The Carboxyl-Terminal Domain of RNA Polymerase II
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病菌学杂志 2005年第11期
The Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, Chicago, Illinois 60637
ABSTRACT
The infected-cell protein 22 (ICP22), a regulatory protein encoded by the 22 gene of herpes simplex virus 1, is required for the optimal expression of a set of late viral proteins that includes the products of the US11, UL38, and UL41 genes. ICP22 has two activities. Thus, ICP22 and the UL13 protein kinase mediate the activation of cdc2 and degradation of its partners, cyclins A and B. cdc2 and its new partner, the DNA polymerase accessory factor (UL42), bind topoisomerase II in an ICP22-dependent manner. In addition, ICP22 and UL13 mediate an intermediate phosphorylation of the carboxyl terminus of RNA polymerase II (RNA POL II). Here we report another function of ICP22. Thus, ICP22 physically interacts with cdk9, a constitutively active cyclin-dependent kinase involved in transcriptional regulation. A protein complex containing ICP22 and cdk9 phosphorylates in vitro the carboxyl-terminal domain of RNA POL II in a viral US3 protein kinase-dependent fashion. Finally, the carboxyl-terminal domain of RNA POL II fused to glutathione S-transferase is phosphorylated in reaction mixtures containing complexes pulled down with ICP22 or cdk9 immune precipitated from lysates of wild-type parent virus or UL13 but not US3 mutant-infected cells. The experiments described here place ICP22 and cdk9 in a complex with the carboxyl-terminal domain of RNA POL II. At the same time we confirm the requirement of ICP22 and the UL13 protein kinase in the posttranslational modification of RNA POL II that alters its electrophoretic mobility, although US3 kinase appears to play a role in a cell-type-dependent fashion.
INTRODUCTION
Earlier studies from this laboratory have shown that infected-cell protein 22 (ICP22), a product of the 22 gene of herpes simplex virus 1 (HSV-1), mediates the activation of cdc2 and the degradation of its partners, cyclins A and B (2, 3). cdc2 acquires a new partner, the viral DNA polymerase-associated factor encoded by the UL42 open reading frame (4). The complex of cdc2 and UL42 binds topoisomerase II in an ICP22-dependent manner (5). Finally, ICP22 and the protein kinase encoded by the UL13 open reading frame are required both for the activation of cdc2 and for the optimal expression of a subset of late viral proteins exemplified by three proteins encoded by the UL38, UL41, and US11 genes (3, 23, 27). Independently, Spencer and associates reported that ICP22 and the UL13 protein kinase mediate a posttranslational modification of the RNA polymerase (POL) II that is reflected in an "intermediate" electrophoretic mobility between that of hyperphosphorylated (RNA POL IIo) and hypophosphorylated (RNA POL IIa) states (12, 14, 26, 29). Both the recruitment of topoisomerase II and the modification of RNA POL II could account for optimization of the synthesis of the subset of late proteins. To resolve the question further, it became desirable to investigate the nature of the interaction between ICP22 and RNA POL II.
Cyclin-dependent kinases (cdk's) can be broadly categorized into two subsets, one for cell cycle control and the other for transcriptional regulation (7, 17). Those involved in cell cycle control include cdc2 (cdk1), cdk2, and cdk4 (9, 11, 13, 25, 28). The activation of such cdk's varies and controls cell cycle progression. Interestingly, viruses, including HSV-1, manipulate the cell cycle and their associated cdk's to optimize viral replication. The other set of cdk's involved in transcriptional control includes cdk7, cdk8, and cdk9 (7, 9, 15, 16). Unlike cell cycle cdk's, the transcriptional cdk's are active throughout the cell cycle. They do, however, share the property of "traditional" cdk's in that they have associated cyclins that bind to them (18). All three kinases have been reported to be able to phosphorylate the carboxyl-terminal domain of RNA POL II (CTD) (6, 18). cdk7 is a subunit of TFIIH and is involved in the switch from transcription initiation to transcription elongation through phosphorylation of CTD. cdk9 has a similar function. cdk9 and its partners, the T cyclins, are components of P-TEFb (15, 16, 21). P-TEFb interacts with the human immunodeficiency virus tat-encoded protein to form the trimeric complex Tat-cdk9-cyclin T, which, upon binding to the response element TAR, causes a significant enhancement of elongation of viral transcripts (30).
cdk9 and cyclin T have been shown to colocalize within the nonnucleolar nucleoplasm in nuclear speckles and show only limited colocalization with RNA POL II. The complex does, however, colocalize with several splicing factors, indicating that nuclear speckles might be sites of P-TEFb activity (10).
In the studies described here we show that cdk9, but not cdk7, bound to ICP22 and that the complex containing cdk9 and ICP22 phosphorylated the CTD in a US3 protein kinase-dependent manner. This is the first evidence of an interaction of ICP22 with cdk9 and the formation of a complex that interacts with and modifies the CTD of RNA POL II.
MATERIALS AND METHODS
Cells and viruses. HEp-2 cells were initially obtained from the American Type Culture Collection and maintained in Dulbecco modified Eagle medium (DMEM) with 10% (vol/vol) newborn calf serum. HSV-1(F) is the prototype HSV-1 wild-type strain used in this laboratory (8). The recombinant viruses R325, lacking the carboxyl-terminal domain of ICP22; R7356, lacking the UL13 gene; R7041, lacking the US3 gene; and R7353, lacking both UL13 and US3 genes, were described elsewhere (19, 20, 22, 23, 24).
