The Phosphorylation Status of the Serine-Rich Regi
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病菌学杂志 2005年第3期
Department of Cancer Biology, Abramson Family Cancer Research Institute, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
ABSTRACT
The 86-kDa major immediate-early protein (IE2/IEP86) of human cytomegalovirus (HCMV) contains a serine-rich region (amino acids 258 to 275) with several consensus casein kinase II (CKII) sites. We performed extensive mutational analysis of this region, changing serines to alternating alanines and glycines. Mutation of the serines between amino acids 266 and 275 eliminated in vitro phosphorylation by CKII. In vitro CKII phosphorylation of the serines between amino acids 266 and 269 or between amino acids 271 and 275 inhibited the ability of IE2/IEP86 to bind to TATA-binding protein. Correspondingly, nonphosphorylatable mutants in these regions showed increased activation of specific HCMV gene promoters in transfection studies. Viruses containing mutations of the serines throughout the entire region (amino acids 258 to 275) or the second half (amino acids 266 to 275) of the region showed delayed expression of all viral proteins tested and, correspondingly, delayed growth compared to wild-type HCMV. Mutation of the serines in the first half of the serine-rich region (amino acids 258 to 264) or between amino acids 266 and 269 propagated very slowly and has not been further studied. In contrast, mutation of the serines between amino acids 271 and 275 resulted in accelerated virus growth and accelerated temporal expression of viral proteins. These results suggest that the serine-rich region is structurally complex, possibly affecting multiple functions of IE2/IEP86. The data show that the phosphorylation state of the serine-rich region, particularly between amino acids 271 and 275, modulates the temporal expression of viral genes.
INTRODUCTION
Transcription of the major immediate-early (MIE) gene of human cytomegalovirus (HCMV) produces a number of alternatively spliced and polyadenylated mRNAs that encode several MIE proteins (MIEPs) (25, 31, 32, 34). Two of these, IE1 (72 kDa; also called IE72, IE1491aa, or ppUL123) and IE2 (86 kDa; also called IEP86, IE2579aa, or ppUL122a) appear in abundance in lytic infections and have been extensively examined. These proteins alter the transcriptional activity of viral and cellular promoters and control temporal expression of the HCMV genes (7, 9, 10, 13, 15-17, 21-23, 26, 29, 30, 33, 36, 38). The MIEPs have also been shown to affect cell cycle control and apoptosis (20, 37, 39).
We have previously mapped the major phosphorylation sites of IE2/IEP86 (11) (Fig. 1). One of these sites is a serine-rich region spanning amino acids 258 to 275 (Fig. 1) that contains several consensus sites for casein kinase II (CKII). To determine the role of this region and its phosphorylation in the function of IE2/IEP86, we have made a number of mutations through the region, changing the serines to alternating alanines and glycines. Studies of mutant viruses show that some of these mutations are deleterious to viral gene expression and virus growth, while others accelerate these processes, suggesting that the role of the serine-rich region in IE2/IEP86 function is complex, affecting multiple functions. Our studies establish that one function modulated by the phosphorylation state of the serine-rich region is the temporal expression of viral genes.
MATERIALS AND METHODS
Cells and plasmids. The glioblastoma-astrocytoma cell line U373MG was maintained at passage numbers less than 30 in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, Glutamax, and antibiotics. Life-extended human foreskin fibroblasts (LEHFFs) (5) were maintained at passage numbers less than 15 in DMEM supplemented with 10% fetal calf serum, Glutamax, and antibiotics.
Plasmid pRSV86 Towne contains a cDNA encoding IE2/IEP86 utilizing the complete Towne sequence (3) under the control of the Rous sarcoma virus long terminal repeat. Mutations in the serine-rich region (between amino acids 266 and 275, 266 and 269, and 271 and 275) were made by site-directed mutagenesis by using the QuickChange kit (Stratagene) and the following oligonucleotides: for mutant 266-275, the 5' oligonucleotide was 5'-GCGGTGCGGCTGGGGACGCGGAGGGTGAGGCCGAGGAG-3', and the 3' oligonucleotide was 5'-CTCCTCGGCCTCACCCTCCGCGTCCCCAGCCGCACCGC-3'; for mutant 266-269, the 5' oligonucleotide was 5'-CTTCCTCCTGCGGTGCGGCTGCGGACTCGG-3', and the 3' oligonucleotide was 5'-CTCTCCGAGTCCGCAGCCGCACCGCAGGAGG-3'; for mutant 271-275, the 5' oligonucleotide was 5'-GCAGTTCGGCTTCGGACGCGGAGGGTGAGGCCGAGGAG-3' and the 3' oligonucleotide was 5'-CTCCTCGGCCTCACCCTCCGCGTCCGAAGCCGAACTGC-3'. Mutations in amino acids 258 to 264 and 258 to 275 were made by using a splicing overlap extension protocol (11). First, mutant 258-264 was made by using the 5' oligonucleotide 5'-GCAGGAGCAGGAGACGGAGAGGCAGAGGGAGAGGAGATGAAATGC-3' and the 3'oligonucleotide 5'-CTCCCTCTGCCTCTCCGTCTCCTGCTCCTGCGCATCCTGCTCCTGCT-3'. Then the 258-275 mutant was made by using the 258-264 mutant as a template with the 5' oligonucleotide 5'-GGAGCAGGAGCAGGAGCAGGATGCAGTTCGGCTTCG-3' and the 3' oligonucleotide 5'-GCATCCTGCTCCTGCTCCTGCTCCATCTTCATCGGGCCG-3'. All mutations were verified by sequence analysis.
Plasmid pRL43a contains the genomic MIE region from the Towne strain of HCMV (18, 27). To introduce IE2/IEP86 mutations into pRL43a, we partially digested the plasmid with SmaI, followed by StuI digestion. The SmaI-StuI fragment containing the serine-rich region in pRL43a was replaced with the SmaI-StuI fragment isolated from pRSV86 plasmids containing the mutations in an IE2/IEP86 cDNA.
Reporter plasmids contained the luciferase gene under control of the following promoters: the promoter of the HCMV UL112-113 early genes, plasmid pHM142 (1); and the promoter of the delayed-early gene UL44 (ICP36), plasmid pICP36 (19).
Plasmids expressing glutathione S-transferase (GST) fusion proteins with the full-length IE2/IEP86 coding region and with the IE2/IEP86 fragment encoding amino acids 252 to 367 were prepared containing both the wild-type (WT) and serine region mutations by using the pGEX3X vector (Pharmacia).
Antibodies. For Western analyses, IE1/IE72 and IE2/IEP86 were detected by using anti-exon2/3, a polyclonal antiserum prepared for this laboratory by Cocalico Biologicals, Inc. (Reamstown, Pa.); it recognizes the common 85 amino acids in all known HCMV MIEPs (11). Monoclonal antibody MAb810 (Chemicon), which recognizes the N-terminal region of IE1/IE72 and IE2/IEP86, was used to immunoprecipitate the MIE proteins. Mouse monoclonal antibodies against the p28 tegument protein (UL99), p65 tegument protein (UL83), glycoprotein B (gB or UL55) and polymerase accessory factor (p52 or UL44) were purchased from Advanced Biotechnologies Inc.
Transfections, luciferase assays, immunoprecipitations, and Western analyses. U373MG cells were transfected by using Fugene (Roche) as previously described (3). Briefly, for luciferase assays 2.5 x 105 cells were plated per well in 12-well plates. A transfection mixture was made containing 0.2 μg of the green fluorescent protein-expressing plasmid pCMS-EGFP (Clontech); 0.5 μg of reporter plasmid; and 0.1, 0.5, or 1.2 μg of IE2/IEP86-expressing plasmids plus control plasmid pRSV3/BglII to bring the expression plasmid input to 1.2 μg. One-quarter of each transfection mixture was added to four different wells of the 12-well plate. Three wells were used for luciferase assays, and the fourth was used to obtain total extract to assay for IE2/IEP86 protein expression. Luciferase activity was assayed by using the Promega luciferase assay system, with 12 μg of transfected cell extract harvested according to the manufacturer's instructions, and measured on a Berthold 9501 Lumat luminometer.
