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Previous Article | Next Article 
Molecular and Cellular Biology, March 2003, p. 2055-2067, Vol. 23, No. 6
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.6.2055-2067.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Tumor Virology Division, New England Regional Primate Research Center, Southborough, Massachusetts 01772-9102,1 Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021,2 Institute of Molecular Immunology, GSF-National Research Center for Environment and Health, Munich, Germany3
Received 6 November 2002/ Returned for modification 10 December 2002/ Accepted 18 December 2002
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ABSTRACT |
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INTRODUCTION |
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RNA polymerase II is found in a large holoenzyme complex containing several general transcription factors and the Mediator (32). Mediator is a large complex composed of polypeptides that range in size from 10 to 240 kDa. Several mammalian Mediator activities were discovered that specifically supported (TRAP/SMCC, ARC, DRIP, and Srb/Mediator) or repressed (NAT) the function of activators (30, 32). This complex functions as an interface between sequence-specific transcription factors and the general transcriptional apparatus. For example, the TRAP complex interacts with p53, VP16, NF-
B, and E1A to recruit RNA polymerase II and general transcription factors to form a functional preinitiation complex at the promoter (20). More specifically, the TRAP220 subunit of this complex is known to interact with nuclear receptors, including the thyroid receptor, vitamin D receptor, estrogen receptor, and glucocorticoid receptor; the TRAP150ß subunit is likely an integrator of the E1A and RAS signaling pathways; and the TRAP80 subunit interacts directly with the p53 and VP16 activation domains (6, 18, 21, 40, 48). Thus, TRAP/Mediator/SMCC, a multifunctional complex, contains diverse subunits that serve as specific targets for distinct activators.
Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8, is thought to be an etiologic agent of Kaposi's sarcoma (9). It is also associated with two diseases of B-cell origin, primary effusion lymphoma and an immunoblast variant of Castleman's disease (5, 7). The genomic sequence indicates that KSHV is a gamma herpesvirus that is closely related to Epstein-Barr virus, herpesvirus saimiri, rhesus monkey rhadinovirus, and murine gammaherpesvirus 68 (2, 9, 35, 37, 43).
An important step in the herpesvirus life cycle is the switch from latency to lytic replication. KSHV RTA has been shown to play a central role in the switch of the viral life cycle from latency to lytic replication. Ectopic expression of RTA is sufficient to disrupt viral latency and activate lytic replication to completion (15, 29, 42). As a typical transcription activator, KSHV RTA contains an N-terminal basic DNA-binding domain and a C-terminal acidic activation domain. Its N-terminal DNA-binding domain is well conserved with that of Epstein-Barr virus RTA and other gammaherpesvirus RTA homologs and shows a sequence-specific DNA-binding activity (8, 27, 38). While it is less conserved, a carboxyl acidic activation domain exhibits strong transactivation activity in the heterologous context with the Saccharomyces cerevisiae GAL4 transcription factor (16, 28). It has been shown that RTA activates the expression of numerous viral genes in the KSHV lytic cycle, including its own promoter, polyadenylated nuclear (PAN) RNA, ORF57, vOX-2, viral G protein-coupled receptor, and vIRF1 (10, 12, 13, 22, 36, 38).
While the detailed mechanism of RTA-mediated transcription activation remains unclear, several pieces of evidence suggest that RTA activates its target promoter activity through both direct binding to the specific sequence and interaction with various cellular transcriptional factors. In fact, numerous cellular proteins, including Oct-1, Stat3, novel cellular protein MGC2663, CBP, and RBP J, have been found to interact with and synergize with RTA (16, 17, 26, 36, 45).
Despite extensive studies of RTA-mediated transcriptional activation of viral lytic genes, details of the mechanism are mostly unknown. To delineate the molecular mechanism of RTA-mediated lytic gene expression, we purified proteins that bound to RTA. Mass spectrometry demonstrated that RTA recruits cellular SWI/SNF and TRAP/Mediator complexes through its carboxy-terminal short acidic sequence. Recruitment of SWI/SNF and TRAP/Mediator complexes by RTA into the viral lytic promoters is essential for their gene expression and thus for KSHV reactivation. Furthermore, genetic ablation of these interactions abolishes KSHV viral lytic replication. These results demonstrate that the molecular mechanisms that underlie RTA-mediated transcriptional activation require a large number of transcriptional cofactors and that their actions ultimately direct well-controlled viral gene expression and thereby viral lytic reactivation.
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MATERIALS AND METHODS |
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Protein purification and mass spectrometry. To identify RTA-binding proteins, [35S]methionine- and [35S]cysteine-labeled Raji cells (1 x 107 cells) or 20 liters of Raji cells were resuspended with lysis buffer (0.15 M NaCl, 0.5% Nonidet P-40, and 50 mM HEPES buffer [pH 8.0]) containing protease and phosphatase inhibitors. Precleared lysates were mixed with glutathione beads containing glutathione S-transferase (GST) and GST-RTA fusion protein for 4 h, and the beads were washed extensively with lysis buffer. Proteins bound to glutathione beads were eluted and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands isolated from SDS-PAGE were analyzed by ion-trap mass spectrometry, and the amino acid sequence was determined by tandem mass spectrometry and database search.