Immunoblotting. Cells were harvested as follows. The medium was removed, and the cells were rinsed with phosphate-buffered saline (PBS), scraped into PBS, pelleted by centrifugation, solubilized in high-salt lysis buffer (20 mM Tris, pH 8.0; 1 mM EDTA; 0.5% NP-40; 400 mM NaCl; 0.1 mM sodium orthovanadate; 10 mM NaF; 2 mM dithiothreitol; 100 μg each of phenylmethylsulfonyl fluoride, tosylsulfonyl phenylalanyl chloromethyl ketone, and leupeptin per ml), and stored on ice for 1 h. Insoluble materials were removed by centrifugation as described above. Samples (75 μg/sample) were mixed with equal volumes of a gel loading buffer (2% sodium dodecyl sulfate, 50 mM Tris, pH 6.8, 2.75% sucrose, 5% ?-mercaptoethanol, bromophenol blue), subjected to electrophoresis on a 10% bisacrylamide gel, transferred to nitrocellulose membranes, blocked for 2 h with 5% nonfat dry milk, and reacted with the appropriate antibody. The blots were incubated in AP buffer (100 mM Tris, pH 9.5, 100 mM NaCl, 5 mM MgCl2), followed by AP buffer containing BCIP (5-bromo-4-chloro-3-indolylphosphate) and nitroblue tetrazolium. The reaction was stopped by immersing the blot in a solution containing 100 mM Tris (pH 7.6) and 10 mM EDTA.
Antibodies. Rabbit antibodies to cyclin T and cdk9 (Santa Cruz, CA) were used at 1:250 dilutions. The rabbit antibody to ICP22 (1) was used at a 1:500 dilution in PBS with 1% bovine serum albumin and 0.05% Tween 20. Bound antibody was detected by using secondary antibody diluted 1:3,000 (goat anti-rabbit antibody conjugated to alkaline phosphatase; Bio-Rad).
Production of GST fusion proteins. DNA sequences encoding cdc2, cdk7, or cdk9 were amplified by PCR and cloned into pGEX4T-1. Escherichia coli BL21 cells were transformed with plasmids encoding the above glutathione S-transferase (GST) fusion proteins or GST alone, grown at 30°C until the optical density at 600 nm reached a value of 0.6 to 0.8, and induced with 100 μM isopropyl-?-D-thiogalactosidase for 2 h. Bacteria were collected by centrifugation, resuspended in PBS, lysed by sonication, and mixed with Triton X-100 (1% final concentration). After the cell debris was removed by centrifugation, GST fusion proteins were adsorbed to glutathione-agarose beads (Sigma). The beads were collected and rinsed in PBS. The fusion proteins were eluted with 10 mM glutathione in 50 mM Tris (pH 8.0) and dialyzed against PBS. Protein production was assessed by electrophoresis in denaturing gels followed by Coomassie brilliant blue staining. The GST-CTD plasmid was obtained from Scott Petersen (University of Colorado) and grown as described above, with the exception that the bacteria were induced with 500 μM isopropyl-?-D-thiogalactosidase for 10 h.
Cell infection. HEp-2 cells grown in 25-cm2 flasks were exposed to 2 x 107 PFU of appropriate virus in 1.2 ml of 199V (mixture 199 supplemented with 1% calf serum) on a rotary shaker at 37°C. After 2 h, the inoculum was replaced with 4 ml of fresh DMEM supplemented with 10% serum. Flasks were incubated at 37°C until the cells were harvested at the time points indicated in Results. Time zero is defined as the time at which viral inoculum was added to the cells.
[35S]Met pulse-chase linked to GST pull-down. HEp-2 cells were exposed to virus as above. After 1 h, the inoculum was removed and the cells were starved for 1 h in 199V minus methionine at 37°C. The medium was then replaced with fresh 199V lacking methionine but supplemented with 100 μCi [35S]Met. After 4 h at 37°C, the medium was replaced with 5 ml of DMEM supplemented with 10% serum and incubated at 37°C until harvested at times indicated in Results. Equivalent amounts of protein per sample were brought up to 400 μl in high-salt lysis buffer (final concentration, 400 mM NaCl). Samples were precleared with 50 μl of a 50% slurry of glutathione beads for 2 h at 4°C. The precleared supernatant fluid was reacted for 3 h at 4°C with 10 μl of a 50% slurry of glutathione beads bound to GST alone, cdc2, cdk7, or cdk9. The beads were pelleted by centrifugation and rinsed three times in low-salt lysis buffer (final concentration, 200 mM NaCl). The beads were resuspended in 40 μl of gel loading buffer and subjected to electrophoresis in a denaturing gel and autoradiography.
In vitro kinase assay. HEp-2 cells were exposed to virus as described above, harvested 18 h after infection, lysed, precleared with preimmune serum at 4°C for 2 h, and then reacted with protein A Sepharose beads for 1 h (200 μg/sample). Samples were then centrifuged at 3,000 rpm for 5 min, and the supernatant fluid was collected and reacted with 4 μl of anti-cdk9 antibody overnight at 4°C. The beads were collected by centrifugation and rinsed twice with low-salt buffer (20 mM Tris [pH 8.0], 1 mM EDTA, 0.5% NP-40, 200 mM NaCl, 2 mM dithiothreitol) and twice with incomplete kinase buffer (50 mM Tris [pH 7.4], 10 mM MgCl2, 5 mM dithiothreitol). The beads were then resuspended in 40 μl of complete kinase buffer (20 mM Tris [pH 8.0], 1 mM EDTA, 0.5% NP-40, 1 mM NaCl, 2 mM dithiothreitol, 10 μM ATP, 20 μCi of [-32P]ATP, 2 μg GST-CTD) and incubated at 30°C for 30 min. The reactions were terminated by the addition of gel loading buffer. The samples were then subjected to electrophoresis in a denaturing gel and autoradiography. Quantification of 32P phosphorylation of the substrate was done with the aid of a Molecular Dynamics PhosphorImager (Storm 860).
