|Home | About | Journals | Submit | Contact Us | Français|
Varicella-zoster virus (VZV) open reading frame (ORF) 63 is abundantly transcribed in latently infected human ganglia and encodes a 278-amino-acid protein, IE63, with immediate-early kinetics. IE63 is expressed in the cytoplasm of neurons during VZV latency and in both the cytoplasm and the nucleus during productive infection; however, the mechanism(s) involved in IE63 nuclear import and retention has remained unclear. We constructed and identified a recombinant monoclonal antibody to detect a posttranslationally modified form of IE63. Analysis of a series of IE63 truncation and substitution mutants showed that amino acids 186 to 195 are required for antibody binding. Synthetic peptides corresponding to this region identified IE63 S186 as a target for casein kinase II phosphorylation. In addition, acidic charges supplied by E194 and E195 were required for antibody binding. Immunofluorescence analysis of VZV-infected MeWo cells using the recombinant monoclonal antibody detected IE63 exclusively in the nuclei of infected cells, indicating that casein kinase II phosphorylation of S186 occurs in the nucleus and possibly identifying an initial molecular event operative in VZV reactivation.
Primary infection with the human varicella-zoster virus (VZV) typically causes varicella (chickenpox), after which the virus becomes latent in ganglionic neurons along the entire neuraxis. Reactivation decades later usually produces zoster (shingles) (15). During latency, transcripts mapping to VZV open reading frames (ORFs) 62, 63, and 66 and proteins corresponding to ORFs 21, 29, 62, 63, and 66 have been detected in human ganglia obtained at autopsy (7, 9, 12, 16, 19, 20, 25, 26, 36). Quantitative PCR has shown that ORF 63 is the most prevalent and abundant VZV transcript detected during latency (8).
VZV ORF 63, which is located within the terminal and internal repeat regions of the virus genome and is present as two copies, encodes the immediate-early (IE) protein IE63. This 278-amino-acid protein is present in the cytoplasm during latency but is located predominantly in the nucleus during productive infection (13, 26).
During productive infection in tissue culture cells, VZV IE63 is phosphorylated by casein kinase I (CKI), casein kinase II (CKII), and cyclin-dependent kinase 1 (CDK1) (2, 4, 17, 35, 37). In vitro phosphorylation assays have shown that IE63 is phosphorylated by CKII, CDK1, and a protein kinase encoded by ORF 47 (2, 4, 17, 22). Sequence analysis has suggested 19 potential phosphorylation sites (2, 4). IE63 amino acid S224 is phosphorylated by CDK1 (17), while S165, S173, and S185 are phosphorylated in vitro by serine/threonine kinase, as indicated by alanine substitution mutation of IE63 (2).
Previous studies have shown that phosphorylation of IE63 is required for efficient VZV replication in cell culture (2, 6). Using an antibody that recognizes a posttranslationally modified (PTM) form of IE63, we found that IE63 S186 is phosphorylated by CKII and that phosphorylated S186 is present only in the nuclei of VZV-infected cells.
VZV isolated from a zoster lesion (European strain) was propagated by cocultivating virus-infected MeWo melanoma cells with uninfected cells at a ratio of 1:25 (10) in Dulbecco modified Eagle's medium (Sigma, St. Louis, MO) supplemented with 9% fetal bovine serum (Sigma), penicillin (100 U/liter), streptomycin (0.1 mg/ml), and amphotericin B (250 ng/ml).
IE63 truncation mutants were designed to express protein in Escherichia coli or human embryonic kidney cells (HEK 293; Freestyle cells; Invitrogen, Carlsbad, CA). For expression of IE63 in bacteria, ORF 63 plasmids were cloned into pBAD myc/His A (Invitrogen). For expression of IE63 in mammalian cells, ORF 63 was inserted into pCI-neo (Invitrogen).
