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Journal of Virology, February 2007, p. 1980-1989, Vol. 81, No. 4
0022-538X/07/$08.00+0     doi:10.1128/JVI.02265-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mapping of Key Functions of the Herpes Simplex Virus 1 US3 Protein Kinase: the US3 Protein Can Form Functional Heteromultimeric Structures Derived from Overlapping Truncated Polypeptides{triangledown}

Alice P. W. Poon and Bernard Roizman*

Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, 910 East 58th Street, Chicago, Illinois 60637

Received 16 October 2006/ Accepted 22 November 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Earlier studies have shown that the herpes simplex virus (HSV) US3 encodes two transcriptional units directing the synthesis of the US3 (residues 1 to 481) and US3.5 (residues 77 to 481) protein kinases. Both kinases phosphorylate histone deacetylase 1 (HDAC1) and HDAC2 and enable the expression of genes cotransduced into U2OS cells by recombinant baculoviruses, an activity designated the "helper function." The two kinases differ with respect to antiapoptotic activity. In the studies reported here, we made a series of FLAG-tagged amino- and carboxyl-terminal truncations of US3 and these were tested for antiapoptotic activity, phosphorylation of HDAC1, and the helper function. We report the following. (i) HDAC1 phosphorylation and the helper function were expressed in cells transduced by the truncation encoding residues 182 to 481 but not in cells transduced by the truncation encoding residues 189 to 481 or the amino-terminal polypeptides encompassing the first 188 amino acids. (ii) The self-posttranslational modification requires residues 164 to 481. (iii) The antiapoptotic activity requires both the amino-terminal and the carboxyl-terminal domains, of which the truncated protein containing residues 1 to 163 and that containing residues 164 to 481, respectively, were the smallest fragments tested to be effective. The two domains need not be on the same molecule, but they must overlap. The smallest overlapping pair tested was the fragment containing residues 1 to 181 and that containing residues 164 to 481. Consistent with the hypothesis that the effective overlapping truncations form a heteromultimeric structure, antibody to FLAG coprecipitated untagged US3 from lysates of cells cotransduced with FLAG-tagged, truncated US3 constructs. Although US3 has been reported to be a monomeric enzyme, the results indicate that it can form enzymatically active multimeric structures.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The US3 open reading frame (ORF) encodes a protein that was identified as a protein kinase initially on the basis of its sequence (11) and subsequently through biochemical studies (5, 20). A related kinase encoded by pseudorabies virus was reported to be a serine-threonine kinase targeting the sequence (R)nX(S/T)YY, where n is ≥3, X can be Arg, Ala, Val, Pro, or Ser, and Y can be any of these residues except an acidic residue (21, 22). The US3 protein kinase is dispensable for virus growth in cells in culture (20). The {Delta}US3 virus exemplified by the R7041 mutant (10) yields only 10-fold less virus than the wild-type parent (12). The US3 kinase appears to be essential in experimental animal systems. The {Delta}US3 mutant virus (R7041) had a PFU/50% lethal dose value of 108, compared to 102 for the wild-type parent. The virus established latency, but recovery of virus on explantation was less efficient than that of the wild-type parent (12).

In recent years, interest in the functions of the US3 protein kinase peaked on the basis of several newly discovered functions. Briefly, the following has been observed.

(i) Wild-type virus-infected cells accumulate two mRNAs (25). We recently reported that the longer mRNA encodes the 481-residue US3 protein kinase. The shorter RNA encodes a less abundant, truncated protein beginning with methionine 77. We have designated the truncated protein kinase US3.5. In cells infected with a mutant lacking the {alpha}22 ORF encoding infected-cell protein no. 22 (ICP22), the predominant mRNA is that encoding US3.5. Both US3 and US3.5 protein kinases are expressed in cells transduced by baculoviruses containing the US3 ORF driven by the cytomegalovirus (CMV) immediate-early promoter (19). Both US3 and US3.5 proteins are present in nuclei and cytoplasm and associate with mitochondria (16).

(ii) The US3 protein kinase blocks apoptosis induced by replication-incompetent viruses (e.g., {Delta}ICP4 virus), exogenous agents (e.g., sorbitol), or overexpression of proapoptotic proteins (1, 7, 9, 13-15). In contrast, the US3.5 protein kinase does not block apoptosis (16).

(iii) The expression of mammalian or herpes simplex virus (HSV) genes driven by the immediate-early CMV promoter and introduced into baculoviruses requires the maintenance of the transduced cells in a medium containing sodium butyrate (3). In U2OS cells, cotransduction of either US3 or US3.5 supplants the requirement for sodium butyrate and enables the cotransduced gene to be expressed. This function, designated the helper function, correlates with the phosphorylation of histone deacetylase 1 (HDAC1) and HDAC2 (17).

(iv) Lastly, Roller and associates reported that the US3 protein kinase plays a key role in the maturation of HSV. In the absence of the US3 protein kinase, capsids are retained in nuclei. Envelopment appears to be limited and occurs at the inner nuclear membrane invaginated into the nucleus (23, 24, 27). The presence of similar structures in cells infected with a mutant expressing only the US3.5 protein kinase suggests that US3.5 is less efficient in enabling the restructuring of the nuclear envelope to enable the release of capsids from nuclei (16).