RESULTS
cdk9 pulls down from lysates of HSV-1-infected cells two proteins labeled after infection. In this series of experiments GST-cdc2, GST-cdk7, and GST-cdk9 bound to glutathione beads were reacted with lysates of HEp-2 cells infected with HSV-1(F) and incubated in medium containing [35S]methionine as described in Materials and Methods. The proteins bound to the chimeric proteins on the beads were solubilized and subjected to electrophoresis and autoradiography. All of the procedures were as described in Materials and Methods. The chimeric proteins bound to the beads were subjected to electrophoresis in a denaturing gel and stained with Coomassie blue (Fig. 1A). Each of the samples eluted from the glutathione beads yielded a single major band. The molecular weights of the proteins bound to the glutathione beads were of the expected values of the GST-cdc2, GST-cdk7, and GST-cdk9 chimeric proteins. The autoradiographic images of the labeled infected-cell proteins bound to the chimeric proteins are shown in Fig. 1B. Each of the three chimeric proteins, but not GST alone, pulled down labeled proteins. GST-cdc2 pulled down a protein that was found to be UL42 (4). Of these only GST-cdk9 brought down two proteins with apparent relative molecular weights (Mrs) of 69,000 and 150,000.
The 69,000-Mr protein interacting with GST-cdk9 is ICP22. Two series of experiments indicated that the 69,000-Mr protein pulled down by the GST-cdk9 chimeric protein is ICP22. In the first series, lysates of cells harvested 6 h after mock infection or infection with wild-type or the R325 mutant viruses were reacted with GST-cdk9 as described above. The chimeric proteins bound to the beads were solubilized, subjected to electrophoresis in denaturing gels, transferred to a nitrocellulose membrane, and reacted with anti-ICP22 antibody. Figure 2 shows the reactivity of the antibody with aliquots of whole-cell lysates and the proteins eluted from the GST-cdk9 beads. As shown in Fig. 2 (lane 5), the antibody reacted with a protein band which comigrated with that present in lysate of wild-type virus-infected cells. This band was absent from lysate of R325 mutant-infected cells or the proteins pulled down by GST-cdk9 from lysates of cells infected with the R325 mutant.
In the second series of experiments, GST-cdk9 bound to beads was reacted with lysates of HEp-2 cells either mock infected or infected with HSV-1(F) or with the R325, R7356, or R7041 mutant virus; radiolabeled; and processed as described in the experiments summarized in Fig. 1. The proteins bound to the GST-cdk9 beads were solubilized, subjected to electrophoresis in a denaturing polyacrylamide gel, transferred to a nitrocellulose sheet, and subjected to autoradiography. As shown in Fig. 3 lanes 2 and 4, GST-cdk9 chimeric protein pulled down from lysates of cells infected with wild-type virus or a mutant lacking the UL13 protein kinase the same set of proteins as those pulled down from lysates of wild-type virus-infected cells (Fig. 1). The significant findings were that GST-cdk9 did not pull down a 69,000-Mr protein from lysates of cells infected with the R325 mutant and that the amount of 69,000-Mr protein pulled down from lysates of cells infected with the US3 mutant was significantly lower than those pulled down from wild-type virus-infected cells. We should note that a faint band containing a faster-migrating protein pulled down by GST-cdk9 from lysates of R325-infected cells comigrated with a protein pulled down from lysates of mock-infected cells.
We conclude from these experiments that the 69,000-Mr protein is ICP22, that the functional interaction of ICP22 with cdk9 requires the carboxyl-terminal 220 amino acids absent from lysates of R325-infected cells, and that the interaction is at least partially dependent on the presence of the US3 kinase.
The levels of cdk9 and cyclin T, a partner of cdk9, do not change during the HSV-1 replicative cycle. Earlier studies have shown that, in HSV-1-infected cells, cdc2 is activated but its partners, cyclins A and B, are degraded in an ICP22- and UL13-dependent fashion (2-4). To determine the status of cdk9 and cyclin T in infected cells, replicate cultures of HEp-2 cells were mock infected or infected with HSV-1(F) or R325; harvested at 3, 6, 9, or 16 h after infection; solubilized, subjected to electrophoresis in denaturing gels, and probed with antibody to cdk9 or cyclin T. As shown in Fig. 4, the levels of cdk9 (top panel) or cyclin T (bottom panel) did not change significantly during the course of the HSV-1 replicative cycle. In preliminary experiments anti-cdk9 antibody pulled down a protein that reacted with anti-cyclin T antibody from both infected and mock-infected cells (data not shown).
The protein complex phosphorylating RNA POL II CTD contains ICP22 and cdk9. The objective of the experiments described in this section was to determine whether cdk9-ICP22 complex mediates the phosphorylation of the RNA POL II CTD. In these experiments, lysates of mock-infected HEp-2 cells or cells infected with wild-type HSV-1(F) or mutant virus R7041 (US3), R7356 (UL13), R7353 (US3/UL13), or R325 were reacted with antibody to cdk9 or ICP22. The precipitates were reacted with CTD of RNA POL II in the presence of [-32P]ATP as described in Materials and Methods. The proteins were then solubilized, electrophoretically separated on denaturing gels, and subjected to autoradiography. Panels A and D of Fig. 5 show the autoradiographic images of the RNA POL II CTD reacted with precipitates obtained with cdk9 and ICP22 antibodies, respectively. The amount of radioactivity as measured with the aid of a Molecular Dynamics 860 phosphorimager is shown in Fig. 5C and E. Panel B shows a photograph of the Ponceau S-stained bands containing the GST-RNA POL II CTD as evidence that equal amounts of substrate were present in each reaction mixture. The results show the following: RNA POL II CTD was phosphorylated by immune precipitates brought down by either anti-ICP22 or anti-cdk9 antibody from lysates of wild-type virus-infected cells or cells infected with the UL13 mutant virus. The RNA POL II CTD was not phosphorylated by precipitates from lysates of cells mock infected or infected with US3 or US3/UL13 mutant viruses. The results are consistent with those presented above showing that cdk9 and ICP22 form a complex at least partially dependent on the US3 protein kinase. They also indicate that the complex can phosphorylate the CTD of RNA POL II.