Plasmid bFL-IE63 was constructed to express full-length IE63 in bacteria. ORF 63 was amplified by PCR using VZV DNA extracted from the MeWo cultures as the template in a reaction mixture containing 0.4 μM primers NHM1 and NHM2 (Table (Table1).1). PCR conditions were as follows: 30 cycles of denaturation (94°C for 1 min), annealing (62°C for 1 min), and extension (72°C for 1 min), followed by a final extension cycle at 72°C for 10 min. The PCR product was electrophoresed in a 1% agarose Tris-acetate-EDTA (TAE) gel, and the ~850-bp amplification product was excised and purified (Qiaquick gel extraction kit; Qiagen, Valencia, CA). The PCR fragment and pBAD myc/His A vector were digested with HindIII and either PciI or NcoI, respectively, and electrophoresed on a 1% agarose TAE gel, and the DNA fragments were extracted. The purified linear DNAs were ligated at 25°C for 5 min (Quick ligase; New England Biolabs, Ipswich, MA) and transformed into competent E. coli TOP 10 cells (Invitrogen). Insertion of IE63 into pBAD myc/His A was confirmed by DNA sequencing.
Plasmid eFL-IE63 was constructed to express full-length IE63 in HEK 293 cells. Clone eFL-IE63 was constructed by PCR amplification using bFL-IE63 DNA as the template and primers NHM9 and NHM28 (Table (Table1).1). PCR conditions consisted of 30 cycles of denaturation (95°C for 30 s), annealing (58°C for 30 s), and extension (72°C for 2 min), followed by a final extension cycle (72°C for 10 min). The ~850-bp PCR product was gel purified, digested with NheI and XbaI, and extracted from a 1% agarose TAE gel. The eukaryotic expression vector pCI-neo (Invitrogen) was digested with NheI and XbaI, dephosphorylated with Antarctic phosphatase (New England Biolabs), and purified from a 1% agarose TAE gel. The two DNA fragments were ligated and transformed into E. coli TOP 10 cells as described above. Plasmid DNA was used for transfection of HEK 293 cells.
Plasmids were constructed to express truncation mutants of IE63 in HEK 293 cells. E196 and E199, encoding the first 196 or 199 amino acids of IE63, were derived by PCR amplification using eFL-IE63 plasmid DNA as the template with forward primer NHM9 and reverse primer NHM111 or NHM71, respectively. PCR conditions were the same as those used to construct plasmid eFL-IE63. Gel-purified PCR products were digested with NheI and EcoRI, and the DNA was ligated into the unique NheI and EcoRI sites within pCI-neo. V193, encoding the first 193 amino acids of IE63, was constructed by PCR amplification using eFL-IE63 plasmid DNA as the template with primers NHM9 and NHM95 (Table (Table1).1). PCR conditions were changed to 25 cycles of denaturation (94°C for 1 min), annealing (57°C for 1 min), and extension (72°C for 1 min), followed by a final 10-min extension at 72°C. The purified PCR product and pCI-neo vector were digested with NheI and EcoRI, gel purified, ligated, and transformed into E. coli DH5α cells (Invitrogen).
Specific amino acid substitutions in IE63 were constructed and expressed in HEK 293 cells. The S197A clone, encoding IE63 with a serine-to-alanine substitution at residue 197, was constructed by two-step PCR amplification. The first PCR, which used plasmid eFL-IE63 DNA as the template with primer pairs NHM9-NHM69 and NHM70-NHM28 (Table (Table1),1), was carried out under conditions identical to those used to construct plasmid eFL-IE63. In the second step, the gel-purified PCR products were used as the template for overlap PCR with primers NHM9 and NHM28. The purified overlap PCR product (~850 bp) was digested with NheI and XbaI, gel purified, ligated to NheI- and XbaI-digested pCI-neo, and transformed into TOP 10 cells. Similarly, the E5A clone, encoding IE63 with five alanine substitutions of glutamate residues 194, 195, 196, 198, and 199, was constructed with primer pairs NHM9-NHM102 and NHM101-NHM73. Gel-purified PCR products were used in overlap PCR with primers NHM9 and NHM73. The purified overlap PCR product was digested with NheI and EcoRI, gel purified, ligated to pCI-neo, and transformed into DH5α cells. The E194A and E196A clones, encoding full-length IE63 with an alanine substitution at either residue E194 or residue E196, were constructed using the PCR-dependent Phusion site-directed mutagenesis kit according to the manufacturer's instructions (New England Biolabs) with reverse primer NHM119 and forward primer NHM121 or NHM123, respectively. The PCR products were gel purified, self-ligated, and transformed into TOP 10 cells.