The US3 protein kinase thus appears to be a multifunctional enzyme that plays an important role in the biology of HSV. The objective of the studies reported here was to map the functions associated with the US3 protein. We report that the posttranslational modifications of HDAC1 are associated with functions mapping carboxyl terminal to residue 182 but are not expressed by a truncated protein containing residues 189 to 481 or the amino-terminal polypeptides encompassing the first 188 amino acids. Coincidentally, the truncated protein containing residues 182 to 481 has the helper functions described above, but that containing residues 189 to 481 does not. In contrast, the antiapoptotic activity of the US3 protein kinase requires both the amino-terminal domain (residues 1 to 163) and the carboxyl-terminal domain (residues 164 to 481), which could be located on separate molecules, provided that their amino acid sequences overlap. Consistent with the hypothesis that the overlapping truncated proteins form multimeric structures, we showed that antibody to FLAG-tagged, truncated US3 proteins also pulled down untagged US3 protein kinase from lysates of doubly transduced cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and viruses. U2OS (human osteosarcoma) cells were obtained from the American Type Culture Collection and maintained in McCoy's 5A medium (Gibco) supplemented with 10% fetal bovine serum (FBS). Insect cell line Sf9 (Spodoptera frugiperda) was obtained from PharMingen and was maintained in Grace's medium supplemented with 10% fetal bovine serum.

The baculoviruses used in this study are listed in Fig. 1. For the purpose of identification, they are also designated by numbers following "BC." Those expressing full-length US3 (untagged [BC2600], FLAG tagged [BC2602], and K220N mutant [BC2621]), US3.5 (FLAG tagged [BC2608]), or US11 (BC2618) had been described previously (16, 17, 19, 28). Baculoviruses expressing FLAG-tagged N-terminally or C-terminally truncated US3 proteins were generated for this study.


Figure 1
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  		FIG. 1. Schematic representation of HSV-1(F) US3 (line 1), US3.5 (line 2), C- or N-terminal US3 fragments (lines 3 to 9), and the K220N mutant (line 10) expressed by recombinant baculoviruses and their functions. One wild-type US3 construct and all the truncated constructs were tagged with the FLAG epitope at the carboxyl terminus of the open reading frame (lines 1 to 9). In line 10, the filled circle represents replacement of Lys-220 with Asn in the K220N US3 mutant. Constructs shown in lines 5, 6, and 7 were expressed at very low levels in U2OS cells exposed to 2 PFU of recombinant viruses per cell but better in cells exposed to >10 PFU/cell or in the presence of 6 mM sodium butyrate.

 
Generation of recombinant baculoviruses. Purified DNA fragments encoding FLAG-tagged US3 truncations amplified by PCR were cloned into baculovirus transfer vector. In all cases, the US3 domains in the resultant plasmids were verified by sequencing.

Recombinant baculoviruses were generated using the PharMingen baculovirus expression system as described previously (6, 13). Briefly, plasmid DNA containing the FLAG-tagged US3 truncation was cotransfected into Sf9 insect cells together with the BaculoGold baculovirus DNA (PharMingen) according to the manufacturer's instructions. Supernatant containing the recombinant virus was collected and cleared by centrifugation at 2,500 rpm for 10 min 4 to 6 days after transfection, and virus was amplified in Sf9 cells grown in a 150-cm2 flask. The titers of the baculoviruses were determined on Sf9 cells grown in six-well plates.

Preparation of cell lysates, electrophoretic separation of proteins, and immunoblotting. Cell cultures transduced with baculoviruses were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum in the presence or absence of 6 mM sodium butyrate (Sigma). Cells were harvested 22 h after transduction or at the times indicated in Results, rinsed three times with phosphate-buffered saline containing protease inhibitor cocktail (Roche), and then solubilized in 150 µl of disruption buffer (50 mM Tris-HCl [pH 7], 2% sodium dodecyl sulfate, 710 mM ß-mercaptoethanol, 3% sucrose). Fifty-microliter portions of lysates were boiled for 5 min, and the solubilized proteins were subjected to electrophoresis in 11% or 12% denaturing polyacrylamide gel, transferred to a nitrocellulose sheet, blocked with 5% nonfat milk, reacted with primary antibody followed by appropriate secondary antibody conjugated to alkaline phosphatase (Bio-Rad), and visualized according to the manufacturer's instructions.

Antibodies. The rabbit polyclonal antibody against US3 and the mouse monoclonal antibody against US11 had been described previously (13, 26). The polyclonal antibody against HDAC1 and the monoclonal antibody for the FLAG epitope were purchased from Sigma. The polyclonal antibody for poly-ADP ribose polymerase (PARP) was obtained from Santa Cruz Biotechnology Inc.

Immunoprecipitation. Cells were harvested, rinsed with phosphate-buffered saline containing protease inhibitor cocktail (Roche), and then solubilized in lysis buffer (50 mM Tris [pH 8], 150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% sodium dodecyl sulfate). After incubation on ice for more than 20 min, the lysate was clarified by centrifugation at 12,000 rpm for 5 min. The supernatant was collected and reacted with monoclonal anti-FLAG antibody overnight at 4°C. The immune complex was captured by protein G agarose and then washed five times using wash buffer (50 mM Tris [pH 8], 100 mM NaCl, 0.1% NP-40, 1 mM EDTA). The immunoprecipitate was resuspended in 150 µl of disruption buffer. Fifty-microliter portions were boiled for 5 min, subjected to electrophoresis in 11% or 15% denaturing polyacrylamide gel, transferred to a nitrocellulose sheet, and reacted with anti-FLAG or anti-US3 antibody.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enumeration of functions examined and major features of experimental design. As noted in the introduction, the focus of this report is on three key functions mediated by the US3 protein kinase. These are (i) antiapoptotic activity, (ii) phosphorylation of HDAC1 and HDAC2, and (iii) enablement of the expression of the cotransduced gene. The objective of these studies was to identify on the linear sequence of the US3 kinase the domains required for the execution of these functions. The experimental design adapted to attain this objective was as follows.