In light of the publications showing that ICP22 mediates the formation of the RNA POL IIi in a UL13-dependent manner we took numerous steps to ascertain that these results were reproducible. The experiments were repeated numerous times with independently produced batches of mutant viruses. Moreover, since laboratory contaminations are not unheard of, we verified that the mutants lacked the appropriate protein kinases.
The involvement of UL13 protein kinase in the posttranslational generation of the RNA POL IIi form. The studies on the involvement of ICP22 and UL13 protein kinase were originally done on Vero cells. The studies reported here were done in human cells. One possible explanation for the observation that the CTD of RNA POL II is phosphorylated in both a UL13- and a US3-dependent fashion is that the protein kinase involved in this reaction is cell type dependent. To test this hypothesis, we examined the electrophoretic mobility of RNA POL II in Vero, rabbit skin, and HEp-2 cell lines mock infected or infected with wild-type or mutant viruses as described in Materials and Methods. The results shown in Fig. 6 were as follows. In the case of rabbit skin or HEp-2 cells, the accumulation of RNA POL IIi form was dependent on UL13 protein kinase and not on the US3 protein kinase. In the experiment shown in Fig. 6A, the accumulation of the RNA POL IIi form in infected Vero cells was dependent on both kinases. In other experiments done on Vero cells, the role of the US3 protein kinase was less evident (data not shown). We are led to conclude that the posttranslational modification of RNA POL II underlying the change in mobility is primarily ICP22 and UL13 protein kinase dependent and that under certain physiologic conditions the US3 protein kinase may also be required.
DISCUSSION
An interesting aspect of the biology of HSV is that its proteins are multifunctional and that one fundamental strategy of the virus is to attain its objective in more than one way. ICP22 is an example of both properties. First, it encodes multiple functions, some mapping in the 200-amino-acid sequence at the carboxyl-terminal domain and some, as yet poorly defined, in the residual amino-terminal domain. As exemplified by the recombinant virus R325, the carboxyl-terminal domain appears to be required in a cell-type-specific manner for the synthesis of a subset of proteins exemplified by the products of UL38, UL41, and US11 genes (23). Independently, ICP22 has been shown to have two activities that also map in its carboxyl-terminal domain and which correlate with the optimal synthesis of UL38, UL41, and US11 proteins. One such activity described by this laboratory involves a series of metabolic events resulting in the degradation of cyclins A and B and acquisition by their partner, cdc2, of a novel partner, the DNA polymerase accessory factor encoded by UL42 (2-4). This cdc2 kinase in this partnership is activated and binds the topoisomerase II, both in an ICP22-dependent manner (5). The tie between activation of cdc2 and the formation of the cdc2-UL42-topoisomerase II complex and the synthesis of the subset of late 2 proteins rests on the observation that these proteins are synthesized from mRNA made late in infection, presumably transcribed from progeny DNA. Late in infection the progeny DNA consists of tangles of concatemers. Topoisomerase II could play a role in rendering transcription more efficient by untangling the concatemers, but in addition, topoisomerase II has been reported to play a role in transcription (31). The other property of ICP22 related to transcription is based on reports that ICP22 mediates in a UL13-dependent manner the phosphorylation of the carboxyl-terminal domain of RNA POL II. It should be stressed that connections between the formation of cdc2-UL42-topoisomerase II complex and the phosphorylation of the carboxyl-terminal domain of RNA POL II and the efficiency of synthesis of the subset of late proteins described above are covariant properties dependent on ICP22. To investigate these properties of ICP22, we decided to dissect further the interaction between RNA POL II and ICP22. Our approach was based on the evidence that cdk9 is related to cdc2 and that the involvement of cdk9 in the transcription of viral genes, namely, those of HIV, has already been established (15, 16, 21, 30). The results presented in this report indicate that cdk9 interacts with ICP22, that this interaction is to a large extent dependent on the US3 protein kinase but not on the UL13 protein kinase, and that complexes containing cdk9 and ICP22 phosphorylated the carboxyl-terminal domain of RNA POL II in vitro in a US3- and not in a UL13-dependent manner.
The studies presented in this report confirm the involvement of ICP22 in the posttranslational modification of RNA POL II. They differ in two respects from the earlier studies (29). First, we report that both cdk9 and ICP22 were involved and present in the complex that phosphorylated RNA POL II. In particular we noted that cdk9 alone, in the absence of full-length ICP22, failed to perform this task. Second, we report that the in vitro phosphorylation of RNA POL II was dependent on the presence of the US3 protein kinase and not on the UL13 protein kinase. At the same time we have shown that in the cell-virus system we were using the posttranslational modification of RNA POL II results in the formation of the "intermediate" phosphorylated form of the enzyme.
The hypothesis that we would like to propose is based on the evidence that ICP22 is posttranslationally modified by either one or both of the UL13 and US3 protein kinases (23, 24) and that it performs multiple and potentially diverse functions depending on the state of phosphorylation of the protein. It is conceivable that its functions are determined by its protein partner, the selection of which is, in turn, determined by the nature of the posttranslational modifications. Thus, activation of cdc2 may require phosphorylation of ICP22 by UL13 protein kinase whereas interaction with cdk9 requires phosphorylation by the US3 protein kinase. We have established that the cdk9-ICP22 complex formed in a US3-dependent manner phosphorylated RNA POL II. What remains to be defined is both the nature of the phosphorylation and the effect of this modification on RNA POL II.
ACKNOWLEDGMENTS
We thank Scott Petersen for the plasmid encoding the CTD of RNA POL II and Ralph Weichselbaum for invaluable discussion.