Transfection-infection studies utilized a transfection mixture containing 0.2 μg of red fluorescent protein-expressing plasmid (pDsRed1-C1) and 1 μg of reporter plasmid. One-third of the mixture was added to three wells of a 12-well plate. At 24 h posttransfection, the cells were infected with WT or mutant viruses at a multiplicity of infection (MOI) of 5 in 500 μl of medium; 5 h later the inocula were removed, the cells were washed twice with phosphate-buffered saline, and 1 ml of medium was added. At 48 h postinfection the cells were harvested, and luciferase activity was assayed as described above.
Approximately 1.6 x 106 U373MG cells were plated on 60-mm plates for transfections used for immunoprecipitation experiments. Cells were transfected with 1.8 μg of the IE2/IEP86-expressing plasmids plus 0.2 μg of pCMS-EGFP (Clontech). Total cell extracts were prepared by lysing the cells in radioimmunoprecipitation assay buffer (1% NP-40, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 150 mM NaCl, 10 nM Na phosphate [pH 7.2], 2 mM EDTA) plus 1 μg of leupeptin per ml, 0.7 μg of pepstatin per ml, 1 mM phenylmethylsulfonyl fluoride, 10 μg of aprotinin per ml, 2.5 μg of E64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane] per ml, 4 mN NaF, and 0.1 mM sodium orthovanadate. Lysates were centrifuged for 30 min at 4°C; supernatants were used for immunoprecipitation by overnight incubation of 1 mg of extract, 1 μl of MAb810, and 45 μl of protein G Sepharose beads (75% slurry) at 4°C. The samples were then washed, boiled in SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer, and separated by SDS-8% PAGE. The gel was transferred to nitrocellulose and probed with polyclonal antibody anti-exon 2/3, followed by incubation with the anti-rabbit secondary antibody. The transfer was developed by using enhanced chemiluminescence (Amersham).
In vitro kinase assays and binding assays. The GST fusion proteins were made in Escherichia coli BL21(DE3) pLysS grown in Terrific broth. Fusion protein expression was induced for 1 h with IPTG (isopropyl-?-D-thiogalactopyranoside), bacteria were collected, and fusion proteins were prepared and purified by using glutathione-Sepharose beads (Sigma) as previously described (21). Equivalent amounts (1 to 5 μg) of fusion proteins (quantitated by Sypro red staining) were used for in vitro kinase experiments and in vitro binding experiments.
For in vitro CKII assays, fusion proteins were incubated for 30 min at 30°C with 25 U of CKII (New England Biolabs) in 1x CKII buffer (20 mM Tris-HCl [pH 7.5], 50 mM KCl, and 10 mM MgCl2) (New England Biolabs) containing 0.2 mM ATP and 20 μCi of [-32P]ATP. Assays with other kinases were performed following the manufacturers' instructions. The phosphorylated fusion proteins were purified by using gluthatione-Sepharose beads, boiled in SDS-PAGE loading buffer, and separated by SDS-10% PAGE.The gels were stained with Sypro red to quantify the total amounts of protein, dried, and exposed to a PhophorImager screen to quantitate phosphorylation.
For in vitro factor binding assays, GST fusion proteins were pretreated with nonradioactive ATP and either CKII or heat-inactivated CKII, as described above. The treated proteins were incubated for 30 min at 4°C with 7.5 x 104 cpm of in vitro transcribed and translated [35S]methionine-labeled TATA binding protein (TBP) in 1 ml of NETN buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) containing 3% bovine serum albumin plus protease and phosphatase inhibitors (5 mM NaF, 0.1 mM Na3VO4, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). A total of 45 μl of a 33% glutathione-agarose bead slurry were added to the mix for a 1-h incubation at 4°C. Beads were collected and washed five times with NETN plus protease and phosphatase inhibitors. The in vitro translated factors bound to the GST fusion proteins on the beads were eluted by boiling in SDS-PAGE loading buffer and separated by SDS-PAGE. The gels were dried, and labeled proteins were detected by autoradiography and quantified by using a PhosphorImager.
Mutant virus construction. Virus stocks containing the serine-rich region mutations were made by homologous recombination in LEHFFs. LEHFFs were coelectroporated with 4 μg of BAC-Towne-UL122 DNA, a bacterial artificial chromosome containing the Towne strain of HCMV with a deletion of ORF UL122 (12, 24), 5 μg of Hind III-linearized pRL43a plasmid containing the serine-rich region mutations, and 2 μg of pp71-expressing plasmid pCGNpp71 (24). Electroporation was performed by using a Bio-Rad Gene Pulser with 0.4-cm cuvettes at 260V and 950 μF. Medium was changed the day after electroporation. Since IE2/IEP86 is essential for viral growth, the only viruses which grow are those in which a viable mutant IE2/IEP86 was inserted by homologous recombination. Mutations were confirmed by sequence analysis of viral DNA harvested from infections. In addition, viral DNA extracted from cells infected with the mutant viruses was cleaved with EcoRI, separated on 0.7% agarose gels, and visualized by Southern analysis, with plasmid pRSV86 used as a probe, to check for improper recombination events within the MIE region (results not shown).
Virus stocks were made by collecting viruses from media and sonicated cells, followed by a 10-min centrifugation at 4,000 rpm in a Sorvall RT7 centrifuge to remove cellular debris. Viruses were concentrated by ultracentrifugation onto a 20% sorbitol cushion by using a Beckman SW28 rotor at 19,000 rpm as previously described (2, 14). Pelleted virus was resuspended in serum-free medium.
Viral infection studies. For virus growth curves and infection time courses, 2 x 105 LEHFFs were plated per well in six-well plates, 3 days before infection. Cells were infected by using 1 ml of medium containing input virus at an MOI of 3 or 0.1. Four hours after infection the virus inoculum was removed, the cells were washed twice with phosphate-buffered saline, and 2.5 ml of fresh medium was added. For virus growth curves, viruses were collected either from medium (infection at an MOI of 0.1) or from medium and sonicated cells (infection at an MOI of 3). Virus titers were obtained by using the median tissue culture infectious dose (TCID50) method (4, 28). For Western analysis, infected cells were lysed in radioimmunoprecipitation assay buffer; 10 to 30 μg of total extract was separated by SDS-8% PAGE. The gels were transferred to nitrocellulose and probed with antibodies as described above.
RESULTS
In vitro analysis of IE2/IEP86 phosphorylation with purified kinases. Figure 1A shows a map of IE2/IEP86; the regions denoted 2/3, 5A, 5B, 5C, UR, and 6 are domains of the protein which we previously made as GST fusion proteins (21). The arrows under domains 5A and 5C indicate that these domains bound transcription factors (e.g., TBP and Tef-1) in vitro (21).
We previously mapped and mutated major phosphorylation sites in IE2/IEP86 at amino acids T27, S144, and T233/S234 (11, 12). In vitro phosphorylation of these sites in GST fusion proteins by purified kinases focused on serum-inducible kinases, particularly the mitogen-activated protein kinases ERK2 and JNK1 (11). To develop a more complete picture of the phosphorylation of IE2/IEP86, other purified serine/threonine kinases were tested for their ability to phosphorylate both WT and phosphorylation site mutant GST fusion substrates (Fig. 1A), including the WT and mutated serine-rich region discussed in detail below (Fig. 2). The additional kinases tested were p34Cdc2-cyclin B (Cdc2), a key kinase involved in G2/M transition; CKII, a constitutive nuclear kinase; and two second-messenger-regulated kinases, cyclic AMP-regulated protein kinase A (PKA) and protein kinase C (PKC).