(i) Both the intact and the truncated domains of the US3 kinase shown in Fig. 1 were cloned into baculoviruses. In all cases, the promoter for expression in human cells was the immediate-early human cytomegalovirus promoter. The reason for transducing cells with baculoviruses is to maximize the number of transduced cells, usually close to 100% of the cell population, and to control the multiplicity of infection, i.e., the amount of recombinant baculovirus DNA transduced into cells. Another benefit of the procedure is that unlike with DNA transfections, the products of the transgene may be detected as early as 4 h after transduction. In all experiments, unless otherwise stated, the cells were transduced with 2 PFU of baculoviruses per cell.

(ii) We selected the U2OS cell line for transduction. The rationale for selecting this cell line was based on the observation that in most cell lines, the expression of the transgenes cloned into baculoviruses requires inhibition of HDACs by sodium butyrate. In U2OS cells, transgenes introduced by transduction with baculoviruses are expressed poorly if at all in the absence of sodium butyrate. Exceptions are the protein kinases US3 and US3.5. Moreover, concurrent or sequential transduction of U2OS cells with the US3 or US3.5 kinase enabled the expression of other transgenes even in the absence of sodium butyrate. We have designated this property of the US3 and US3.5 protein kinases "helper functions" (17). Thus, the objective of these studies was to determine (i) which domains of the US3 kinases enabled self-expression in U2OS cells in the absence of sodium butyrate, (ii) which domains enabled cotransduced transgenes to be expressed, and (iii) whether the phosphorylation of HDAC1 and the execution of the helper functions were covariant properties.

(iii) The polyclonal rabbit antibody to the US3 protein kinase was raised against a polypeptide containing residues 98 to 364. In consequence, the antibody does not react with the polypeptide containing residues 1 to 76 and very poorly with polypeptides lacking 150 N-terminal residues. In order to monitor transgene expression, all constructs except the K220N mutant of the US3 protein kinase were tagged with the FLAG epitope. Usually, the immunoblots were probed with both anti-US3 and anti-FLAG antibody.

(iv) Lastly, in an earlier report, we showed that unlike US3, the US3.5 protein kinase does not block apoptosis induced by the proapoptotic protein BAD or by MG132, a proteasome inhibitor (16). In the studies reported here, we noted that sodium butyrate induced the cleavage of PARP, an indicator of apoptosis, and that the US3 protein kinase blocked the cleavage, whereas the US3.5 kinase did not. Since sodium butyrate was used to enable the expression of transduced genes, it was convenient to use sodium butyrate also to test whether the various truncated forms of the US3 kinase blocked cleavage of PARP induced by the drug.

Figure 1 shows the results obtained from analyses of cells transduced with either intact or truncated forms of the US3 kinase. It is convenient to consider each function enumerated in Fig. 1 separately.

Accumulation of the products of transduced domains of the US3 protein kinase. One striking observation noted in these studies is that the transduced cells differed with respect to the accumulation of the product of transduction. In the experiments supporting these conclusions, U2OS cells were exposed to 2 PFU or 5 PFU of recombinant baculovirus (the construct encoding residues 1 to 163) per cell in the presence or absence of sodium butyrate. As shown in Fig. 2B, E, G, and H, in the absence of sodium butyrate, we could readily demonstrate the accumulation of the intact US3 kinase (residues 1 to 481) (Fig. 2E, lane 7), the intact US3.5 protein kinase (residues 77 to 481) (Fig. 2E, lane 9), the K220N mutant (Fig. 2E, lane 17), and the truncated proteins containing residues 1 to 181 (Fig. 2B, lane 1), 1 to 188 (Fig. 2B, lane 3), 164 to 481 (Fig. 2E, lane 11, and G and H, lane 21), and 182 to 481 (Fig. 2G and H, lane 23). The protein containing residues 1 to 163 was expressed in trace amounts (Fig. 2G and H, lane 27). We did not detect the accumulation of the products of transduction with the construct encoding residues 1 to 76 (not shown) or that encoding residues 189 to 481 (Fig. 2E, lane 15, and G and H, lane 25). In all three instances, the constructs encoding residues 189 to 481 and 1 to 163 (Fig. 2G and H, lanes 26 and 28) were expressed in cells transduced and maintained in the presence of 6 mM sodium butyrate. All other constructs, with the exception of the US3 and US3.5 protein kinases, were expressed at higher levels in cells transduced and maintained in the presence of sodium butyrate. When cells were transduced with 10 or more baculovirus PFU/cell (Fig. 3), we could detect the expression of the truncated protein containing residues 1 to 163 (Fig. 3, lane 5) but only trace amounts of the polypeptides containing residues 1 to 76 (Fig. 3, lane 3) and 189 to 481 (not shown) in the absence of sodium butyrate. The polypeptides containing residues 1 to 76 and 1 to 163 were both expressed in readily detectable amounts in cells transduced and maintained in the presence of sodium butyrate (Fig. 3, lanes 4 and 6, respectively). The summary of the data shown in Fig. 1 suggests that in the absence of other viral gene products or sodium butyrate, the accumulation of a product encoded by the intact or truncated US3 gene requires the presence of at least a portion of residues 164 to 188.