These studies were aided by grants from the National Cancer Institute (CA78766, CA71933, CA83939, CA87661, and CA88860), United States Public Health Service.
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ABSTRACT
The infected-cell protein 22 (ICP22), a regulatory protein encoded by the 22 gene of herpes simplex virus 1, is required for the optimal expression of a set of late viral proteins that includes the products of the US11, UL38, and UL41 genes. ICP22 has two activities. Thus, ICP22 and the UL13 protein kinase mediate the activation of cdc2 and degradation of its partners, cyclins A and B. cdc2 and its new partner, the DNA polymerase accessory factor (UL42), bind topoisomerase II in an ICP22-dependent manner. In addition, ICP22 and UL13 mediate an intermediate phosphorylation of the carboxyl terminus of RNA polymerase II (RNA POL II). Here we report another function of ICP22. Thus, ICP22 physically interacts with cdk9, a constitutively active cyclin-dependent kinase involved in transcriptional regulation. A protein complex containing ICP22 and cdk9 phosphorylates in vitro the carboxyl-terminal domain of RNA POL II in a viral US3 protein kinase-dependent fashion. Finally, the carboxyl-terminal domain of RNA POL II fused to glutathione S-transferase is phosphorylated in reaction mixtures containing complexes pulled down with ICP22 or cdk9 immune precipitated from lysates of wild-type parent virus or UL13 but not US3 mutant-infected cells. The experiments described here place ICP22 and cdk9 in a complex with the carboxyl-terminal domain of RNA POL II. At the same time we confirm the requirement of ICP22 and the UL13 protein kinase in the posttranslational modification of RNA POL II that alters its electrophoretic mobility, although US3 kinase appears to play a role in a cell-type-dependent fashion.
INTRODUCTION
Earlier studies from this laboratory have shown that infected-cell protein 22 (ICP22), a product of the 22 gene of herpes simplex virus 1 (HSV-1), mediates the activation of cdc2 and the degradation of its partners, cyclins A and B (2, 3). cdc2 acquires a new partner, the viral DNA polymerase-associated factor encoded by the UL42 open reading frame (4). The complex of cdc2 and UL42 binds topoisomerase II in an ICP22-dependent manner (5). Finally, ICP22 and the protein kinase encoded by the UL13 open reading frame are required both for the activation of cdc2 and for the optimal expression of a subset of late viral proteins exemplified by three proteins encoded by the UL38, UL41, and US11 genes (3, 23, 27). Independently, Spencer and associates reported that ICP22 and the UL13 protein kinase mediate a posttranslational modification of the RNA polymerase (POL) II that is reflected in an "intermediate" electrophoretic mobility between that of hyperphosphorylated (RNA POL IIo) and hypophosphorylated (RNA POL IIa) states (12, 14, 26, 29). Both the recruitment of topoisomerase II and the modification of RNA POL II could account for optimization of the synthesis of the subset of late proteins. To resolve the question further, it became desirable to investigate the nature of the interaction between ICP22 and RNA POL II.
Cyclin-dependent kinases (cdk's) can be broadly categorized into two subsets, one for cell cycle control and the other for transcriptional regulation (7, 17). Those involved in cell cycle control include cdc2 (cdk1), cdk2, and cdk4 (9, 11, 13, 25, 28). The activation of such cdk's varies and controls cell cycle progression. Interestingly, viruses, including HSV-1, manipulate the cell cycle and their associated cdk's to optimize viral replication. The other set of cdk's involved in transcriptional control includes cdk7, cdk8, and cdk9 (7, 9, 15, 16). Unlike cell cycle cdk's, the transcriptional cdk's are active throughout the cell cycle. They do, however, share the property of "traditional" cdk's in that they have associated cyclins that bind to them (18). All three kinases have been reported to be able to phosphorylate the carboxyl-terminal domain of RNA POL II (CTD) (6, 18). cdk7 is a subunit of TFIIH and is involved in the switch from transcription initiation to transcription elongation through phosphorylation of CTD. cdk9 has a similar function. cdk9 and its partners, the T cyclins, are components of P-TEFb (15, 16, 21). P-TEFb interacts with the human immunodeficiency virus tat-encoded protein to form the trimeric complex Tat-cdk9-cyclin T, which, upon binding to the response element TAR, causes a significant enhancement of elongation of viral transcripts (30).
cdk9 and cyclin T have been shown to colocalize within the nonnucleolar nucleoplasm in nuclear speckles and show only limited colocalization with RNA POL II. The complex does, however, colocalize with several splicing factors, indicating that nuclear speckles might be sites of P-TEFb activity (10).
In the studies described here we show that cdk9, but not cdk7, bound to ICP22 and that the complex containing cdk9 and ICP22 phosphorylated the CTD in a US3 protein kinase-dependent manner. This is the first evidence of an interaction of ICP22 with cdk9 and the formation of a complex that interacts with and modifies the CTD of RNA POL II.
MATERIALS AND METHODS
Cells and viruses. HEp-2 cells were initially obtained from the American Type Culture Collection and maintained in Dulbecco modified Eagle medium (DMEM) with 10% (vol/vol) newborn calf serum. HSV-1(F) is the prototype HSV-1 wild-type strain used in this laboratory (8). The recombinant viruses R325, lacking the carboxyl-terminal domain of ICP22; R7356, lacking the UL13 gene; R7041, lacking the US3 gene; and R7353, lacking both UL13 and US3 genes, were described elsewhere (19, 20, 22, 23, 24).