GST pulldown assays. RTA and TRAP subunits were in vitro transcribed and translated with a T7-coupled transcription-translation system (Promega, Madison, Wis.). The labeled proteins were incubated with GST fusion protein-saturated resin in binding buffer (20 mM HEPES [pH 7.4], 100 mM NaCl, and 0.1% NP-40 supplemented with protease inhibitors). The reaction mixture was incubated at 4°C for 2 h. The beads were then washed four times with binding buffer, SDS-PAGE sample buffer was added, and the proteins were analyzed by SDS-PAGE and visualized by PhosphorImager (BAS-1500; Fuji Film Co., Tokyo, Japan).
Flow cytometry. Cells (5 x 105) were washed with complete medium and stained with unconjugated K8.1 primary antibody, followed by fluorescein isothiocyanate-conjugated secondary antibody at 4°C. After a final wash, the cells were fixed with 2% paraformaldehyde, and flow cytometry was performed with a fluorescence-activated cell scan/sorter (Becton Dickinson, Mountain View, Calif.).
ATPase assay. Reactions were carried out at 30°C with 2 µM ATP in 12 mM sodium HEPES (pH 7.9)-60 mM KCl-7 mM MgCl2-60 ng of bovine serum albumin per µl-6% glycerol, as described previously (34). ATP hydrolysis was measured with affinity-purified resin in the presence or absence of 20 nM plasmid DNA (5.6 kb). A PhosphorImager was used to quantitate the ratio of inorganic phosphate to ATP at each time point.
In vitro transcription, TRAP/Mediator purification, and immunodepletion of TRAP/Mediator from nuclear extracts. Detailed procedures for in vitro transcription, TRAP/Mediator purification, and immunodepletion of TRAP/Mediator were described previously (4).
Chromatin immunoprecipitation.
The chromatin immunoprecipitation assay was performed according to the manufacturer's (Upstate Biotech) instructions with several modifications. Briefly, T75 culture dishes were treated with 1% formaldehyde for 10 min at room temperature. After a brief sonication, immunoprecipitation was performed with the appropriate antibody. After several washes, the immunocomplexes were eluted with 50 mM Tris (pH 8.0)-1 mM EDTA-1% SDS at 65°C for 10 min, adjusted to 200 mM NaCl, and incubated at 65°C for 5 h to reverse the crosslinks. After successive treatments with 10 µg of RNase A and 20 µg of proteinase K per ml, the samples were extracted with phenol-chloroform and precipitated with ethanol. One tenth of the immunoprecipitated DNAs was analyzed by PCR with the primer sets for Rp (nucleotides 71221 to 71550, KSHV GenBank accession number U75698), Mp (nucleotides 81661 to 81920), vOX-2p (nucleotides 127684 to 127911), PANp (nucleotides 28727 to 29043), vIRFp (nucleotides 85589 to 85709), gBp (nucleotides 8341 to 8642), and POLp (nucleotides 10602 to 11162). Amplifications (26 cycles) were performed in the presence of 5 µCi of [
-32P]dCTP, and the PCR products were analyzed in 5% polyacrylamide gels. For the chromatin reimmunoprecipitation (reChip) assays, after washing of protein G-Sepharose beads from the primary immunoprecipitation, the complexes were eluted by incubation with 10 mM dithiothreitol at 37°C for 30 min and diluted to 40 times the original volume. Eluates were reimmunoprecipitated with the second antibody. The anti-acetyl histone H3 antibody was purchased from Upstate Biotechnology.
Generation of riboprobe templates and RNase protection assay.
RNA (100 ng/reaction) extracted from KSHV-infected BCBL1 cells was used as the template for cDNA synthesis initiated by random hexamer primers, followed by PCR with a GeneAmp kit (Perkin-Elmer Cetus, Foster City, Calif.) and appropriate primers for the amplification of KSHV-specific DNA fragments. The reverse transcription (RT)-PCR conditions, design of PCR primers, and ligation of the amplified DNA fragments into the pEF1/Myc-His A vector were detailed previously (19). The subclone designations and nucleotide sequence based from KSHV GenBank accession number U75698 were as follows: RTA (nucleotides 68349 to 68680), vIRF-1 (nucleotides 18618 to 18857), and ORF57 (6779 to 6977). A riboprobe template set specific for KSHV RTA, ORF57, and vIRF1 was assembled from EcoRI-linearized and purified subclones. All riboprobe syntheses were driven by T7 bacteriophage RNA polymerase with [-32P]UTP (Amersham, Arlington Heights, Ill.) as the labeling nucleotide. Probe bands were visualized by autoradiography by with a PhosphorImager.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interaction of RTA with SWI/SNIF and TRAP/Mediator complexes. To confirm the interaction of RTA with SWI/SNF and TRAP/Mediator complexes, we repeated the GST pulldown and performed immunoblot analysis with antibodies to SWI/SNF and TRAP/Mediator subunits. This showed that the subunits of SWI/SNF and TRAP/Mediator complexes tested were detected in the GST-RTA complexes, whereas none of them were present in the GST complex (Fig. 2A). Furthermore, CDK8 and cyclin C were also readily detected in the GST-RTA complexes but not in the GST complexes (Fig. 2A).