Figure 2
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  		FIG. 2. Functions of intact and truncated forms of the US3 protein kinase. Replicate cultures of U2OS cells in 25-cm2 flasks were either mock transduced (lanes 5 and 6) or exposed to baculoviruses (2 PFU/cell) expressing FLAG-tagged full-length or truncated wild-type US3 (lanes 1 to 4 and 7 to 16) or the hemagglutinin-tagged K220N US3 mutant (lanes 17 and 18) and maintained in DMEM supplemented with 10% FBS in the absence (lanes 1, 3, 5, 7, 9, 11, 13, 15, and 17) or presence (lanes 2, 4, 6, 8, 10, 12, 14, 16, and 18) of 6 mM sodium butyrate (Na B). The cells were harvested at 23 h after transduction, rinsed three times with phosphate-buffered saline containing protease inhibitor cocktail (Roche), and then solubilized in 150 µl of disruption buffer (50 mM Tris-HCl [pH 7], 2% sodium dodecyl sulfate, 710 mM ß-mercaptoethanol, 3% sucrose). Fifty-microliter portions of lysates were boiled for 5 min, and the solubilized proteins were subjected to electrophoresis in 11% denaturing polyacrylamide gels, transferred to nitrocellulose sheets, blocked with 5% nonfat milk, and reacted with polyclonal antibody to PARP (A and D), US3 (B and E), or HDAC1 (C and F). The PARP cleavage products are identified by the filled circles to the right of the lanes. In a separate experiment (G, H, and I) (lanes 19 to 28), U2OS cells were exposed to different stocks of baculoviruses (2 PFU/cell) expressing C-terminal US3 fragments (lanes 21 to 26) or baculovirus (5 PFU/cell) expressing N-terminal amino acids 1 to 163 (lanes 27 and 28). Cells were harvested at 22 h after transduction and processed as described above. The electrophoretically separated proteins were reacted with polyclonal antibody to US3 (G) or HDAC1 (I). The monoclonal antibody for the FLAG epitope was also used to detect the expression of truncated US3 proteins (H). The designations to the left of panel B and to the right of panels G and H identify the bands marked by bars.

 

Figure 3
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  		FIG. 3. Functions of intact and truncated forms of the US3 protein kinase. U2OS cells were exposed to baculovirus (>10 PFU/cell) expressing FLAG-tagged N-terminal fragments containing residues 1 to 76 (lanes 3 and 4) or 1 to 163 (lanes 5 and 6) (10 PFU/cell) or baculovirus expressing FLAG-tagged US3 (lanes 7 and 8) (2 PFU/cell). Cells were maintained in DMEM supplemented with 10% FBS in the presence (lanes 2, 4, 6, and 8) or absence (lanes 1, 3, 5, and 7) of 6 mM sodium butyrate, harvested 23 h after transduction, and processed as described in the legend to Fig. 2. The electrophoretically separated proteins were reacted with polyclonal antibody to US3, PARP, or HDAC1 or monoclonal antibody to the FLAG epitope. The PARP cleavage products are identified by the filled circles to the right of the lanes.

 
In the course of these experiments, we noticed that the truncated proteins containing residues 164 to 481, 182 to 481, and especially 189 to 481 reacted poorly to the anti-US3 antibody (Fig. 2E, lanes 11 to 16, and G, lanes 23 to 26, and Fig. 4, lanes 6 to 10). The accumulation of these proteins was readily detectable with the anti-FLAG antibody.


Figure 4
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  		FIG. 4. Domains required for the helper function coincide with those required for HDAC1 modification. At –5 h, replicate cultures of U2OS cells in 25-cm2 flasks were either mock transduced (lanes 1 to 3); exposed to baculoviruses (1 PFU/cell) expressing FLAG-tagged US3, US3.5, or C-terminal fragments containing residues 164 to 481 or 182 to 481 (lanes 4 to 7); or exposed to baculovirus (1 to 4 PFU/cell as indicated) expressing the C-terminal fragment containing residues 189 to 481 (lanes 8 to 10), the N-terminal fragment containing residues 1 to 188 (lanes 11 to 13), or the hemagglutinin-tagged K220N US3 mutant (lanes 14 to 16) and maintained in DMEM supplemented with 10% FBS in the presence of 6 mM sodium butyrate (lanes 2 to 16). At zero hour, the cells were mock transduced (lane 2) or exposed to baculovirus expressing US11 (1 PFU/cell). The cells were maintained in DMEM supplemented with 10% FBS in the presence of 6 mM sodium butyrate (lanes 2 to 16), harvested 22 h later, and processed as described in the legend to Fig. 2. The electrophoretically separated proteins were reacted with polyclonal antibody to US3, PARP, or HDAC1 or monoclonal antibody to US11 or to the FLAG epitope.

 
The antiapoptotic activity of the US3 protein kinase. In the experiments described above, we determined that transduced cells had the inherent capacity to accumulate the products of transduced genes by comparing untreated transduced cells with cells transduced and maintained in the presence of sodium butyrate. In the course of these studies, we noted that sodium butyrate induced the cleavage of PARP in U2OS cells (Fig. 2D, lane 6, and Fig. 3, lane 2). We took advantage of this observation to score the abilities of truncated US3 proteins to block PARP cleavage. The results are summarized in Fig. 1. In these series of experiments, only the intact, full-length, functional US3 protein kinase (Fig. 2D, lane 8, Fig. 3, lane 8, and Fig. 4, lane 4) blocked the accumulation of cleaved PARP in transduced cells maintained in the presence of sodium butyrate. The results obtained with the US3.5 kinase containing residues 77 to 481 or the K220N mutant (Fig. 2D, lanes 10 and 18) are consistent with those published earlier (16). The fundamental conclusion for the results presented here, to be reinforced in experiments shown later, is that the antiapoptotic functions reside both at the amino terminus of the protein and at a site carboxyl terminal to residue 188, inasmuch as neither the US3.5 protein nor the truncated protein containing residues 1 to 188 alone was able to block apoptosis (Fig. 2A, lane 4, and D, lane 10).