Immunoblotting. Cells were harvested as follows. The medium was removed, and the cells were rinsed with phosphate-buffered saline (PBS), scraped into PBS, pelleted by centrifugation, solubilized in high-salt lysis buffer (20 mM Tris, pH 8.0; 1 mM EDTA; 0.5% NP-40; 400 mM NaCl; 0.1 mM sodium orthovanadate; 10 mM NaF; 2 mM dithiothreitol; 100 μg each of phenylmethylsulfonyl fluoride, tosylsulfonyl phenylalanyl chloromethyl ketone, and leupeptin per ml), and stored on ice for 1 h. Insoluble materials were removed by centrifugation as described above. Samples (75 μg/sample) were mixed with equal volumes of a gel loading buffer (2% sodium dodecyl sulfate, 50 mM Tris, pH 6.8, 2.75% sucrose, 5% ?-mercaptoethanol, bromophenol blue), subjected to electrophoresis on a 10% bisacrylamide gel, transferred to nitrocellulose membranes, blocked for 2 h with 5% nonfat dry milk, and reacted with the appropriate antibody. The blots were incubated in AP buffer (100 mM Tris, pH 9.5, 100 mM NaCl, 5 mM MgCl2), followed by AP buffer containing BCIP (5-bromo-4-chloro-3-indolylphosphate) and nitroblue tetrazolium. The reaction was stopped by immersing the blot in a solution containing 100 mM Tris (pH 7.6) and 10 mM EDTA.
Antibodies. Rabbit antibodies to cyclin T and cdk9 (Santa Cruz, CA) were used at 1:250 dilutions. The rabbit antibody to ICP22 (1) was used at a 1:500 dilution in PBS with 1% bovine serum albumin and 0.05% Tween 20. Bound antibody was detected by using secondary antibody diluted 1:3,000 (goat anti-rabbit antibody conjugated to alkaline phosphatase; Bio-Rad).
Production of GST fusion proteins. DNA sequences encoding cdc2, cdk7, or cdk9 were amplified by PCR and cloned into pGEX4T-1. Escherichia coli BL21 cells were transformed with plasmids encoding the above glutathione S-transferase (GST) fusion proteins or GST alone, grown at 30°C until the optical density at 600 nm reached a value of 0.6 to 0.8, and induced with 100 μM isopropyl-?-D-thiogalactosidase for 2 h. Bacteria were collected by centrifugation, resuspended in PBS, lysed by sonication, and mixed with Triton X-100 (1% final concentration). After the cell debris was removed by centrifugation, GST fusion proteins were adsorbed to glutathione-agarose beads (Sigma). The beads were collected and rinsed in PBS. The fusion proteins were eluted with 10 mM glutathione in 50 mM Tris (pH 8.0) and dialyzed against PBS. Protein production was assessed by electrophoresis in denaturing gels followed by Coomassie brilliant blue staining. The GST-CTD plasmid was obtained from Scott Petersen (University of Colorado) and grown as described above, with the exception that the bacteria were induced with 500 μM isopropyl-?-D-thiogalactosidase for 10 h.
Cell infection. HEp-2 cells grown in 25-cm2 flasks were exposed to 2 x 107 PFU of appropriate virus in 1.2 ml of 199V (mixture 199 supplemented with 1% calf serum) on a rotary shaker at 37°C. After 2 h, the inoculum was replaced with 4 ml of fresh DMEM supplemented with 10% serum. Flasks were incubated at 37°C until the cells were harvested at the time points indicated in Results. Time zero is defined as the time at which viral inoculum was added to the cells.
[35S]Met pulse-chase linked to GST pull-down. HEp-2 cells were exposed to virus as above. After 1 h, the inoculum was removed and the cells were starved for 1 h in 199V minus methionine at 37°C. The medium was then replaced with fresh 199V lacking methionine but supplemented with 100 μCi [35S]Met. After 4 h at 37°C, the medium was replaced with 5 ml of DMEM supplemented with 10% serum and incubated at 37°C until harvested at times indicated in Results. Equivalent amounts of protein per sample were brought up to 400 μl in high-salt lysis buffer (final concentration, 400 mM NaCl). Samples were precleared with 50 μl of a 50% slurry of glutathione beads for 2 h at 4°C. The precleared supernatant fluid was reacted for 3 h at 4°C with 10 μl of a 50% slurry of glutathione beads bound to GST alone, cdc2, cdk7, or cdk9. The beads were pelleted by centrifugation and rinsed three times in low-salt lysis buffer (final concentration, 200 mM NaCl). The beads were resuspended in 40 μl of gel loading buffer and subjected to electrophoresis in a denaturing gel and autoradiography.
In vitro kinase assay. HEp-2 cells were exposed to virus as described above, harvested 18 h after infection, lysed, precleared with preimmune serum at 4°C for 2 h, and then reacted with protein A Sepharose beads for 1 h (200 μg/sample). Samples were then centrifuged at 3,000 rpm for 5 min, and the supernatant fluid was collected and reacted with 4 μl of anti-cdk9 antibody overnight at 4°C. The beads were collected by centrifugation and rinsed twice with low-salt buffer (20 mM Tris [pH 8.0], 1 mM EDTA, 0.5% NP-40, 200 mM NaCl, 2 mM dithiothreitol) and twice with incomplete kinase buffer (50 mM Tris [pH 7.4], 10 mM MgCl2, 5 mM dithiothreitol). The beads were then resuspended in 40 μl of complete kinase buffer (20 mM Tris [pH 8.0], 1 mM EDTA, 0.5% NP-40, 1 mM NaCl, 2 mM dithiothreitol, 10 μM ATP, 20 μCi of [-32P]ATP, 2 μg GST-CTD) and incubated at 30°C for 30 min. The reactions were terminated by the addition of gel loading buffer. The samples were then subjected to electrophoresis in a denaturing gel and autoradiography. Quantification of 32P phosphorylation of the substrate was done with the aid of a Molecular Dynamics PhosphorImager (Storm 860).