The E195A clone, encoding full-length IE63 with an alanine substitution at glutamate residue 195, was constructed as follows. First, the shuttle vector pFA-N2SP was constructed by annealing 10 μg/μl of NHM117 and NHM118 (Table (Table1)1) at 65°C for 10 min, followed by slow cooling at room temperature. The annealed product was ligated into plasmid pFA-CMV at the unique BamHI and EcoRI sites. Target DNAs for insertion into the shuttle vector were constructed by PCR amplification of eFL-IE63 plasmid DNA using primers NHM9 and NHM129. The ~600-bp amplification product contains N-terminal sequences of ORF 63 with both an E195A mutation and a silent mutation resulting in a ScaI site encompassing residues S200 and T201. The DNA was inserted into pFA-N2SP at unique NheI and ScaI sites to yield plasmid pFA-N63. Plasmid pFA-63I was constructed by PCR amplification of eFL-IE63 DNA using primers NHM73 and NHM130 to amplify C-terminal sequences of ORF 63. The gel-purified ~250-bp PCR product was digested with PmeI and EcoRI and was ligated to unique PmeI and EcoRI sites within pFA-N63 to construct the plasmid. The PmeI restriction site in plasmid pFA-63I was removed by digesting the plasmid with ScaI, followed by religation. The modified ORF 63 containing E195A and a silent mutation at residues 200 and 201 was shuttled into pCI-neo using the unique NheI and EcoRI sites. The resulting E195A plasmid was used to express ORF 63 with the E195A substitution in HEK 293 cells.
Five additional clones were constructed to fuse green fluorescent protein (GFP) to ORF 63 truncated at various sites within the N terminus. All constructs were produced using PCR amplification of plasmid eFL-IE63 template DNA with primer pair NHM128-NHM124 (plasmid GFP-T103), NHM128-NHM125 (plasmid GFP-S150), NHM128-NHM126 (plasmid GFP-I176), NHM128-NHM136 (plasmid GFP-S186), or NHM128-NHM127 (plasmid GFP-D187). ORF 63-specific PCR products were gel purified, digested with BsrGI and NotI, directionally cloned in the plasmid RSV-EGFP (27), and transformed into TOP 10 E. coli cells.
Recombinant antibody (rAb) 63E4 was constructed as described for rAb 63P4 (31). With approval from the Institutional Animal Care and Use Committee at the University of Colorado—Denver, purified Flag-tagged IE63 expressed from pF63-CEP was used to immunize two 4-week-old BALB/c mice over the course of 2 months. After 60 days, mice were euthanized, and their spleens were harvested, snap-frozen in liquid nitrogen, and stored at −80°C. RNA from spleen cells was extracted using TriReagent (Molecular Research Center, Cincinnati, OH) and enriched for mRNA using oligo(dT) affinity chromatography (Nucleo Trap minikit; BD Biosciences, San Jose, CA). First-strand cDNAs were prepared using reverse transcriptase (Transcriptor; Roche Diagnostics, Mannheim, Germany). Mouse immunoglobulin G(κ) [IgG(κ)] light-chain variable and constant regions, along with heavy-chain variable and constant framework region 1, were PCR amplified and cloned into the pComb3H vector for phage expression (3). Phage particles specific for IE63 expressed in mammalian cells were selected and enriched by panning as described previously (5). Panning was repeated three times to obtain IE63-specific phage. DNA heavy- and light-chain sequences from these phage, along with protein secretion leader sequences, were PCR amplified and shuttled into pIgG1-Flag and pCEP-4, respectively. DNA from the resulting clones was transfected into HEK 293 Freestyle cells, and full-length recombinant IgG1 was affinity purified from the medium 4 days later.