The posttranslational modification of HDAC1. Elsewhere, we reported that both the full-length US3 and the truncated US3.5 protein kinase phosphorylate HDAC1 and 2 and that the K220N mutant did not express this function (16, 17, 19). The objective of these studies was to map the boundaries of the truncated US3 kinase capable of mediating the phosphorylation of HDAC1. A summary of the results observed on analyses of the various domains is shown in Fig. 1. In brief, the results presented in Fig. 2F, lanes 7 to 14, and I, lanes 21 to 24, and Fig. 4, lanes 4 to 7, show that HDAC1 was posttranslationally modified in cells transduced with baculoviruses encoding residues 164 to 481 or 182 to 481. The truncated protein containing residues 189 to 481 did not mediate the posttranslational modification of HDAC1 even in cells transduced with 4 PFU/cell and maintained in the presence of sodium butyrate (Fig. 4, lane 10). It is particularly important to note that while the amounts of the polypeptides containing residues 189 to 481 (Fig. 4, lane 10) and 182 to 481 (Fig. 4, lane 7) accumulating in the transduced cells were similar, the construct containing residues 182 to 481 mediated the phosphorylation of HDAC1, whereas that containing residues 189 to 481 did not. None of the other truncated US3 constructs mediated the posttranslational modification of HDAC1 (Fig. 2C, lanes 1 to 4, and I, lanes 27 and 28, Fig. 3, lanes 3 to 6, and Fig. 4, lanes 11 to 13). We conclude that the phosphorylation of HDAC1 is associated with functions mapping carboxyl terminal to residue 182.

The expression of the helper function of the US3 protein kinase. Earlier, we reported that the US3 and US3.5 protein kinases enabled the expression of cotransduced genes and that this function required a functional kinase inasmuch as the K220N mutant was unable to act as a helper. The level of expression of the cotransduced gene was higher when the transduction of the US3 helper preceded that of the target gene by several hours (17). In the experiment described in this report, we used the HSV-1 US11 gene as the target for amplified expression. Earlier studies have shown that the US3 protein kinase enables the accumulation of higher levels of US11 protein than 6 mM sodium butyrate (17) (Fig. 4, compare lanes 3 and 4). In the experiment whose results are shown in Fig. 4, U2OS cells were transduced with US3 or truncated portions of the gene 5 h before the transduction of US11. To ensure the expression of the K220N mutant and of the constructs containing residues 1 to 188 and 189 to 481, cells were maintained in the presence of sodium butyrate and were also transduced with higher ratios of recombinant baculoviruses expressing these three constructs. As shown in Fig. 4, the functions required for the expression and accumulation of the US11 protein could not be differentiated from those required for posttranslational modification of HDAC1. Again, we conclude that the helper function maps carboxyl terminal to residue 182 and is covariant with phosphorylation of HDAC1.

The self-dependent modification of truncated forms of the US3 protein kinase. The US3 protein kinase is extensively posttranslationally modified (19). In the course of studies on the expression of US3 in infected and transduced cells, we noted that the US3 protein kinase transduced into cells forms several bands, suggesting posttranslational modification by itself or by cellular enzymes. The accumulation of several bands reacting with anti-US3 antibody or anti-FLAG antibody is clearly seen in numerous lanes in Fig. 2 to 4. The objective of this experiment was to define the US3 sequence requirements for posttranslational processing of the protein. In this experiment, U2OS cells were transduced at –5 h with US3 protein kinase and at zero hour with baculoviruses encoding the truncated proteins containing residues 164 to 481, 182 to 481, 189 to 481, or 1 to 163. This set of transductions was done with pretransduced cells, cells exposed to sodium butyrate during and after transduction, or cells that were neither pretransduced with the US3 protein kinase nor treated with sodium butyrate. As shown in Fig. 5, the proteins containing residues 164 to 481 were posttranslationally modified in both the presence and the absence of the US3 protein kinase (Fig. 5, lanes 4 to 6). In contrast, truncated proteins containing residues 182 to 481 or 189 to 481 were posttranslationally modified in cells cotransduced with the US3 protein kinase, and they were not modified in the absence of the kinase (Fig. 5, lanes 7 to 12). Lastly, the protein containing residues 1 to 163 was not posttranslationally modified by itself or by US3 (Fig. 5, lanes 13 to 15).


Figure 5
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  		FIG. 5. Posttranslational modifications of US3 domains. Replicate cultures of U2OS cells in 25-cm2 flasks were either mock transduced (lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, and 15) or exposed to baculovirus (1 PFU/cell) expressing untagged US3 (lanes 1, 4, 7, 10, and 13) and maintained in DMEM supplemented with 10% FBS. Five hours later, cells were mock transduced (lanes 1 to 3) or exposed to baculovirus expressing FLAG-tagged C-terminal residues 164 to 481, 182 to 481, or 189 to 481 (lanes 4 to 12) (1 PFU/cell) or N-terminal fragments containing residues 1 to 163 (lanes 13 to 15) (5 PFU/cell). The cells were maintained in DMEM supplemented with 10% FBS in the presence (lanes 3, 6, 9, 12, and 15) or absence (lanes 1, 2, 4, 5, 7, 8, 10, 11, 13, and 14) of 6 mM sodium butyrate, harvested 22 h later, and processed as described in the legend to Fig. 2. The electrophoretically separated proteins were reacted with polyclonal antibody to US3 or monoclonal antibody to the FLAG epitope. The arrow marked "A" indicates a self-modified polypeptide, and arrows marked "B" indicate polypeptides modified by US3.

