum (FBS; SeraCare Life Sciences), 10 mM HEPES and 50 μg gentamicin (Gibco) ml−1 unless otherwise noted.

    The 108 strain of BoHV-1 was used in all experiments (Misra et al., 1983). Infections were performed by rocking 85–90 % confluent cell monolayers with BoHV-1 in a minimal volume of DMEM supplemented with 5 % FBS for 1 h, followed by incubation at 37 °C with an additional 10 ml DMEM supplemented with 5 % FBS.

    Gene isolation and cloning.

    The US3 gene was obtained from a cDNA library constructed from BoHV-1-infected MDBK cells, and cloned into a vector encoding a double-HA tag, resulting in pUS3–HA, as described previously (Labiuk et al., 2009). A construct expressing ECFP-labelled US3 was also generated. Briefly, based on the GenBank sequence for BoHV-1 US3 (NP_045368), the full-length gene was amplified by PCR from a cDNA library (HotStar HiFidelity Polymerase kit; Qiagen). The forward primer incorporated an NheI restriction endonuclease site (5′-TAGCTAGCACGACCCGACGTTCTTG-3′) and the reverse primer, a PstI site (5′-TACTGCAGAGAGGCCGCACCGAAGA-3′; italics indicating restriction sites). The PCR product was cloned into the NheI and PstI sites of pECFP-N1 (Clontech) in-frame with the gene encoding ECFP at the 3′ end. The correct sequence of the resulting plasmid (pUS3–ECFP) was confirmed. A minor difference from the US3 sequence in GenBank (a composite of strains K22, P8-2, 34, Jura and Cooper) was noted, such that in strain 108, bases 319, 324, 325 and 327 are guanine instead of adenine. However, this generally did not affect the amino acid sequence with the exception of two lysines at positions 107 and 109, which were glutamates.

    The UL49 gene was obtained from the pVP22–YFP plasmid described previously (Zheng et al., 2005) and subcloned into the EcoRV site of pFLAG–CMV-2 (Sigma), in-frame with the gene encoding the FLAG peptide at the 5′ end. Similarly, the UL49 gene was subcloned into the NdeI site of pET15b (Novagen), in-frame with the gene encoding a His6 tag at the 5′ end. The correct sequences of the resulting plasmids, pFLAG–VP22 and pET15b–VP22, were confirmed.

    Antibodies.

    VP22-specific polyclonal antibodies were produced using the VP22–His6 fusion protein. Briefly, Escherichia coli Tuner Competent Cells (Novagen) were transformed with pET15b–VP22, and recombinant protein expression was induced with 0.5 mM IPTG at 37 °C. Cells were lysed under denaturing conditions and purified on Ni Sepharose 6 Fast Flow resin (Amersham) according to the method described in the manual ‘ProBond Purification System for Purification of Polyhistidine-Containing Recombinant Proteins' (Invitrogen). The elutions were analysed by SDS-PAGE on 10 % gels and by Western blotting. A distinct protein band of the correct molecular mass was observed, which was further purified by extraction from the SDS-PAGE gel (Retamal et al., 1999). Rabbits were immunized four times with 20 μg purified VP22 formulated with 20 μg CpG ODN 2007 and 10 % Emulsigen (MVP Laboratories) as described previously (Ioannou et al., 2003).

    Polyclonal antibodies specific for US3 were prepared as described previously (Labiuk et al., 2009) by using a synthetic peptide corresponding to the 27 aa at the N terminus of US3. Monoclonal anti-casein kinase 2β clone 6D5 (anti-CK2), monoclonal anti-FLAG clone M2 and monoclonal anti-HA clone HA-7 were obtained from Sigma. Anti-GFP rabbit IgG polyclonal antibodies were obtained from Molecular Probes.

    Preparation of lysates.

    Cos-7 or FBT cell monolayers were transfected with plasmid DNA (16 μg per 150 cm2) by using Plus Reagent and Lipofectamine (Invitrogen) in Optimem according to the manufacturer's instructions. Cells were incubated for 48 h in DMEM containing 10 % FBS. In the case of BoHV-1-infected FBT cells, infection was initiated 10 h prior to lysis. To harvest, cells were washed twice with PBS, pH 7.3, then gently rocked for 2–3 min with 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. Lysates were collected on ice, stored at −20 °C and cleared by centrifugation at 1600 g for 10 min at 4 °C prior to use.

    Immunoprecipitations and Western blot analysis.