E. coli cultures containing an IE63 expression plasmid were incubated for 4 h at 37°C in 100 ml Luria broth containing 100 μg/ml ampicillin. At an optical density at 600 nm of ~0.5, recombinant protein expression was induced with l-arabinose at a final concentration of 0.02%. After an additional 4-h incubation, bacteria were collected by centrifugation (3,000 × g for 10 min at 4°C) and stored at −20°C. Full-length and modified IE63 proteins were expressed in HEK 293 cells according to the supplier's (Invitrogen) protocol with minor modifications. Briefly, two 1-ml tubes of medium (Optimem; Invitrogen) were prepared, mixed with either 20 μg of DNA or 40 μl of lipid transfection reagent (293 Transfectin; Invitrogen), and incubated for 5 min at 25°C. Tube contents were combined and incubated for an additional 30 min at 25°C. The DNA mixture was added to 2 × 107 HEK 293 cells in suspension cultures and incubated for 4 days at 37°C under 8% CO2. Cells were collected by centrifugation (1,000 × g for 5 min at room temperature) and stored at −80°C in 1 ml phosphate-buffered saline (PBS).
Recombinant proteins from cell lysates were mixed 1:1 (vol/vol) with 2× Laemmli sample loading buffer containing 50 mM dithiothreitol, and the mixture was heated to 100°C for 5 min. Denatured proteins were loaded onto precast 4-to-20% acrylamide gradient gels (Tris-HEPES sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels; Pierce, Rockford, IL), separated by electrophoresis (at 150 V for 45 min at room temperature), and transferred (at 225 mA for 2 h at 4°C) to nitrocellulose membranes (Millipore, Billerica, MA) in a Trans-blotter (Bio-Rad). Membranes were blocked for 1 h in PBS-casein buffer (Pierce) and were incubated overnight with rocking at 4°C with one of the following anti-63 antibodies diluted in PBS-casein: mouse rAb 63E4 (5 μg/ml) or 63P4 (5 μg/ml), mouse monoclonal antibody 9A12 (dilution, 1:1,000), or rabbit polyclonal anti-63 (dilution, 1:50,000). Membranes were washed twice for 5 min with 0.1% Tween 20 in PBS (PBST), incubated with a secondary antibody (a horseradish peroxidase-conjugated rabbit anti-mouse [dilution, 1:100,000] or goat anti-rabbit [dilution, 1:50,000] antibody) for 3 h in PBS-casein at room temperature, washed in PBST four times for 5 min each time, and developed using enhanced chemiluminescence (Millipore). Western blotting was repeated three or more times.
Target peptides corresponding to IE63 sequences between amino acids 185 and 195 (inclusive) along with a triglycine linker were synthesized for CKII-dependent phosphorylation (Genscript, Piscataway, NJ). Unmodified peptide (GGG185SSDGEDFIVEE195) or peptides methylated at E194, E195, or both were suspended in Tris-buffered saline containing 50 mM ammonium bicarbonate at a final concentration of 5 mg/ml. Synthetic peptides were phosphorylated in 10-μl reaction mixtures containing 5 μg peptide, 1 mM ATP, and 500 U of CKII (New England Biolabs) in the manufacturer's buffer and were incubated at 30°C for 1.5 h. Peptides were blotted onto nitrocellulose membranes, air dried, and analyzed for binding to rAb 63E4 by Western blotting. In vitro CKII assays were repeated twice.