 
Relevant to these studies are two conclusions. First, we noted that the electrophoretic mobility of the truncated protein containing residues 1 to 163 is much slower than expected: it resembled that of the much larger protein containing residues 164 to 481 (Fig. 2G and H, lane 28, and Fig. 5, lane 13). The truncated proteins containing residues 1 to 181 and 1 to 188 behaved similarly (Fig. 2B, lanes 1 to 4). The nucleotide sequences of the genes encoding these truncated proteins were verified by sequencing and are correct; their slow electrophoretic mobilities remain a puzzle. The second relevant conclusion is that the truncated protein containing residues 164 to 481 is modified differently from the truncated protein containing residues 182 to 481 or 189 to 481. Thus, all three truncated forms were modified, but only one, i.e., the construct containing residues 164 to 481, did not require the presence of US3 protein kinase in trans for the posttranslational modification. We conclude that the function responsible for the auto-modification maps carboxyl terminal to residue 164. Moreover, at least one site of posttranslational modification of the US3 protein kinase maps carboxyl terminal to residue 189.

The ineffective N-terminal and C-terminal truncations cooperate to block apoptosis in cotransduced cells, provided that they overlap. In the experiments described above, we noted that the antiapoptotic function of the US3 protein kinase was not expressed by either N-terminal or C-terminal truncations and we concluded that functions expressed by both N-terminal and C-terminal domains are required. To test the hypothesis that these functions can complement each other and need not be expressed by a single molecule, U2OS cells were transduced with mixtures of truncated US3 polypeptides or truncated polypeptides and the intact US3 or US3.5 protein kinase. A summary of the results is shown in Fig. 6. As in the earlier studies, we used 6 mM sodium butyrate to induce PARP cleavage. The control and transduced cells were monitored for PARP cleavage and HDAC1 posttranslational modification. The results of the experiments supporting the conclusions shown in Fig. 6 were as follows.


Figure 6
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  		FIG. 6. Schematic representation of complementation studies. Each set of lines represents a mixture of US3 constructs cloned in recombinant baculoviruses and used for transduction in complementation analyses. In the case of pairs 1, 3, 7, and 8, the US3 constructs totally or partially overlap as indicated schematically in the figure.

 
(i) We first tested three constructs (containing residues 1 to 163, 1 to 181, and 1 to 188). None of these constructs blocked apoptosis by themselves, as shown earlier in Fig. 2 and 4 and also in Fig. 7A, lanes 3 and 13, and B, lanes 2 and 12. However, all three constructs complemented US3.5 in blocking PARP cleavage (Fig. 7A, lanes 5 and 15, and B, lanes 4 and 13).


Figure 7
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  		FIG. 7. Complementation studies of N-terminal fragments containing residues 1 to 163, 1 to 181, and 1 to 188. (A) At –5 h, replicate cultures of U2OS cells in 25-cm2 flasks were either mock transduced (lanes 1, 3, and 13), exposed to baculoviruses (1 PFU/cell) expressing FLAG-tagged full-length or truncated wild-type US3 (lanes 4 to 8, 14, and 15) or untagged US3 (lanes 2 and 12), or exposed to baculovirus (1 to 4 PFU/cell as indicated) expressing the hemagglutinin-tagged K220N US3 mutant (lanes 9 to 11) and maintained in DMEM supplemented with 10% FBS in the presence of 6 mM sodium butyrate. At zero hour, the cells were mock transduced (lane 1) or exposed to baculovirus (1 PFU/cell) expressing N-terminal amino acids 1 to 188 (lanes 2 to 11) or 1 to 181 (lanes 12 to 15). The cells were maintained in DMEM supplemented with 10% FBS in the presence of 6 mM sodium butyrate, harvested 23 h later, and processed as described in the legend to Fig. 2. The electrophoretically separated proteins were reacted with polyclonal antibody to PARP or HDAC1 or monoclonal antibody to the FLAG epitope. (B) At –5 h, U2OS cells were exposed to baculoviruses (1 PFU/cell) expressing FLAG-tagged full-length or truncated wild-type US3 (lanes 3 to 7, 9, 10, and 13 to 16) or the hemagglutinin-tagged K220N US3 mutant (lanes 8 and 11) in the presence of 6 mM sodium butyrate. At zero hour, the cells were transduced with baculovirus expressing N-terminal amino acids 1 to 181 (lanes 12 to 16) (1 PFU/cell) or 1 to 163 (lanes 2 to 8) (5 PFU/cell). The cells were maintained in DMEM supplemented with 10% FBS in the presence of 6 mM sodium butyrate, harvested at 22 h, and processed as described in the legend to Fig. 2. The electrophoretically separated proteins were reacted with polyclonal antibody to PARP, US3, or HDAC1. Filled circles to the right of the lanes in the top (PARP) panels identify mixtures of complementing US3 constructs.