Purified kinases were incubated with the various bacterially expressed, purified GST fusion substrates in the presence of [-32P]ATP as described in Materials and Methods. The results are summarized in Fig. 1A, where the kinases that specifically phosphorylated individual IE2/IEP86 domains are listed below the respective domains. Phosphorylation by those kinases indicated in italics and underlined was inhibited by the phosphorylation site mutation shown above each region. It is interesting that there are several cases of overlapping kinase specificities; for example, T27 appears to be a phosphorylation site for both ERK2 and Cdc2, and T233/S234 appears to be phosphorylated by both ERK2 and PKA. Although major phosphorylation sites have been previously mapped and characterized (11), these data suggest that other sites can be targeted by a number of different kinases. In particular, the serine-rich region is a strong target for CKII; this is characterized below.
Mutations of the serine-rich region. Figure 1B shows the amino acid sequence of the WT serine-rich region; serines that are potential CKII sites are boxed. To study the significance of the phosphorylation of this region, we made a variety of mutations where either all or portions of the serines between amino acids 258 and 275 were mutated to alternating glycines and alanines. These mutations are listed in Fig. 1B (the mutated bases are underlined). Each mutation was introduced into an IE2/IEP86 cDNA expression plasmid. These were transfected into U373MG cells to test the expression and stability of the mutant proteins. At 48 h after transfection the cells were harvested, and IE2/IEP86 was immunoprecipitated by using MAb810, which recognizes the amino-terminal end of the protein. The precipitated proteins were separated by SDS-PAGE and detected by Western analysis by using a polyclonal antibody that recognizes the first 85 amino acids common to all MIEPs. Figure 1C shows that the various mutations in the serine-rich region do not significantly alter the levels of IE2/IEP86 or its sumoylated form in U373MG cells transfected with plasmids expressing cDNA copies of WT or mutated IE2/IEP86.
The serine-rich region is a substrate for phosphorylation by CKII in vitro. To determine which serines in the region were needed for CKII phosphorylation, we inserted each mutation into the GST-5C fusion protein (Fig. 1). Equal amounts of purified GST fusion protein were incubated with CKII and [-32P]ATP (see Material and Methods) and then fractionated by SDS-PAGE. The autoradiograph of the phosphorylated forms is shown at the bottom of Fig. 2; the intensity of phosphorylation was quantitated by PhosphorImager analysis and is shown by the bar graphs. Mutation of all serines between amino acids 258 and 275 and mutation of only the serines between amino acids 266 and 275 dramatically reduced CKII phosphorylation. This result suggests that the potential CKII consensus sites between amino acids 266 and 275 are the targets of phosphorylation by CKII in vitro. Mutation of the first three (between amino acids 266 and 269) or the last three serines (between amino acids 271 and 275) resulted in a 38 and 60% reduction in CKII phosphorylation, respectively, suggesting that CKII utilizes the serines in each region.
The panel on the left of Fig. 2 shows a similar CKII labeling experiment using GST fusions with full-length IE2/IEP86 containing either WT or mutant 258-275 sequences. Mutation of the serines between 258 and 275 resulted in an 80% reduction in full-length IE2/IEP86 phosphorylation by CKII. This indicates that the serine-rich region is the major site for in vitro CKII phosphorylation of IE2/IEP86.
The serine-rich region is also phosphorylated in vivo. Mutant 258-275 was inserted into a full-length IE2/IEP86 cDNA expression plasmid. The plasmid was transfected into U373MG cells that were labeled with 32P; IE2/IEP86 was purified and subjected to two-dimensional tryptic phosphopeptide analysis (data not shown). Compared to WT IE2/IEP86, the mutation of the serine-rich region resulted in a dramatic decrease in the intensity of tryptic peptide D/D', one of the major tryptic phosphopeptides that we previously characterized (11).
In vitro phosphorylation of the serine-rich region by CKII inhibits binding to TBP. We showed previously that regions 5A and 5C (Fig. 1A) of IE2/IEP86 can independently bind TBP (21). Since region 5C contains the serine-rich region, we determined whether TBP binding was affected by CKII phosphorylation. Equal amounts of WT and mutant GST 5C fusion proteins were incubated for 30 min at 30°C with nonradioactive ATP plus CKII or heat-inactivated CKII. The treated proteins were then incubated with in vitro transcribed and translated [35S]methionine-labeled TBP (see Materials and Methods). GST fusion proteins and the labeled factors were purified on glutathione beads, eluted, separated by SDS-PAGE, and detected and quantitated by autoradiography and PhosphorImager analysis. Figure 3 shows that treatment with CKII, but not heat-inactivated CKII, inhibited binding of both the WT and the 258-64 mutant fusion proteins to TBP. However, mutation of serines between amino acids 266 and 275, 266 and 269, and 271 and 275 resulted in fusion proteins that bound TBP regardless of the treatment by CKII. These results indicate that hypophosphorylation of the serines between residues 266 and 269 or 271 and 275 correlates with TBP binding. These data suggest that the phosphorylation status of the latter half of the serine-rich region may regulate protein-protein interactions of IE2/IEP86, which may affect its ability to mediate transcriptional activation.
Mutation of the CKII consensus sites affects the ability of IE2/IEP86 to activate viral promoters. To test how the mutations in the serine-rich region affected IE2/IEP86 transcriptional activation, the IE2/IEP86 cDNA expression plasmids containing the serine-region mutations (Fig. 1B) were cotransfected with promoter-luciferase reporter plasmids into U373MG cells. Promoter activation was measured by luciferase activity 48 h after transfection. The reporter plasmids contained either the promoter of the HCMV early gene UL112-113 (1) or the promoter of the HCMV delayed-early gene ICP36 (UL44) (19). To obtain dose response data, three different amounts of IE2/IEP86-expressing plasmids (0.1, 0.5, and 1.2 μg) were cotransfected with a constant amount of reporter plasmid (Fig. 4).
All of the mutations activated the promoter of the early gene UL112-113 to a greater extent than that of the WT IE2/IEP86 (Fig. 4). However, we noted that the 258-264 and 266-269 mutants were less efficient than the other mutations in this activation. In contrast, we found that while all the mutations activated the promoter for the delayed-early gene UL44 (ICP36), the levels of activation were consistently less than the WT IE2/IEP86 level. These differential effects of the mutations on the two viral promoters suggest that the serine-rich region and its phosphorylation status may play a role in the temporal control of HCMV gene expression. To test this hypothesis we introduced the mutations into viruses.
Introduction of serine-rich region mutations into viruses. We have made mutant viruses by homologous recombination in LEHFFs as described in Materials and Methods. To ascertain that the phenotype of the viruses was due to the mutation in IE2/IEP86 and not to an unwanted spontaneous mutation in another area of the BAC-Towne clone, we separately prepared and characterized at least two independent isolates for each mutation. These are denoted isolates A and B in the following data; in all cases the results of the two isolates were similar.
Viable stocks of viruses containing the mutation of the serines in the entire region (amino acids 258 to 275), serines between amino acids 266 and 275, and serines between amino acids 271 and 275 have been produced. However, the propagation of viruses containing mutations of serines between amino acids 258 and 264 and between amino acids 266 and 269 was extremely slow compared to propagation of the WT and the other mutants; therefore, we have not develop viral stocks of these mutants.
Figures 5 and 6 show analysis of WT Towne and the mutant viruses in LEHFFs infected at an MOI of 3. Viruses from media and cells were collected at various times after infection, and virus titers were determined by the TCID50 method (see Materials and Methods). Figure 5A shows the growth curves of WT Towne and mutants 258-275 and 271-275. Mutation of the serines in the entire region (residues 258 to 275) showed delayed growth kinetics compared with WT Towne virus. In contrast, mutation of the serines between residues 271 and 275 resulted in a virus that grew faster than WT Towne during the early phase of infection and then reached a plateau with similar or slightly reduced yield compared with growth of the WT. In Fig. 5A the input for mutant 271-275 was calculated to be slightly higher than that of the WT; this could account for the faster growth. However, we have repeated this experiment several times under conditions where there is no input discrepancy and seen the same result. For example, Fig. 5B shows faster growth of mutant 271-275 compared to growth of the WT under conditions where the cells were serum starved for 48 h prior to infection. Other experiments, presented below, further document the accelerated growth of mutant 271-275.