VZV-infected and uninfected MeWo cells were propagated on coverslips, harvested 2 days postinfection, fixed in 4% paraformaldehyde for 20 min, washed with PBS, permeabilized with 0.3% Triton X-100 in PBS for 20 min, washed with PBS, and blocked with 2% bovine serum albumin (BSA) in PBS for 30 min. Primary antibody 63P4 (0.5 μg/ml) or 63E4 (1 μg/ml) in PBS containing 2% BSA was added and incubated for 20 min at room temperature. Coverslips were washed in PBS three times for 5 min each time. After further blocking with 2% BSA in PBS for 20 min, coverslips were incubated with a 1:500 dilution of donkey anti-mouse IgG (Alexa Fluor 594; Invitrogen) in PBS containing 2% BSA for 20 min, washed in PBS three times for 5 min each time, and mounted on slides with a 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting medium (Vectashield; Vector Laboratories, Southfield, MI). Coverslips were sealed with clear nail polish. Image analysis was performed as described previously (29). Immunofluorescence images were captured at room temperature on a Nikon Diaphot fluorescence microscope equipped with a SensiCam charge-coupled device camera (Cooke Corporation, Tonawanda, NY) using Slidebook software (Intelligent Imaging Innovations, Inc., Denver, CO). All images were digitally deconvolved using the no-neighbors algorithm (Slidebook), converted to tagged-image file format, and processed by Photoshop (Adobe Systems, Inc., Mountain View, CA). Experiments were repeated at least three times to determine the cellular location of IE63 detected by antibodies.
To identify PTM IE63, rAb 63E4 was raised in mice against IE63 produced in HEK 293 cells. The control antibody was rAb 63P4, which detects an unmodified form of IE63 (31). Western blot analysis (Fig. (Fig.1A)1A) revealed reactivity of rAb 63E4 with IE63 from VZV-infected MeWo cells (lane 2) and from HEK 293 cells transfected with an IE63 expression plasmid (lane 4), as well as some weak reactivity with unmodified IE63 expressed and purified in E. coli (lane 3), whereas rAb 63P4 (Fig. (Fig.1B)1B) detected IE63 in VZV-infected MeWo cells (lane 2) and protein purified from bacterial (lane 3) and mammalian (lane 4) cells. Neither rAb reacted with MeWo cell proteins (Fig. 1A and B, lanes 1). These findings suggest that only rAb 63E4 detects a PTM form of IE63.
To identify potentially phosphorylated IE63 amino acids, we searched the consensus sequences of CKI, CKII, and CDK1. As shown in Fig. Fig.2,2, amino acids T8, S15, S82, S173, S200, and S203 were identified based on the CKI consensus sequence [(S/T)XX(S/T)] as potential targets for phosphorylation, while the CKII consensus sequence [(S/T)XX(E/D)] identified S150, S165, T171, S181, and S186, and the CDK1 consensus sequence [(S/T)PX(R/K)] identified only S224. (Letters in boldface in consensus sequences represent potentially phosphorylated IE63 amino acids.) These potential phosphorylation sites served in constructing IE63 truncation mutants for subsequent identification of the PTM amino acid(s) required for antibody binding.
Several C-terminal IE63 truncation and substitution mutants (Fig. (Fig.3A)3A) were expressed in HEK 293 cells and examined by Western blotting for reactivity with a rabbit anti-IE63 polyclonal antibody (Fig. (Fig.3B),3B), which detected full-length IE63 (lane 1), as well as all mutants of the protein (lanes 2 to 9), and with rAb 63E4 (Fig. (Fig.3C),3C), which detected full-length IE63 (lane 1) and the C-terminally truncated E199 mutant (lane 2), but not IE63 truncated to V193 (lane 3). The six amino acids in IE63 between V193 and E199 include five glutamates and one serine (194EEESEE199). While S197 was not predicted to be phosphorylated by CKI, CKII, or CDK1, and IE63 containing an S197A substitution was detected by rAb 63E4 (Fig. (Fig.3C,3C, lane 4), replacement of the five glutamates with alanines eliminated 63E4 binding to IE63 (lane 5). The 63E4 antibody bound to truncated IE63 when two of the five glutamates (E198 and E199) as well as S197 were deleted (Fig. (Fig.3C,3C, lane 6), indicating that 194EEE196 is required for antibody binding. Further analysis of the 194EEE196 region by alanine substitution showed that the 63E4 antibody did not bind full-length IE63 containing an E194A (Fig. (Fig.3C,3C, lane 7) or E195A (lane 8) mutation but did bind full-length IE63 containing an E196A substitution (lane 9). Thus, while amino acids from E196 to the C terminus of IE63 are not required for PTM of IE63, E194 and E195 are required for rAb 63E4 binding.