 
(ii) Mixtures of N- and C-truncated constructs that overlapped at least in part were able to block cleavage of PARP. These include a mixture of the construct containing residues 1 to 181 or 1 to 188 with the construct containing residues 164 to 481 (Fig. 7A, lane 6, and B, lane 14). An overlap of a few residues (e.g., 1 to 188 and 182 to 481 [Fig. 7A, lane 7]) was not sufficient to block PARP cleavage, and in fact, none of the mixtures of nonoverlapping constructs were able to block apoptosis (e.g., that containing residues 1 to 188 plus that containing residues 189 to 481 [Fig. 7A, lane 8], that containing residues 1 to 163 plus that containing residues 164 to 481 [Fig. 7B, lane 5], and that containing residues 1 to 181 plus that containing residues 182 to 481 [Fig. 7B, lane 15]).

(iii) The truncated construct containing residues 1 to 76 was expressed in the presence of sodium butyrate (Fig. 8, lane 2) or by cotransduction with the US3.5 protein kinase (Fig. 8, lane 3). Although the US3.5 protein kinase enabled the expression of the construct containing residues 1 to 76, the mixture did not block the cleavage of PARP. In contrast, cotransduction of the US3.5 protein kinase and the truncated construct containing residues 1 to 163 blocked the cleavage of PARP (Fig. 8, compare lanes 3 and 4).


Figure 8
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  		FIG. 8. Complementation studies of the N-terminal fragment containing residues 1 to 76. U2OS cells were either mock transduced (lanes 1, 2, and 6) or exposed to baculovirus (1 PFU/cell) expressing FLAG-tagged US3.5 (lanes 3 to 5) for 5 h in the presence of 6 mM sodium butyrate and then transduced with baculovirus (10 PFU/cell) expressing N-terminal amino acids 1 to 163 (lanes 4 and 6) or 1 to 76 (>10 PFU/cell) (lanes 2 and 3). The cells were maintained in DMEM supplemented with 10% FBS in the presence of 6 mM sodium butyrate, harvested 22 h later, and processed as described in the legend to Fig. 2. The solubilized proteins were subjected to electrophoresis in 15% denaturing polyacrylamide gel. The electrophoretically separated proteins were reacted with polyclonal antibody to PARP or HDAC1 or monoclonal antibody to the FLAG epitope.

 
The US3 protein kinase forms multimeric structures. One hypothesis that could explain the results shown in Fig. 7 is that overlapping domains enable the formation of multimeric structures, i.e., the amino-terminal domain interacts with the overlapping carboxyl-terminal domain. To test the hypothesis that the US3 protein kinase can form multimeric structures, we carried out the experiments described in the legend to Fig. 9. Briefly, U2OS cells were transduced at –5.5 h with a baculovirus encoding an untagged US3 kinase. At zero hour, the cells were transduced with either an amino-terminal or a carboxyl-terminal truncation. The cells were harvested at 22 h after the second transduction, lysed, and cleared by centrifugation as described in Materials and Methods. The soluble fraction was reacted with antibody to FLAG. The electrophoretically separated precipitate done in duplicate was reacted with anti-US3 or anti-FLAG antibody. Analyses of proteins in the insoluble and soluble fractions of cell lysates indicate that all FLAG-tagged proteins were expressed, although their distributions differed (data not shown). Two of them (containing residues 182 to 481 and 189 to 481) were recoverable at relatively lower levels in the soluble fraction. The results presented in Fig. 9 show that the anti-FLAG antibody pulled down the cotransduced US3 or US3.5 protein kinase and that the quantities of the pulled-down, untagged US3 kinase were proportional to the quantities of the input FLAG-tagged proteins.


Figure 9
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  		FIG. 9. US3 can form multimeric structures. (A) List of FLAG-tagged N-terminally or C-terminally truncated polypeptides expressed in baculoviruses [Bac-(N) or Bac-(C), respectively] tested for interaction with full-length US3. (B) Experimental design. At –5.5 h, replicate cultures of U2OS cells in 25-cm2 flasks were exposed to baculovirus (1 PFU/cell) expressing untagged US3 and maintained in DMEM supplemented with 10% FBS. At zero hour, the cells were either mock transduced or exposed to baculovirus (1 PFU/cell) expressing a FLAG-tagged C-terminal fragment (Bac-C) or N-terminal fragment (Bac-N) containing residues 1 to 181, 1 to 188 (1 PFU/cell), or 1 to 163 (5 PFU/cell). The cells were maintained in DMEM supplemented with 10% FBS, harvested at 22 h, and lysed as described in Materials and Methods. (C) Immunoprecipitation. Solubilized proteins (Input) were reacted with the monoclonal anti-FLAG antibody as described in Materials and Methods. Proteins from immunoprecipitates (IP) were electrophoretically separated in 11% or 15% denaturing polyacrylamide gel and probed for full-length and truncated US3 proteins.

 
The purpose of the second experiment was to determine whether the FLAG-tagged polypeptide containing the 76 amino-terminal residues could pull down the untagged US3 protein kinase. The experiment was carried out as described above. As illustrated in Fig. 10, the FLAG-tagged construct containing residues 1 to 76 was expressed but failed to pull down the untagged US3/US3.5 protein kinase.


Figure 10
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  		FIG. 10. The 76 N-terminal amino acids are not involved in multimeric structure formation. The experimental design was as described in the legend to Fig. 9. U2OS cells were exposed to baculovirus (>10 PFU/cell) expressing the N-terminal fragment containing residues 1 to 76. To ensure expression of the protein, a replicate, doubly transduced culture was maintained in a medium containing 6 mM sodium butyrate (lanes 4 and 8). Cells were harvested and processed as described in the legend to Fig. 9. Proteins were immunoprecipitated using anti-FLAG antibody and analyzed as described in Materials and Methods.