In Fig. 5C the appearance of viral proteins during the time course was determined. A total of 30 μg of total cell extract was separated by SDS-PAGE, and viral proteins were analyzed by Western analysis. The slow growth of the 258-275 mutant (Fig. 5A) correlated with delayed and reduced expression of the MIEPs, the delayed-early protein p52 (UL44), and late proteins gB and p28 (Fig. 5C). In contrast, the accelerated growth of the 271-275 mutant correlated with an early appearance of gB and p28 in comparison to their appearance in the WT, while expression of the MIEPs and p52 were similar to expression in the WT (Fig. 5C).
Figure 6 shows a time course of the appearance of viral proteins from mutant 266-275 compared with mutant 258-275 and WT Towne. The 266-275 mutant virus was isolated later than the other two and was analyzed separately. In the Western analyses shown, we have additionally analyzed the late protein pp65 and the precursor and cleaved forms of gB. Similar to mutant 258-275, mutant 266-275 showed delayed appearance of viral proteins relative to their appearance in WT Towne.
In Fig. 7A growth rates of WT Towne and mutants 258-275, 266-275 and 271-275 were compared at an MOI of 0.1 to determine whether the effects of the mutations were accentuated at a lower MOI. Virus titers in the culture medium were determined, and cells were harvested for protein analysis. Similar to results with the higher MOI infections (Fig. 5), mutant 271-275 grew faster than the WT during the early phase of infection and then reached a plateau with a slightly reduced yield compared to the WT yield. Also in agreement with results of the experiment with a higher MOI, mutant 258-275 grew considerably more slowly than the WT. The growth rate of mutants 266-275 was similar to that of mutant 258-275, which is in agreement with the delayed appearance of viral proteins indicated in Fig. 6.
Proteins extracted from the cells infected for 7 and 11 days at an MOI of 0.1 were subjected to Western analysis for the expression of immediate-early, early, and late proteins (Fig. 7B). These data are consistent with the growth curves and the previous protein production data. At day 7 postinfection mutant 271-275, which initially grew faster than the WT, showed levels of p52, p28, and pp65 that were increased relative to levels in WT Towne. By day 11 these increased levels were still noticeable; the most striking difference was a significantly elevated level of gB compared to the WT level. Overall, there appeared to be a general acceleration and increase in viral gene expression from mutant 271-275. In contrast, expression of all viral proteins was much lower than WT levels at day 7 from mutants 258-275 and 266-275. Expression increased by day 11 but still lagged significantly behind the level in the WT Towne virus.
The dramatic differences in growth rates between the different mutations do not correlate well with the modest effects of the various mutations on promoter activation seen in the cotransfection experiments shown in Fig. 4. This may indicate that other viral factors cooperate with mutated IEP86 to cause the observed growth phenotypes. Thus, we performed transfection-infection experiments where the mutant IE2/IEP86 would be produced from the infecting virus along with other viral factors. The same luciferase reporter plasmids, with UL112-113 and ICP36 as promoters, were transfected (1 μg) for 24 h prior to infection with WT Towne or the various serine region mutant viruses. Luciferase activity was determined 48 h after infection (Fig. 8). The activation of the UL112-113 promoter by all of the IE2/IEP86 mutants was greater than activation by WT IE2/IEP86 as noted in the cotransfection experiments. However, each of the mutants activated the ICP36 promoter to a greater extent than the WT, unlike the cotransfection experiments, where the activation levels of the mutants were lower than the WT level. Thus, neither of the experiments examining promoter activation by using reporter plasmids can explain the viral growth phenotypes of the various mutations in the serine-rich region (see Discussion).
DISCUSSION
The data presented above suggest that the serine-rich region between amino acids 258 and 275 affects the ability of HCMV IE2/IEP86 to control HCMV temporal gene expression. However, the data also suggest that the region is complex, as shown by the summary in Fig. 9. Mutation of all serines in this region (amino acids 258 to 275) or only of the serines in the second half of the region (266 to 275) resulted in viable viruses with delayed gene expression and slow growth compared to the WT Towne virus. However, mutation of the serines in the first half of the region (amino acids 258 to 264) or just the serines between amino acids 266 and 269 resulted in very slow propagation of viruses.
At this point we cannot explain why the entire serine-rich region can be mutated and still propagate a virus while mutations in smaller segments of the region appear to be more deleterious. One possibility is that the serine-rich region has interrelated subregions affecting interrelated functions, resulting in a phenotype that may only be detected during the course of the viral infection. Thus, mutating specific parts of the serine-rich region (e.g., amino acids 258 to 264 or 266 to 269) may compromise the interrelated functions in a way that results in the inhibition of growth, whereas mutation of the entire region, removing all the interrelated subregions and functions, is less deleterious, resulting in a slow-growing virus.
The ability of the serine-rich region to affect multiple functions of IE2/IEP86 may also arise from the effect of phosphorylation on the conformation of the protein. Our published modeling of the C-terminal half of IE2/IEP86 suggests that IE2/IEP86 has a dynamic tertiary structure in which subtle changes, caused by amino acid variations, phosphorylation, or sumoylation, may mediate significant changes in the functions of the protein (3). Thus, phosphorylation of the serine-rich region may alter the conformation and change activities in distant regions of the protein suspected to be involved in the transcriptional activation of early and late viral promoters (35).
The in vitro TBP binding and promoter activation data do not help to explain the puzzling phenotypes of the serine region mutants, especially that of the slow-growing mutants. Yet it is difficult to consider that alterations in the transcriptional functions of IE2/IEP86 do not contribute, at least in part, to the phenotypes of the mutants during viral infection. In this regard it should be considered that the phenotypic consequence of inappropriate transcriptional activation might be manifested in inappropriate formation of virions, e.g., inappropriate tegument formation. Thus, the phenotypes noted may result from the incoming virion's being defective rather than from an effect of the mutant IE2/IEP86 synthesized during the course of the infection. This possibility is most readily supported by the data from mutant 258-275 (Fig. 5) and mutant 266-275 (Fig. 6), which show delayed production of the MIEPs, suggesting that the activation of the MIE promoter may be delayed, an effect that would be most readily explained by malformation of the tegument of the infecting virion (6).
Although the serine-rich region is complex and may affect multiple functions, our studies of the 271-275 mutant provide the most straightforward conclusions about one function of the region. These studies clearly showed that hypophosphorylation of the serines between amino acids 271 and 275 resulted in the accelerated appearance of both early and late gene products and, correspondingly, the faster appearance of progeny viruses during the early phase of the growth curve. Thus, the activities of the kinases and phosphatases that target the serine-rich region may significantly control viral gene expression and the progression of viral growth. CKII is potentially such a kinase; recent data from Epstein-Barr virus suggest that hypophosphorylation of ZEBRA at its CKII sites abolished ZEBRA's capacity to repress Rta-mediated activation of Epstein-Barr virus late genes but did not alter ZEBRA's ability to synergize with Rta for the activation of early lytic cycle genes (8). Similarly, hypophosphorylation of the CKII sites between amino acids 271 and 275 may relieve repression of selected HCMV delayed-early and late genes (35) while maintaining the activation of early genes; hence mutation of these sites would result in premature expression of these genes, as we have observed. This model suggests that the viral infection induces cellular or viral kinases and phosphatases at appropriate times in order to mediate temporal expression of viral genes as needed for appropriate virion formation during the course of the infection.
ACKNOWLEDGMENTS
The authors thank Tom Shenk for reagents for construction of viruses, Hua Zhu for the BAC Towne UL122 clone, Wolfram Brune for his helpful suggestions, Sherri Adams for critical reading of the manuscript, and the members of the Alwine laboratory for helpful discussion and critical evaluation of the data. Cheers to all.
This work was supported by Public Health Service grant CA28379 awarded to J.C.A. by the National Cancer Institute.