    Monoclonal anti-HA agarose conjugate (clone HA-7; Sigma) (40 μl resin per 4 ml Cos-7 lysate or 10 ml FBT lysate), anti-VP22 (10 μl) or anti-GFP (10 μl) antibodies were incubated with cell lysates overnight at 4 °C. Samples with anti-VP22 or anti-GFP antibodies were additionally incubated with protein A–Sepharose CL-4B (GE Healthcare Bio-Sciences) for 2 h. Samples were washed three times with 1 ml wash buffer (150 mM NaCl, 50 mM Tris/HCl, pH 7.4) unless otherwise indicated. Bound proteins were eluted by boiling in 100 μl SDS-PAGE sample buffer for 5 min. Twenty microlitres of each sample was analysed on 10 % SDS-PAGE gels, and the proteins were transferred to nitrocellulose membranes and then incubated with the appropriate antibodies at dilutions of 1 : 2000. Subsequently, membranes were incubated with alkaline phosphatase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG diluted at 1 : 2000 (KPL), and Western blots were developed using SigmaFAST BCIP/NBT (Sigma).

    Confocal microscopy.

    MDBK cells were cultured on Permanox 2 chamber slides (Lab-Tek) in minimal essential medium (MEM) supplemented with 1 % FBS. After reaching approximately 90 % confluency, the cells were infected with BoHV-1 or BoHV-1-UL49–EYFP, which expresses VP22 as an EYFP fusion protein, at the indicated m.o.i. in MEM supplemented with 1 % FBS and BoHV-1 neutralizing serum (diluted 1 : 250). After incubation, the cells were washed three times with PBS, incubated with 4 % paraformaldehyde for 30 min and then washed again three times with PBS. The cells were permeabilized with ice-cold acetone for 2 min at −20 °C, washed again with PBS, and then incubated with 5 % normal goat serum in PBS for 2 h at room temperature. Finally, the cells were incubated with rabbit anti-US3 serum at a 1 : 1000 dilution for 2 h at room temperature, followed by Alexa Fluor 488 or Alexa Fluor 633 goat anti-rabbit IgG (Invitrogen) at a 1 : 500 dilution for 1 h at room temperature. Slides were washed with PBS and then de-ionized water, air-dried and mounted using ProLong Gold Antifade Reagent with DAPI (Invitrogen) prior to examination on a Zeiss LSM410 confocal microscope equipped with external argon ion 488 nm laser.

    Mutagenesis.

    The QuikChange Site-Directed Mutagenesis kit (Stratagene) was used according to the manufacturer's directions to produce kinase-null US3 mutant versions of the pUS3–HA plasmid. The sense and antisense primers used to change the K195 or K282 codons are listed in Supplementary Table S1 (available in JGV Online). Altered plasmids were sequenced to confirm the presence of the correct mutations and to confirm that no other mutations along the gene had been inadvertently produced.

    Kinase assays.

    Cos-7 cells were transfected with pUS3–HA or pFLAG–VP22 and lysed. Anti-FLAG M2 affinity gel (20 μl) or anti-HA agarose (20 μl) was washed according to the manufacturer's instructions and incubated with 1 ml of the appropriate lysate overnight at 4 °C. Resin with bound protein was washed at least five times with 1 ml wash buffer (50 mM Tris/HCl, 150 mM NaCl, pH 7.4). If multiple samples of the same protein were required for a particular experiment, the incubations and washes were all performed as one batch, and then the beads were split equally for the assay, thus ensuring identical treatment for all samples.

    Kinase assays were adapted from those described previously (Takashima et al., 1999). Briefly, 5 μl of 0.3 μCi μl−1 (11.1 kBq) [γ-32P]ATP (Perkin Elmer) was added to the substrate mixture consisting of 20 μl FLAG–VP22 on anti-FLAG M2 affinity gel or Gibco double-distilled water, 5 μl 10× kinase buffer (500 mM MgCl2, 1 % Triton X-100, 10 mM 2-mercaptoethanol, 500 mM Tris/HCl, pH 8.0), 1 μl 3× FLAG peptide (Sigma) and double-distilled water (Gibco) to total 40 μl. The substrate mixtures plus [γ-32P]ATP were added to US3–HA bound to anti-HA agarose conjugate or anti-HA agarose conjugate without US3–HA as control (20 μl), and incubated for 30 min at room temperature. Some samples contained the kinase inhibitors InSolution Casein Kinase II Inhibitor, DMAT at 10 μM, InSolution Casein Kinase I Inhibitor D4476 at 20 μM, PKC inhibitor Ro-32-0432 at 10 μM or Kenpaullone at 50 μM (Calbiochem), or one of the protein kinase A (PKA) inhibitors KT5720 at 20 μM and myristoylated PKI 14-22 amide (14-22 Amide) at 20 μM (Murray, 2008). Before loading onto 10 % SDS-PAGE gels, samples were boiled with 70 μl SDS-PAGE loading dye. After separation of proteins, gels were dried and exposed to Imaging Screen K for visualization on a Molecular Imager FX (Bio-Rad). In the experiments with the US3 mutants, the proteins were transferred to nitrocellulose so that equal loading could be verified by immunoblot analysis prior to exposure to Imaging Screen K.

    Acknowledgments

    The authors wish to acknowledge the valued technical assistance and advice of Jennifer McIntosh, Natalia Vasilenko, Shirley Lam 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. 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. 554.

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