RESULTS
cdk9 pulls down from lysates of HSV-1-infected cells two proteins labeled after infection. In this series of experiments GST-cdc2, GST-cdk7, and GST-cdk9 bound to glutathione beads were reacted with lysates of HEp-2 cells infected with HSV-1(F) and incubated in medium containing [35S]methionine as described in Materials and Methods. The proteins bound to the chimeric proteins on the beads were solubilized and subjected to electrophoresis and autoradiography. All of the procedures were as described in Materials and Methods. The chimeric proteins bound to the beads were subjected to electrophoresis in a denaturing gel and stained with Coomassie blue (Fig. 1A). Each of the samples eluted from the glutathione beads yielded a single major band. The molecular weights of the proteins bound to the glutathione beads were of the expected values of the GST-cdc2, GST-cdk7, and GST-cdk9 chimeric proteins. The autoradiographic images of the labeled infected-cell proteins bound to the chimeric proteins are shown in Fig. 1B. Each of the three chimeric proteins, but not GST alone, pulled down labeled proteins. GST-cdc2 pulled down a protein that was found to be UL42 (4). Of these only GST-cdk9 brought down two proteins with apparent relative molecular weights (Mrs) of 69,000 and 150,000.
The 69,000-Mr protein interacting with GST-cdk9 is ICP22. Two series of experiments indicated that the 69,000-Mr protein pulled down by the GST-cdk9 chimeric protein is ICP22. In the first series, lysates of cells harvested 6 h after mock infection or infection with wild-type or the R325 mutant viruses were reacted with GST-cdk9 as described above. The chimeric proteins bound to the beads were solubilized, subjected to electrophoresis in denaturing gels, transferred to a nitrocellulose membrane, and reacted with anti-ICP22 antibody. Figure 2 shows the reactivity of the antibody with aliquots of whole-cell lysates and the proteins eluted from the GST-cdk9 beads. As shown in Fig. 2 (lane 5), the antibody reacted with a protein band which comigrated with that present in lysate of wild-type virus-infected cells. This band was absent from lysate of R325 mutant-infected cells or the proteins pulled down by GST-cdk9 from lysates of cells infected with the R325 mutant.
In the second series of experiments, GST-cdk9 bound to beads was reacted with lysates of HEp-2 cells either mock infected or infected with HSV-1(F) or with the R325, R7356, or R7041 mutant virus; radiolabeled; and processed as described in the experiments summarized in Fig. 1. The proteins bound to the GST-cdk9 beads were solubilized, subjected to electrophoresis in a denaturing polyacrylamide gel, transferred to a nitrocellulose sheet, and subjected to autoradiography. As shown in Fig. 3 lanes 2 and 4, GST-cdk9 chimeric protein pulled down from lysates of cells infected with wild-type virus or a mutant lacking the UL13 protein kinase the same set of proteins as those pulled down from lysates of wild-type virus-infected cells (Fig. 1). The significant findings were that GST-cdk9 did not pull down a 69,000-Mr protein from lysates of cells infected with the R325 mutant and that the amount of 69,000-Mr protein pulled down from lysates of cells infected with the US3 mutant was significantly lower than those pulled down from wild-type virus-infected cells. We should note that a faint band containing a faster-migrating protein pulled down by GST-cdk9 from lysates of R325-infected cells comigrated with a protein pulled down from lysates of mock-infected cells.
We conclude from these experiments that the 69,000-Mr protein is ICP22, that the functional interaction of ICP22 with cdk9 requires the carboxyl-terminal 220 amino acids absent from lysates of R325-infected cells, and that the interaction is at least partially dependent on the presence of the US3 kinase.
The levels of cdk9 and cyclin T, a partner of cdk9, do not change during the HSV-1 replicative cycle. Earlier studies have shown that, in HSV-1-infected cells, cdc2 is activated but its partners, cyclins A and B, are degraded in an ICP22- and UL13-dependent fashion (2-4). To determine the status of cdk9 and cyclin T in infected cells, replicate cultures of HEp-2 cells were mock infected or infected with HSV-1(F) or R325; harvested at 3, 6, 9, or 16 h after infection; solubilized, subjected to electrophoresis in denaturing gels, and probed with antibody to cdk9 or cyclin T. As shown in Fig. 4, the levels of cdk9 (top panel) or cyclin T (bottom panel) did not change significantly during the course of the HSV-1 replicative cycle. In preliminary experiments anti-cdk9 antibody pulled down a protein that reacted with anti-cyclin T antibody from both infected and mock-infected cells (data not shown).
The protein complex phosphorylating RNA POL II CTD contains ICP22 and cdk9. The objective of the experiments described in this section was to determine whether cdk9-ICP22 complex mediates the phosphorylation of the RNA POL II CTD. In these experiments, lysates of mock-infected HEp-2 cells or cells infected with wild-type HSV-1(F) or mutant virus R7041 (US3), R7356 (UL13), R7353 (US3/UL13), or R325 were reacted with antibody to cdk9 or ICP22. The precipitates were reacted with CTD of RNA POL II in the presence of [-32P]ATP as described in Materials and Methods. The proteins were then solubilized, electrophoretically separated on denaturing gels, and subjected to autoradiography. Panels A and D of Fig. 5 show the autoradiographic images of the RNA POL II CTD reacted with precipitates obtained with cdk9 and ICP22 antibodies, respectively. The amount of radioactivity as measured with the aid of a Molecular Dynamics 860 phosphorimager is shown in Fig. 5C and E. Panel B shows a photograph of the Ponceau S-stained bands containing the GST-RNA POL II CTD as evidence that equal amounts of substrate were present in each reaction mixture. The results show the following: RNA POL II CTD was phosphorylated by immune precipitates brought down by either anti-ICP22 or anti-cdk9 antibody from lysates of wild-type virus-infected cells or cells infected with the UL13 mutant virus. The RNA POL II CTD was not phosphorylated by precipitates from lysates of cells mock infected or infected with US3 or US3/UL13 mutant viruses. The results are consistent with those presented above showing that cdk9 and ICP22 form a complex at least partially dependent on the US3 protein kinase. They also indicate that the complex can phosphorylate the CTD of RNA POL II.