Present address: Department of Neurology, Yale University School of Medicine, New Haven, CT 06520.
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ABSTRACT
The 86-kDa major immediate-early protein (IE2/IEP86) of human cytomegalovirus (HCMV) contains a serine-rich region (amino acids 258 to 275) with several consensus casein kinase II (CKII) sites. We performed extensive mutational analysis of this region, changing serines to alternating alanines and glycines. Mutation of the serines between amino acids 266 and 275 eliminated in vitro phosphorylation by CKII. In vitro CKII phosphorylation of the serines between amino acids 266 and 269 or between amino acids 271 and 275 inhibited the ability of IE2/IEP86 to bind to TATA-binding protein. Correspondingly, nonphosphorylatable mutants in these regions showed increased activation of specific HCMV gene promoters in transfection studies. Viruses containing mutations of the serines throughout the entire region (amino acids 258 to 275) or the second half (amino acids 266 to 275) of the region showed delayed expression of all viral proteins tested and, correspondingly, delayed growth compared to wild-type HCMV. Mutation of the serines in the first half of the serine-rich region (amino acids 258 to 264) or between amino acids 266 and 269 propagated very slowly and has not been further studied. In contrast, mutation of the serines between amino acids 271 and 275 resulted in accelerated virus growth and accelerated temporal expression of viral proteins. These results suggest that the serine-rich region is structurally complex, possibly affecting multiple functions of IE2/IEP86. The data show that the phosphorylation state of the serine-rich region, particularly between amino acids 271 and 275, modulates the temporal expression of viral genes.
INTRODUCTION
Transcription of the major immediate-early (MIE) gene of human cytomegalovirus (HCMV) produces a number of alternatively spliced and polyadenylated mRNAs that encode several MIE proteins (MIEPs) (25, 31, 32, 34). Two of these, IE1 (72 kDa; also called IE72, IE1491aa, or ppUL123) and IE2 (86 kDa; also called IEP86, IE2579aa, or ppUL122a) appear in abundance in lytic infections and have been extensively examined. These proteins alter the transcriptional activity of viral and cellular promoters and control temporal expression of the HCMV genes (7, 9, 10, 13, 15-17, 21-23, 26, 29, 30, 33, 36, 38). The MIEPs have also been shown to affect cell cycle control and apoptosis (20, 37, 39).
We have previously mapped the major phosphorylation sites of IE2/IEP86 (11) (Fig. 1). One of these sites is a serine-rich region spanning amino acids 258 to 275 (Fig. 1) that contains several consensus sites for casein kinase II (CKII). To determine the role of this region and its phosphorylation in the function of IE2/IEP86, we have made a number of mutations through the region, changing the serines to alternating alanines and glycines. Studies of mutant viruses show that some of these mutations are deleterious to viral gene expression and virus growth, while others accelerate these processes, suggesting that the role of the serine-rich region in IE2/IEP86 function is complex, affecting multiple functions. Our studies establish that one function modulated by the phosphorylation state of the serine-rich region is the temporal expression of viral genes.
MATERIALS AND METHODS
Cells and plasmids. The glioblastoma-astrocytoma cell line U373MG was maintained at passage numbers less than 30 in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, Glutamax, and antibiotics. Life-extended human foreskin fibroblasts (LEHFFs) (5) were maintained at passage numbers less than 15 in DMEM supplemented with 10% fetal calf serum, Glutamax, and antibiotics.
Plasmid pRSV86 Towne contains a cDNA encoding IE2/IEP86 utilizing the complete Towne sequence (3) under the control of the Rous sarcoma virus long terminal repeat. Mutations in the serine-rich region (between amino acids 266 and 275, 266 and 269, and 271 and 275) were made by site-directed mutagenesis by using the QuickChange kit (Stratagene) and the following oligonucleotides: for mutant 266-275, the 5' oligonucleotide was 5'-GCGGTGCGGCTGGGGACGCGGAGGGTGAGGCCGAGGAG-3', and the 3' oligonucleotide was 5'-CTCCTCGGCCTCACCCTCCGCGTCCCCAGCCGCACCGC-3'; for mutant 266-269, the 5' oligonucleotide was 5'-CTTCCTCCTGCGGTGCGGCTGCGGACTCGG-3', and the 3' oligonucleotide was 5'-CTCTCCGAGTCCGCAGCCGCACCGCAGGAGG-3'; for mutant 271-275, the 5' oligonucleotide was 5'-GCAGTTCGGCTTCGGACGCGGAGGGTGAGGCCGAGGAG-3' and the 3' oligonucleotide was 5'-CTCCTCGGCCTCACCCTCCGCGTCCGAAGCCGAACTGC-3'. Mutations in amino acids 258 to 264 and 258 to 275 were made by using a splicing overlap extension protocol (11). First, mutant 258-264 was made by using the 5' oligonucleotide 5'-GCAGGAGCAGGAGACGGAGAGGCAGAGGGAGAGGAGATGAAATGC-3' and the 3'oligonucleotide 5'-CTCCCTCTGCCTCTCCGTCTCCTGCTCCTGCGCATCCTGCTCCTGCT-3'. Then the 258-275 mutant was made by using the 258-264 mutant as a template with the 5' oligonucleotide 5'-GGAGCAGGAGCAGGAGCAGGATGCAGTTCGGCTTCG-3' and the 3' oligonucleotide 5'-GCATCCTGCTCCTGCTCCTGCTCCATCTTCATCGGGCCG-3'. All mutations were verified by sequence analysis.
Plasmid pRL43a contains the genomic MIE region from the Towne strain of HCMV (18, 27). To introduce IE2/IEP86 mutations into pRL43a, we partially digested the plasmid with SmaI, followed by StuI digestion. The SmaI-StuI fragment containing the serine-rich region in pRL43a was replaced with the SmaI-StuI fragment isolated from pRSV86 plasmids containing the mutations in an IE2/IEP86 cDNA.
Reporter plasmids contained the luciferase gene under control of the following promoters: the promoter of the HCMV UL112-113 early genes, plasmid pHM142 (1); and the promoter of the delayed-early gene UL44 (ICP36), plasmid pICP36 (19).
Plasmids expressing glutathione S-transferase (GST) fusion proteins with the full-length IE2/IEP86 coding region and with the IE2/IEP86 fragment encoding amino acids 252 to 367 were prepared containing both the wild-type (WT) and serine region mutations by using the pGEX3X vector (Pharmacia).
Antibodies. For Western analyses, IE1/IE72 and IE2/IEP86 were detected by using anti-exon2/3, a polyclonal antiserum prepared for this laboratory by Cocalico Biologicals, Inc. (Reamstown, Pa.); it recognizes the common 85 amino acids in all known HCMV MIEPs (11). Monoclonal antibody MAb810 (Chemicon), which recognizes the N-terminal region of IE1/IE72 and IE2/IEP86, was used to immunoprecipitate the MIE proteins. Mouse monoclonal antibodies against the p28 tegument protein (UL99), p65 tegument protein (UL83), glycoprotein B (gB or UL55) and polymerase accessory factor (p52 or UL44) were purchased from Advanced Biotechnologies Inc.
Transfections, luciferase assays, immunoprecipitations, and Western analyses. U373MG cells were transfected by using Fugene (Roche) as previously described (3). Briefly, for luciferase assays 2.5 x 105 cells were plated per well in 12-well plates. A transfection mixture was made containing 0.2 μg of the green fluorescent protein-expressing plasmid pCMS-EGFP (Clontech); 0.5 μg of reporter plasmid; and 0.1, 0.5, or 1.2 μg of IE2/IEP86-expressing plasmids plus control plasmid pRSV3/BglII to bring the expression plasmid input to 1.2 μg. One-quarter of each transfection mixture was added to four different wells of the 12-well plate. Three wells were used for luciferase assays, and the fourth was used to obtain total extract to assay for IE2/IEP86 protein expression. Luciferase activity was assayed by using the Promega luciferase assay system, with 12 μg of transfected cell extract harvested according to the manufacturer's instructions, and measured on a Berthold 9501 Lumat luminometer.