 
We conclude from these studies that US3 can form enzymatically active multimeric structures. The results presented in Fig. 9 and 10 suggest that the multimerization sites are within the domain of the US3.5 protein.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The salient features of the results presented in this report are as follows.

(i) The expression of mammalian or viral genes introduced into cells by transduction with recombinant baculoviruses requires maintenance of the transduced cells in a medium containing inhibitors of HDACs (3). The common inhibitor used for this purpose is sodium butyrate. In U2OS cells, the US3 protein kinase is expressed in a multiplicity-dependent fashion in the absence of sodium butyrate (17). In the experiments reported here, we tested the expression of truncated forms of the US3 protein kinase. Our findings show that at 2 PFU of recombinant baculovirus per cell, the US3 and US3.5 protein kinases were expressed to virtually the same level in the presence or absence of sodium butyrate (Fig. 2E, lanes 7 to 10), whereas the protein lacking kinase activity (the K220N construct) was expressed at a much higher level in the presence of sodium butyrate. We also noted that the truncated construct containing residues 164 to 481 accumulated only slightly better in the presence of sodium butyrate than the construct containing residues 182 to 481 (Fig. 2G and H, lanes 21 to 24), even though both constructs expressed similar functions as described below.

(ii) Elsewhere, we reported that the US3 and US3.5 protein kinases phosphorylate HDAC 1 and HDAC2 (16, 18, 19). We also reported that in U2OS cells, both US3 and US3.5 enable the expression of cotransduced cellular or viral genes. We designated this the helper function of US3 protein kinases (17). In the studies reported here, both the phosphorylation of HDAC1 and the helper function (based on studies of the expression of US11, a {gamma}2 HSV-1 gene expressed poorly in the absence of either US3 or sodium butyrate) mapped to a position carboxyl terminal to residue 182. Although the construct containing residues 182 to 481 expressed both functions, whereas the construct containing residues 189 to 481 did not, the data are consistent with the hypothesis that the truncated form containing residues 189 to 481 lacks kinase activity and do not support the hypothesis that it lacks both the kinase activity and the recognition site for HDAC1.

(iii) We also reported elsewhere that the full-length US3 protein kinase blocked apoptosis, whereas the inactive US3 kinase carrying the K220N substitution or US3.5 did not (16). In the studies reported here, we used inhibition of PARP cleavage as an indicator of antiapoptotic activity. Attempts to complement the US3.5 kinase with a construct containing the first 76 residues of US3 that are missing from US3.5 failed, but truncated constructs containing residues that overlapped with those of the US3.5 sequence blocked PARP cleavage. The smallest pair of truncated constructs tested that blocked PARP cleavage contained residues 1 to 181 and 164 to 481. The results suggested that (i) the US3 kinase can form higher-order structures and (ii) the antiapoptotic activity requires at the very least an activity mapping at the amino terminus and the kinase activity mapping carboxyl terminally to residue 182. The significance of these results stems from two considerations.

First, although Daikoku et al. (4) reported that the monomeric enzyme was active, their data did not exclude the possibility that US3 forms multimeric structures. Immune precipitations using antibody to the FLAG epitope-tagged, truncated constructs pulled down untagged US3 kinase from lysates of doubly transduced cells. The results unambiguously indicate that US3 can form a multimeric kinase. In light of the report by Daikoku et al., two questions arise: (i) do the monomeric and multimeric enzymes have identical or dissimilar functions, and (ii) what conditions determine the structure of the kinase?

The second consideration stems from the observation that the US3 protein kinase is a multifunctional enzyme that targets a large number of diverse proteins (8, 14, 16, 18, 19, 21, 22). Earlier, this laboratory reported that the phosphorylation site of the US3 protein kinase is similar to that of protein kinase A (PKA) and that antibody to the residues phosphorylated by PKA also reacts with proteins phosphorylated by US3 (2, 16). Unlike those of PKA with its multiple subunits, all of the activities associated with the US3 protein kinase are associated with two polypeptides of the 481 (US3) and 405 (US3.5) residues. Since the two differ with respect to antiapoptotic activity, a simple explanation of the results obtained so far is that the US3 kinases contain both the recognition sites for their targets and the protein kinase sites and that the interaction of US3 and its targets depends on the structure (481 versus 405 residues), posttranslational modifications, and secondary structure. It seems clear from the data presented in this and preceding reports that the recognition site for the target of the antiapoptotic activity maps at the amino terminus, whereas the recognition site for HDAC1 is carboxyl terminal to residue 182. Finally, our results indicate that the amino-terminal activity does not need to be on the same polypeptide as the kinase activity and our interpretation is that the antiapoptotic function of US3 is or can be executed by the multimeric US3 enzyme. Both the structure of the multimeric enzyme and the target of the antiapoptotic activity remain to be defined.


   ACKNOWLEDGMENTS
 
These studies were aided by National Cancer Institute grants CA115662, CA83939, CA71933, CA78766, and CA88860.


   FOOTNOTES
 
* Corresponding author. Mailing address: The Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, 910 East 58th Street, Chicago, IL 60637. Phone: (773) 702-1898. Fax: (773) 702-1631. E-mail: bernard.roizman{at}bsd.uchicago.edu. Back

{triangledown} Published ahead of print on 6 December 2006. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Virology, February 2007, p. 1980-1989, Vol. 81, No. 4
0022-538X/07/$08.00+0     doi:10.1128/JVI.02265-06
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