In light of the publications showing that ICP22 mediates the formation of the RNA POL IIi in a UL13-dependent manner we took numerous steps to ascertain that these results were reproducible. The experiments were repeated numerous times with independently produced batches of mutant viruses. Moreover, since laboratory contaminations are not unheard of, we verified that the mutants lacked the appropriate protein kinases.
The involvement of UL13 protein kinase in the posttranslational generation of the RNA POL IIi form. The studies on the involvement of ICP22 and UL13 protein kinase were originally done on Vero cells. The studies reported here were done in human cells. One possible explanation for the observation that the CTD of RNA POL II is phosphorylated in both a UL13- and a US3-dependent fashion is that the protein kinase involved in this reaction is cell type dependent. To test this hypothesis, we examined the electrophoretic mobility of RNA POL II in Vero, rabbit skin, and HEp-2 cell lines mock infected or infected with wild-type or mutant viruses as described in Materials and Methods. The results shown in Fig. 6 were as follows. In the case of rabbit skin or HEp-2 cells, the accumulation of RNA POL IIi form was dependent on UL13 protein kinase and not on the US3 protein kinase. In the experiment shown in Fig. 6A, the accumulation of the RNA POL IIi form in infected Vero cells was dependent on both kinases. In other experiments done on Vero cells, the role of the US3 protein kinase was less evident (data not shown). We are led to conclude that the posttranslational modification of RNA POL II underlying the change in mobility is primarily ICP22 and UL13 protein kinase dependent and that under certain physiologic conditions the US3 protein kinase may also be required.
DISCUSSION
An interesting aspect of the biology of HSV is that its proteins are multifunctional and that one fundamental strategy of the virus is to attain its objective in more than one way. ICP22 is an example of both properties. First, it encodes multiple functions, some mapping in the 200-amino-acid sequence at the carboxyl-terminal domain and some, as yet poorly defined, in the residual amino-terminal domain. As exemplified by the recombinant virus R325, the carboxyl-terminal domain appears to be required in a cell-type-specific manner for the synthesis of a subset of proteins exemplified by the products of UL38, UL41, and US11 genes (23). Independently, ICP22 has been shown to have two activities that also map in its carboxyl-terminal domain and which correlate with the optimal synthesis of UL38, UL41, and US11 proteins. One such activity described by this laboratory involves a series of metabolic events resulting in the degradation of cyclins A and B and acquisition by their partner, cdc2, of a novel partner, the DNA polymerase accessory factor encoded by UL42 (2-4). This cdc2 kinase in this partnership is activated and binds the topoisomerase II, both in an ICP22-dependent manner (5). The tie between activation of cdc2 and the formation of the cdc2-UL42-topoisomerase II complex and the synthesis of the subset of late 2 proteins rests on the observation that these proteins are synthesized from mRNA made late in infection, presumably transcribed from progeny DNA. Late in infection the progeny DNA consists of tangles of concatemers. Topoisomerase II could play a role in rendering transcription more efficient by untangling the concatemers, but in addition, topoisomerase II has been reported to play a role in transcription (31). The other property of ICP22 related to transcription is based on reports that ICP22 mediates in a UL13-dependent manner the phosphorylation of the carboxyl-terminal domain of RNA POL II. It should be stressed that connections between the formation of cdc2-UL42-topoisomerase II complex and the phosphorylation of the carboxyl-terminal domain of RNA POL II and the efficiency of synthesis of the subset of late proteins described above are covariant properties dependent on ICP22. To investigate these properties of ICP22, we decided to dissect further the interaction between RNA POL II and ICP22. Our approach was based on the evidence that cdk9 is related to cdc2 and that the involvement of cdk9 in the transcription of viral genes, namely, those of HIV, has already been established (15, 16, 21, 30). The results presented in this report indicate that cdk9 interacts with ICP22, that this interaction is to a large extent dependent on the US3 protein kinase but not on the UL13 protein kinase, and that complexes containing cdk9 and ICP22 phosphorylated the carboxyl-terminal domain of RNA POL II in vitro in a US3- and not in a UL13-dependent manner.
The studies presented in this report confirm the involvement of ICP22 in the posttranslational modification of RNA POL II. They differ in two respects from the earlier studies (29). First, we report that both cdk9 and ICP22 were involved and present in the complex that phosphorylated RNA POL II. In particular we noted that cdk9 alone, in the absence of full-length ICP22, failed to perform this task. Second, we report that the in vitro phosphorylation of RNA POL II was dependent on the presence of the US3 protein kinase and not on the UL13 protein kinase. At the same time we have shown that in the cell-virus system we were using the posttranslational modification of RNA POL II results in the formation of the "intermediate" phosphorylated form of the enzyme.
The hypothesis that we would like to propose is based on the evidence that ICP22 is posttranslationally modified by either one or both of the UL13 and US3 protein kinases (23, 24) and that it performs multiple and potentially diverse functions depending on the state of phosphorylation of the protein. It is conceivable that its functions are determined by its protein partner, the selection of which is, in turn, determined by the nature of the posttranslational modifications. Thus, activation of cdc2 may require phosphorylation of ICP22 by UL13 protein kinase whereas interaction with cdk9 requires phosphorylation by the US3 protein kinase. We have established that the cdk9-ICP22 complex formed in a US3-dependent manner phosphorylated RNA POL II. What remains to be defined is both the nature of the phosphorylation and the effect of this modification on RNA POL II.
ACKNOWLEDGMENTS
We thank Scott Petersen for the plasmid encoding the CTD of RNA POL II and Ralph Weichselbaum for invaluable discussion.
These studies were aided by grants from the National Cancer Institute (CA78766, CA71933, CA83939, CA87661, and CA88860), United States Public Health Service.
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