Transfection-infection studies utilized a transfection mixture containing 0.2 μg of red fluorescent protein-expressing plasmid (pDsRed1-C1) and 1 μg of reporter plasmid. One-third of the mixture was added to three wells of a 12-well plate. At 24 h posttransfection, the cells were infected with WT or mutant viruses at a multiplicity of infection (MOI) of 5 in 500 μl of medium; 5 h later the inocula were removed, the cells were washed twice with phosphate-buffered saline, and 1 ml of medium was added. At 48 h postinfection the cells were harvested, and luciferase activity was assayed as described above.
Approximately 1.6 x 106 U373MG cells were plated on 60-mm plates for transfections used for immunoprecipitation experiments. Cells were transfected with 1.8 μg of the IE2/IEP86-expressing plasmids plus 0.2 μg of pCMS-EGFP (Clontech). Total cell extracts were prepared by lysing the cells in radioimmunoprecipitation assay buffer (1% NP-40, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 150 mM NaCl, 10 nM Na phosphate [pH 7.2], 2 mM EDTA) plus 1 μg of leupeptin per ml, 0.7 μg of pepstatin per ml, 1 mM phenylmethylsulfonyl fluoride, 10 μg of aprotinin per ml, 2.5 μg of E64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane] per ml, 4 mN NaF, and 0.1 mM sodium orthovanadate. Lysates were centrifuged for 30 min at 4°C; supernatants were used for immunoprecipitation by overnight incubation of 1 mg of extract, 1 μl of MAb810, and 45 μl of protein G Sepharose beads (75% slurry) at 4°C. The samples were then washed, boiled in SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer, and separated by SDS-8% PAGE. The gel was transferred to nitrocellulose and probed with polyclonal antibody anti-exon 2/3, followed by incubation with the anti-rabbit secondary antibody. The transfer was developed by using enhanced chemiluminescence (Amersham).
In vitro kinase assays and binding assays. The GST fusion proteins were made in Escherichia coli BL21(DE3) pLysS grown in Terrific broth. Fusion protein expression was induced for 1 h with IPTG (isopropyl-?-D-thiogalactopyranoside), bacteria were collected, and fusion proteins were prepared and purified by using glutathione-Sepharose beads (Sigma) as previously described (21). Equivalent amounts (1 to 5 μg) of fusion proteins (quantitated by Sypro red staining) were used for in vitro kinase experiments and in vitro binding experiments.
For in vitro CKII assays, fusion proteins were incubated for 30 min at 30°C with 25 U of CKII (New England Biolabs) in 1x CKII buffer (20 mM Tris-HCl [pH 7.5], 50 mM KCl, and 10 mM MgCl2) (New England Biolabs) containing 0.2 mM ATP and 20 μCi of [-32P]ATP. Assays with other kinases were performed following the manufacturers' instructions. The phosphorylated fusion proteins were purified by using gluthatione-Sepharose beads, boiled in SDS-PAGE loading buffer, and separated by SDS-10% PAGE.The gels were stained with Sypro red to quantify the total amounts of protein, dried, and exposed to a PhophorImager screen to quantitate phosphorylation.
For in vitro factor binding assays, GST fusion proteins were pretreated with nonradioactive ATP and either CKII or heat-inactivated CKII, as described above. The treated proteins were incubated for 30 min at 4°C with 7.5 x 104 cpm of in vitro transcribed and translated [35S]methionine-labeled TATA binding protein (TBP) in 1 ml of NETN buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) containing 3% bovine serum albumin plus protease and phosphatase inhibitors (5 mM NaF, 0.1 mM Na3VO4, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). A total of 45 μl of a 33% glutathione-agarose bead slurry were added to the mix for a 1-h incubation at 4°C. Beads were collected and washed five times with NETN plus protease and phosphatase inhibitors. The in vitro translated factors bound to the GST fusion proteins on the beads were eluted by boiling in SDS-PAGE loading buffer and separated by SDS-PAGE. The gels were dried, and labeled proteins were detected by autoradiography and quantified by using a PhosphorImager.
Mutant virus construction. Virus stocks containing the serine-rich region mutations were made by homologous recombination in LEHFFs. LEHFFs were coelectroporated with 4 μg of BAC-Towne-UL122 DNA, a bacterial artificial chromosome containing the Towne strain of HCMV with a deletion of ORF UL122 (12, 24), 5 μg of Hind III-linearized pRL43a plasmid containing the serine-rich region mutations, and 2 μg of pp71-expressing plasmid pCGNpp71 (24). Electroporation was performed by using a Bio-Rad Gene Pulser with 0.4-cm cuvettes at 260V and 950 μF. Medium was changed the day after electroporation. Since IE2/IEP86 is essential for viral growth, the only viruses which grow are those in which a viable mutant IE2/IEP86 was inserted by homologous recombination. Mutations were confirmed by sequence analysis of viral DNA harvested from infections. In addition, viral DNA extracted from cells infected with the mutant viruses was cleaved with EcoRI, separated on 0.7% agarose gels, and visualized by Southern analysis, with plasmid pRSV86 used as a probe, to check for improper recombination events within the MIE region (results not shown).
Virus stocks were made by collecting viruses from media and sonicated cells, followed by a 10-min centrifugation at 4,000 rpm in a Sorvall RT7 centrifuge to remove cellular debris. Viruses were concentrated by ultracentrifugation onto a 20% sorbitol cushion by using a Beckman SW28 rotor at 19,000 rpm as previously described (2, 14). Pelleted virus was resuspended in serum-free medium.
Viral infection studies. For virus growth curves and infection time courses, 2 x 105 LEHFFs were plated per well in six-well plates, 3 days before infection. Cells were infected by using 1 ml of medium containing input virus at an MOI of 3 or 0.1. Four hours after infection the virus inoculum was removed, the cells were washed twice with phosphate-buffered saline, and 2.5 ml of fresh medium was added. For virus growth curves, viruses were collected either from medium (infection at an MOI of 0.1) or from medium and sonicated cells (infection at an MOI of 3). Virus titers were obtained by using the median tissue culture infectious dose (TCID50) method (4, 28). For Western analysis, infected cells were lysed in radioimmunoprecipitation assay buffer; 10 to 30 μg of total extract was separated by SDS-8% PAGE. The gels were transferred to nitrocellulose and probed with antibodies as described above.
RESULTS
In vitro analysis of IE2/IEP86 phosphorylation with purified kinases. Figure 1A shows a map of IE2/IEP86; the regions denoted 2/3, 5A, 5B, 5C, UR, and 6 are domains of the protein which we previously made as GST fusion proteins (21). The arrows under domains 5A and 5C indicate that these domains bound transcription factors (e.g., TBP and Tef-1) in vitro (21).
We previously mapped and mutated major phosphorylation sites in IE2/IEP86 at amino acids T27, S144, and T233/S234 (11, 12). In vitro phosphorylation of these sites in GST fusion proteins by purified kinases focused on serum-inducible kinases, particularly the mitogen-activated protein kinases ERK2 and JNK1 (11). To develop a more complete picture of the phosphorylation of IE2/IEP86, other purified serine/threonine kinases were tested for their ability to phosphorylate both WT and phosphorylation site mutant GST fusion substrates (Fig. 1A), including the WT and mutated serine-rich region discussed in detail below (Fig. 2). The additional kinases tested were p34Cdc2-cyclin B (Cdc2), a key kinase involved in G2/M transition; CKII, a constitutive nuclear kinase; and two second-messenger-regulated kinases, cyclic AMP-regulated protein kinase A (PKA) and protein kinase C (PKC).