To map the N terminus of the 63E4 PTM epitope, several IE63 truncations were constructed and transfected into HEK 293 cells. Because preliminary experiments indicated that IE63 was unstable when N-terminal truncations were expressed in bacteria or HEK 293 cells, all mutants were fused to the C terminus of GFP and shown to be stable in HEK 293 cells. Western blot analysis using mouse monoclonal antibody 9A12 (Fig. (Fig.3D)3D) revealed reactivity with IE63 deleted from the N terminus to T103 (lane 1), S150 (lane 2), I176 (lane 3), or D187 (lane 4), while use of the 63E4 antibody (Fig. (Fig.3E)3E) detected IE63 deleted from the N terminus to T103 (lane 1), S150 (lane 2), or I176 (lane 3); a band resulting from the probing of an IE63 mutant in which the first 186 amino acids were deleted with the 63E4 antibody was visible upon longer exposure of the blot (Fig. (Fig.3F,3F, lane 4). Together these results map the 63E4 epitope to the amino acids between I176 and E195, a stretch of 20 amino acids (IEFRDSDAESSDGEDFIVEE) that contains two potential CKII phosphorylation sites, S181 and S186.
Homology between the VZV and simian varicella virus (SVV) IE63 proteins was used to design synthetic peptides to fine map the 63E4 epitope. Figure Figure3G3G shows that rAb 63E4 binds SVV IE63. Since SVV IE63 has a proline corresponding to VZV IE63 S181, and S181 is the only potential phosphoreceptor between VZV IE63 I176 and E184, the peptides used for in vitro CKII assays spanned VZV amino acids S185 to E195. These synthetic peptides (VZV IE63 S185 to E195) have a potential CKII phosphorylation site at S186 and two potential methylation sites at E194 and E195. To test these potential PTM sites, the synthetic peptide spanning S185 to E195 was methylated at either E194 or E195, or both, and was incubated in the presence or absence of CKII. Figure Figure44 shows that the 63E4 antibody recognized only the CKII-phosphorylated, unmethylated peptide (Fig. (Fig.4A,4A, bottom), but not the unphosphorylated peptide (Fig. (Fig.4A,4A, top) or the peptide methylated at E194 (Fig. (Fig.4B),4B), E195 (Fig. (Fig.4C),4C), or both (Fig. (Fig.4D),4D), independent of CKII phosphorylation. These results suggested that IE63 is phosphorylated at S186 by CKII and is not methylated at E194 or E195.
To confirm that phosphorylated S186 is the PTM recognized by rAb 63E4, a GFP-fused IE63 mutant N-terminally truncated at S186 was constructed, and antibody binding was compared by Western blot analysis to that of GFP-fused IE63 mutants N-terminally truncated at I176 or D187 (Fig. (Fig.5,5, left). The 63E4 antibody bound the I176 (Fig. (Fig.5,5, left, lane 1) and S186 (lane 3) truncation mutants at equivalent levels but bound the D187 truncation mutant (lane 2) at greatly reduced levels. The same analysis using antibody 9A12 showed that similar amounts of IE63 constructs were analyzed (Fig. (Fig.5,5, right). Together, these results indicate that IE63 is phosphorylated by CKII at S186 in VZV-infected MeWo cells.
Confocal analysis of VZV-infected MeWo cells with the 63E4 and 63P4 antibodies was used to determine the cellular localization of CKII-dependent S186 phosphorylation (Fig. (Fig.6).6). The results show that in VZV-infected cells, rAb 63E4 detected IE63 (Fig. (Fig.6A)6A) that colocalized with DAPI-stained nuclei (Fig. 6B and C), whereas antibody 63P4 detected IE63 (Fig. (Fig.6D)6D) in the cytoplasm but not in the DAPI-stained nuclei (Fig. 6E and F).