Purified kinases were incubated with the various bacterially expressed, purified GST fusion substrates in the presence of [-32P]ATP as described in Materials and Methods. The results are summarized in Fig. 1A, where the kinases that specifically phosphorylated individual IE2/IEP86 domains are listed below the respective domains. Phosphorylation by those kinases indicated in italics and underlined was inhibited by the phosphorylation site mutation shown above each region. It is interesting that there are several cases of overlapping kinase specificities; for example, T27 appears to be a phosphorylation site for both ERK2 and Cdc2, and T233/S234 appears to be phosphorylated by both ERK2 and PKA. Although major phosphorylation sites have been previously mapped and characterized (11), these data suggest that other sites can be targeted by a number of different kinases. In particular, the serine-rich region is a strong target for CKII; this is characterized below.
Mutations of the serine-rich region. Figure 1B shows the amino acid sequence of the WT serine-rich region; serines that are potential CKII sites are boxed. To study the significance of the phosphorylation of this region, we made a variety of mutations where either all or portions of the serines between amino acids 258 and 275 were mutated to alternating glycines and alanines. These mutations are listed in Fig. 1B (the mutated bases are underlined). Each mutation was introduced into an IE2/IEP86 cDNA expression plasmid. These were transfected into U373MG cells to test the expression and stability of the mutant proteins. At 48 h after transfection the cells were harvested, and IE2/IEP86 was immunoprecipitated by using MAb810, which recognizes the amino-terminal end of the protein. The precipitated proteins were separated by SDS-PAGE and detected by Western analysis by using a polyclonal antibody that recognizes the first 85 amino acids common to all MIEPs. Figure 1C shows that the various mutations in the serine-rich region do not significantly alter the levels of IE2/IEP86 or its sumoylated form in U373MG cells transfected with plasmids expressing cDNA copies of WT or mutated IE2/IEP86.
The serine-rich region is a substrate for phosphorylation by CKII in vitro. To determine which serines in the region were needed for CKII phosphorylation, we inserted each mutation into the GST-5C fusion protein (Fig. 1). Equal amounts of purified GST fusion protein were incubated with CKII and [-32P]ATP (see Material and Methods) and then fractionated by SDS-PAGE. The autoradiograph of the phosphorylated forms is shown at the bottom of Fig. 2; the intensity of phosphorylation was quantitated by PhosphorImager analysis and is shown by the bar graphs. Mutation of all serines between amino acids 258 and 275 and mutation of only the serines between amino acids 266 and 275 dramatically reduced CKII phosphorylation. This result suggests that the potential CKII consensus sites between amino acids 266 and 275 are the targets of phosphorylation by CKII in vitro. Mutation of the first three (between amino acids 266 and 269) or the last three serines (between amino acids 271 and 275) resulted in a 38 and 60% reduction in CKII phosphorylation, respectively, suggesting that CKII utilizes the serines in each region.
The panel on the left of Fig. 2 shows a similar CKII labeling experiment using GST fusions with full-length IE2/IEP86 containing either WT or mutant 258-275 sequences. Mutation of the serines between 258 and 275 resulted in an 80% reduction in full-length IE2/IEP86 phosphorylation by CKII. This indicates that the serine-rich region is the major site for in vitro CKII phosphorylation of IE2/IEP86.
The serine-rich region is also phosphorylated in vivo. Mutant 258-275 was inserted into a full-length IE2/IEP86 cDNA expression plasmid. The plasmid was transfected into U373MG cells that were labeled with 32P; IE2/IEP86 was purified and subjected to two-dimensional tryptic phosphopeptide analysis (data not shown). Compared to WT IE2/IEP86, the mutation of the serine-rich region resulted in a dramatic decrease in the intensity of tryptic peptide D/D', one of the major tryptic phosphopeptides that we previously characterized (11).
In vitro phosphorylation of the serine-rich region by CKII inhibits binding to TBP. We showed previously that regions 5A and 5C (Fig. 1A) of IE2/IEP86 can independently bind TBP (21). Since region 5C contains the serine-rich region, we determined whether TBP binding was affected by CKII phosphorylation. Equal amounts of WT and mutant GST 5C fusion proteins were incubated for 30 min at 30°C with nonradioactive ATP plus CKII or heat-inactivated CKII. The treated proteins were then incubated with in vitro transcribed and translated [35S]methionine-labeled TBP (see Materials and Methods). GST fusion proteins and the labeled factors were purified on glutathione beads, eluted, separated by SDS-PAGE, and detected and quantitated by autoradiography and PhosphorImager analysis. Figure 3 shows that treatment with CKII, but not heat-inactivated CKII, inhibited binding of both the WT and the 258-64 mutant fusion proteins to TBP. However, mutation of serines between amino acids 266 and 275, 266 and 269, and 271 and 275 resulted in fusion proteins that bound TBP regardless of the treatment by CKII. These results indicate that hypophosphorylation of the serines between residues 266 and 269 or 271 and 275 correlates with TBP binding. These data suggest that the phosphorylation status of the latter half of the serine-rich region may regulate protein-protein interactions of IE2/IEP86, which may affect its ability to mediate transcriptional activation.
Mutation of the CKII consensus sites affects the ability of IE2/IEP86 to activate viral promoters. To test how the mutations in the serine-rich region affected IE2/IEP86 transcriptional activation, the IE2/IEP86 cDNA expression plasmids containing the serine-region mutations (Fig. 1B) were cotransfected with promoter-luciferase reporter plasmids into U373MG cells. Promoter activation was measured by luciferase activity 48 h after transfection. The reporter plasmids contained either the promoter of the HCMV early gene UL112-113 (1) or the promoter of the HCMV delayed-early gene ICP36 (UL44) (19). To obtain dose response data, three different amounts of IE2/IEP86-expressing plasmids (0.1, 0.5, and 1.2 μg) were cotransfected with a constant amount of reporter plasmid (Fig. 4).
All of the mutations activated the promoter of the early gene UL112-113 to a greater extent than that of the WT IE2/IEP86 (Fig. 4). However, we noted that the 258-264 and 266-269 mutants were less efficient than the other mutations in this activation. In contrast, we found that while all the mutations activated the promoter for the delayed-early gene UL44 (ICP36), the levels of activation were consistently less than the WT IE2/IEP86 level. These differential effects of the mutations on the two viral promoters suggest that the serine-rich region and its phosphorylation status may play a role in the temporal control of HCMV gene expression. To test this hypothesis we introduced the mutations into viruses.
Introduction of serine-rich region mutations into viruses. We have made mutant viruses by homologous recombination in LEHFFs as described in Materials and Methods. To ascertain that the phenotype of the viruses was due to the mutation in IE2/IEP86 and not to an unwanted spontaneous mutation in another area of the BAC-Towne clone, we separately prepared and characterized at least two independent isolates for each mutation. These are denoted isolates A and B in the following data; in all cases the results of the two isolates were similar.
Viable stocks of viruses containing the mutation of the serines in the entire region (amino acids 258 to 275), serines between amino acids 266 and 275, and serines between amino acids 271 and 275 have been produced. However, the propagation of viruses containing mutations of serines between amino acids 258 and 264 and between amino acids 266 and 269 was extremely slow compared to propagation of the WT and the other mutants; therefore, we have not develop viral stocks of these mutants.
Figures 5 and 6 show analysis of WT Towne and the mutant viruses in LEHFFs infected at an MOI of 3. Viruses from media and cells were collected at various times after infection, and virus titers were determined by the TCID50 method (see Materials and Methods). Figure 5A shows the growth curves of WT Towne and mutants 258-275 and 271-275. Mutation of the serines in the entire region (residues 258 to 275) showed delayed growth kinetics compared with WT Towne virus. In contrast, mutation of the serines between residues 271 and 275 resulted in a virus that grew faster than WT Towne during the early phase of infection and then reached a plateau with similar or slightly reduced yield compared with growth of the WT. In Fig. 5A the input for mutant 271-275 was calculated to be slightly higher than that of the WT; this could account for the faster growth. However, we have repeated this experiment several times under conditions where there is no input discrepancy and seen the same result. For example, Fig. 5B shows faster growth of mutant 271-275 compared to growth of the WT under conditions where the cells were serum starved for 48 h prior to infection. Other experiments, presented below, further document the accelerated growth of mutant 271-275.
In Fig. 5C the appearance of viral proteins during the time course was determined. A total of 30 μg of total cell extract was separated by SDS-PAGE, and viral proteins were analyzed by Western analysis. The slow growth of the 258-275 mutant (Fig. 5A) correlated with delayed and reduced expression of the MIEPs, the delayed-early protein p52 (UL44), and late proteins gB and p28 (Fig. 5C). In contrast, the accelerated growth of the 271-275 mutant correlated with an early appearance of gB and p28 in comparison to their appearance in the WT, while expression of the MIEPs and p52 were similar to expression in the WT (Fig. 5C).
Figure 6 shows a time course of the appearance of viral proteins from mutant 266-275 compared with mutant 258-275 and WT Towne. The 266-275 mutant virus was isolated later than the other two and was analyzed separately. In the Western analyses shown, we have additionally analyzed the late protein pp65 and the precursor and cleaved forms of gB. Similar to mutant 258-275, mutant 266-275 showed delayed appearance of viral proteins relative to their appearance in WT Towne.
In Fig. 7A growth rates of WT Towne and mutants 258-275, 266-275 and 271-275 were compared at an MOI of 0.1 to determine whether the effects of the mutations were accentuated at a lower MOI. Virus titers in the culture medium were determined, and cells were harvested for protein analysis. Similar to results with the higher MOI infections (Fig. 5), mutant 271-275 grew faster than the WT during the early phase of infection and then reached a plateau with a slightly reduced yield compared to the WT yield. Also in agreement with results of the experiment with a higher MOI, mutant 258-275 grew considerably more slowly than the WT. The growth rate of mutants 266-275 was similar to that of mutant 258-275, which is in agreement with the delayed appearance of viral proteins indicated in Fig. 6.
Proteins extracted from the cells infected for 7 and 11 days at an MOI of 0.1 were subjected to Western analysis for the expression of immediate-early, early, and late proteins (Fig. 7B). These data are consistent with the growth curves and the previous protein production data. At day 7 postinfection mutant 271-275, which initially grew faster than the WT, showed levels of p52, p28, and pp65 that were increased relative to levels in WT Towne. By day 11 these increased levels were still noticeable; the most striking difference was a significantly elevated level of gB compared to the WT level. Overall, there appeared to be a general acceleration and increase in viral gene expression from mutant 271-275. In contrast, expression of all viral proteins was much lower than WT levels at day 7 from mutants 258-275 and 266-275. Expression increased by day 11 but still lagged significantly behind the level in the WT Towne virus.
The dramatic differences in growth rates between the different mutations do not correlate well with the modest effects of the various mutations on promoter activation seen in the cotransfection experiments shown in Fig. 4. This may indicate that other viral factors cooperate with mutated IEP86 to cause the observed growth phenotypes. Thus, we performed transfection-infection experiments where the mutant IE2/IEP86 would be produced from the infecting virus along with other viral factors. The same luciferase reporter plasmids, with UL112-113 and ICP36 as promoters, were transfected (1 μg) for 24 h prior to infection with WT Towne or the various serine region mutant viruses. Luciferase activity was determined 48 h after infection (Fig. 8). The activation of the UL112-113 promoter by all of the IE2/IEP86 mutants was greater than activation by WT IE2/IEP86 as noted in the cotransfection experiments. However, each of the mutants activated the ICP36 promoter to a greater extent than the WT, unlike the cotransfection experiments, where the activation levels of the mutants were lower than the WT level. Thus, neither of the experiments examining promoter activation by using reporter plasmids can explain the viral growth phenotypes of the various mutations in the serine-rich region (see Discussion).
DISCUSSION
The data presented above suggest that the serine-rich region between amino acids 258 and 275 affects the ability of HCMV IE2/IEP86 to control HCMV temporal gene expression. However, the data also suggest that the region is complex, as shown by the summary in Fig. 9. Mutation of all serines in this region (amino acids 258 to 275) or only of the serines in the second half of the region (266 to 275) resulted in viable viruses with delayed gene expression and slow growth compared to the WT Towne virus. However, mutation of the serines in the first half of the region (amino acids 258 to 264) or just the serines between amino acids 266 and 269 resulted in very slow propagation of viruses.
At this point we cannot explain why the entire serine-rich region can be mutated and still propagate a virus while mutations in smaller segments of the region appear to be more deleterious. One possibility is that the serine-rich region has interrelated subregions affecting interrelated functions, resulting in a phenotype that may only be detected during the course of the viral infection. Thus, mutating specific parts of the serine-rich region (e.g., amino acids 258 to 264 or 266 to 269) may compromise the interrelated functions in a way that results in the inhibition of growth, whereas mutation of the entire region, removing all the interrelated subregions and functions, is less deleterious, resulting in a slow-growing virus.
The ability of the serine-rich region to affect multiple functions of IE2/IEP86 may also arise from the effect of phosphorylation on the conformation of the protein. Our published modeling of the C-terminal half of IE2/IEP86 suggests that IE2/IEP86 has a dynamic tertiary structure in which subtle changes, caused by amino acid variations, phosphorylation, or sumoylation, may mediate significant changes in the functions of the protein (3). Thus, phosphorylation of the serine-rich region may alter the conformation and change activities in distant regions of the protein suspected to be involved in the transcriptional activation of early and late viral promoters (35).
The in vitro TBP binding and promoter activation data do not help to explain the puzzling phenotypes of the serine region mutants, especially that of the slow-growing mutants. Yet it is difficult to consider that alterations in the transcriptional functions of IE2/IEP86 do not contribute, at least in part, to the phenotypes of the mutants during viral infection. In this regard it should be considered that the phenotypic consequence of inappropriate transcriptional activation might be manifested in inappropriate formation of virions, e.g., inappropriate tegument formation. Thus, the phenotypes noted may result from the incoming virion's being defective rather than from an effect of the mutant IE2/IEP86 synthesized during the course of the infection. This possibility is most readily supported by the data from mutant 258-275 (Fig. 5) and mutant 266-275 (Fig. 6), which show delayed production of the MIEPs, suggesting that the activation of the MIE promoter may be delayed, an effect that would be most readily explained by malformation of the tegument of the infecting virion (6).
Although the serine-rich region is complex and may affect multiple functions, our studies of the 271-275 mutant provide the most straightforward conclusions about one function of the region. These studies clearly showed that hypophosphorylation of the serines between amino acids 271 and 275 resulted in the accelerated appearance of both early and late gene products and, correspondingly, the faster appearance of progeny viruses during the early phase of the growth curve. Thus, the activities of the kinases and phosphatases that target the serine-rich region may significantly control viral gene expression and the progression of viral growth. CKII is potentially such a kinase; recent data from Epstein-Barr virus suggest that hypophosphorylation of ZEBRA at its CKII sites abolished ZEBRA's capacity to repress Rta-mediated activation of Epstein-Barr virus late genes but did not alter ZEBRA's ability to synergize with Rta for the activation of early lytic cycle genes (8). Similarly, hypophosphorylation of the CKII sites between amino acids 271 and 275 may relieve repression of selected HCMV delayed-early and late genes (35) while maintaining the activation of early genes; hence mutation of these sites would result in premature expression of these genes, as we have observed. This model suggests that the viral infection induces cellular or viral kinases and phosphatases at appropriate times in order to mediate temporal expression of viral genes as needed for appropriate virion formation during the course of the infection.
ACKNOWLEDGMENTS
The authors thank Tom Shenk for reagents for construction of viruses, Hua Zhu for the BAC Towne UL122 clone, Wolfram Brune for his helpful suggestions, Sherri Adams for critical reading of the manuscript, and the members of the Alwine laboratory for helpful discussion and critical evaluation of the data. Cheers to all.
This work was supported by Public Health Service grant CA28379 awarded to J.C.A. by the National Cancer Institute.
Present address: Department of Neurology, Yale University School of Medicine, New Haven, CT 06520.
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