We report here that VZV IE63 is phosphorylated at S186 by CKII, as discovered using the newly constructed rAb 63E4, whose epitope maps between S186 and E195. Critical features within the 63E4 epitope are CKII-dependent phosphorylation at S186 and the presence of acidic residues E194 and E195. Antibody binding is reduced when S186 is unphosphorylated and is ablated when either E194 or E195 is mutated to alanine or when their acidic charge is neutralized by methylation. The detection of S186-phosphorylated IE63 exclusively by rAb 63E4 in the nuclei of infected cells suggests that CKII is also located in the nucleus.
The accepted requirement for a CKII phosphorylation consensus sequence is (S/T)XX(E/D), where serine or threonine is the receptor for phosphorylation. While acidic residues in close proximity on either side of the consensus sequence may enhance CKII binding or kinase activity owing to the large number of basic residues in the CKII alpha subunits (reviewed in reference 18), these additional acidic residues are not absolutely required for phosphorylation (reviewed in reference 21). For example, a glutamate at position −1 with respect to the consensus CKII sequence has a higher Km (40 μM) than methionine (Km, 20 μM) at the same location, indicating that CKII has a higher affinity for a substrate when the −1 amino acid is not acidic (32). Here we have demonstrated CKII-dependent phosphorylation of S186. While we cannot rule out a decreased phosphorylation of S186 when the acidic charge on E194 or E195 is neutralized, the binding of rAb 63E4 to IE63 with a deletion of the first 186 amino acids (the D187 truncation mutant), albeit at reduced levels, indicates that the acidic charges have a greater effect on antibody affinity than CKII activity. Optimal 63E4 binding requires not only phosphorylated S186 but also the epitope through E195, and thus, 63E4 recognizes a specific linear IE63 epitope and does not recognize all CKII-phosphorylated events.
Putative IE63 phosphorylation sites have been identified by in silico analysis and characterized by mutagenesis. Baiker et al. (2) have reported that IE63 extracted from mammalian (293T) cells is preferentially phosphorylated by cellular kinases at S165, S173, and S185 but not at S186. However, their findings that S165, S173, and S185 were available for phosphorylation in the in vitro kinase assay indicate that these residues were not phosphorylated by cellular kinases in 293T cells. Furthermore, their finding that A186S substitution did not reduce the level of IE63 phosphorylation in vitro suggests that S186 was already phosphorylated in mammalian cells, a conclusion supported by our results. In another study (4), in silico analysis of IE63 identified amino acids S150, S165, T171, S181, and S186 as possible targets for CKII phosphorylation, and transient transfection of a eukaryotic expression vector with mutants in which these residues were replaced with alanine led to reduced nuclear localization of IE63. However, several studies have shown that phosphorylation of IE63 is not required for nuclear import (1, 2, 6, 37). Thus, our detection of IE63 localized in the nucleus and phosphorylated at S186 suggests that CKII phosphorylation occurs in the nucleus, with resultant nuclear retention of IE63.
Reactivation of both VZV (14, 24, 34) and SVV (23, 28, 33) is also associated with exposure to ionizing radiation. Moreover, VZV DNA and infectious virus are present in the saliva of astronauts during and shortly after space flight (11, 30), and preliminary investigations suggest that ionizing radiation is the major cause of VZV reactivation in astronauts (S. K. Mehta, personal communication). The cellular localization of CKII catalytic subunits is known to be altered by ionizing radiation (38). Our finding that CKII-dependent phosphorylation of IE63 S186 is located in the nucleus may identify an initial molecular mechanism involved in VZV reactivation, in which altered IE63 cellular localization, i.e., translocation from the cytoplasm to the nucleus, represents the critical step. Further studies are needed to elaborate the involvement of cellular kinases in VZV reactivation.
This study was supported in part by Public Health Service grants NS32623 (to R.J.C. and D.G.), AG032958 (to R.J.C. and D.G.), and AG006127 (to D.G.) from the National Institutes of Health. N.H.M. is supported by Public Health Service grant NS07321 from the National Institutes of Health.
We thank Sebastien Bontems (University of Liège; Liège, Belgium) for the 9A12 antibody, Marina Hoffman for editorial review, and Cathy Allen for manuscript preparation.
Published ahead of print on 16 September 2009.