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Specific region of RTA required for SWI/SNF and TRAP interaction. To define the specific region of RTA required for the interaction with SWI/SNF and TRAP/Mediator complexes, additional GST-RTA fusion proteins, GST-RTA(C598-691), GST-RTA(C627-691), and GST-RTA(C598-644), were generated (Fig. 3A). Pulldown assays showed that the GST-RTA(C598-691) and GST-RTA(C598-644) fusion proteins interacted with the SWI/SNF and TRAP complexes as efficiently as GST-RTA(C581-691) (Fig. 3C). In contrast, GFT-RTA(C627-691) was not capable of interacting with the SWI/SNF and TRAP/Mediator complexes under the same conditions (Fig. 3C). Immunoblot analysis confirmed that cellular proteins associated with GST-RTA(C598-691) and GST-RTA(C598-644) were part of the SWI/SNF and TRAP/Mediator complexes (data not shown). This indicates that a sequence of 47 amino acids of RTA is necessary for interacting with SWI/SNF and TRAP complexes.
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To test whether these sequences are involved in the interaction with SWI/SNF and TRAP complexes, we generated GST-RTA fusions containing mutations at these sequences as follows. The two glutamic acids at residues 612 and 613 were replaced with alanines to generate GST-RTA(C598-691 DD-AA), the ILQ residues at 619 to 621 were replaced with alanines to generate GST-RTA(C598-691 ILQ-AAA), and 10 amino acids at residues 612 to 621 were deleted to generate GST-RTA(C598-691 
DD-ILQ) (Fig. 3A and 3C). Interestingly, GST-RTA(C598-691 ILQ-AAA) migrated in SDS-PAGE with a slightly altered rate, suggesting a conformation change (bottom of Fig. 3C). Similar amounts of GST-RTA fusion proteins were used for the pulldown assay with 35S-labeled Raji cell lysates. We found that all three point mutations or deletions at the conserved sequences of the RTA carboxyl region abolished its interaction with SWI/SNF and TRAP/Mediator complexes (Fig. 3C). This result was further confirmed by immunoblot assay with antibodies to SWI/SNF and TRAP/Mediator complexes (data not shown). These results indicate that a region of 47 amino acids of RTA is necessary for interacting with SWI/SNF and TRAP/Mediator complexes and that the conserved sequences of this region appear to play an important role in this interaction.
Role of SWI/SNF and TRAP/Mediator complexes in RTA-mediated transcriptional activation.
To elucidate the role of the RTA interaction with SWI/SNF and TRAP/Mediator complexes, we examined the level of transcriptional activity of RTA(DD-AA), RTA(ILQ-AAA), and RTA(DD-ILQ) mutants, which do not bind to SWI/SNF and TRAP complexes, as shown in Fig. 2B. As a control, we included RTA AD, which contains a deletion of the carboxy-terminal transcriptional activation domain (amino acids 598 to 691) (16). While wild-type RTA activated its own promoter (Rp) and the ORF57 promoter (Mp) by 30- to 50-fold, the RTA(DD-AA), RTA(ILQ-AAA), and RTA(DD-ILQ) mutants exhibited a marked reduction of transcriptional activation activity (Fig. 3D). The RTA(DD-ILQ) mutant in particular exhibited very little transcriptional activation activity compared to wild-type RTA, whereas the RTA AD deletion mutant had no activity (Fig. 3D). RTA and its mutants were expressed at equivalent levels (Fig. 3D). These results indicate that mutations at the conserved sequences which cripple the interaction with SWI/SNF and TRAP/Mediator complexes drastically reduce the RTA-mediated activation of Rp and Mp promoter activity, suggesting an important role of SWI/SNF and TRAP complexes in RTA-mediated transcription of Rp and Mp.
SWI/SNF complex facilitates RTA-mediated transcriptional activation. Since RTA interacts with the chromatin-remodeling SWI/SNF complex that contains DNA-dependent ATPase activity, we examined ATPase activity in the affinity-purified RTA complexes. After incubation with DNase and ethidium bromide to exclude any potential contamination by chromosomal DNA, crude extracts from Raji B cells were mixed with GST or GST-RTA(C581-691). After extensive washing, affinity-purified GST and GST-RTA(C581-691) complexes were assayed for ATPase activity. Purified GST-RTA(C581-691) complexes contained an approximately threefold-higher basal level of ATPase activity than purified GST complexes (Fig. 4A). In addition, this ATPase activity was significantly enhanced by the addition of template plasmid DNA (Fig. 4A). In contrast, GST complexes did not contain a significant level of ATPase activity, nor did the addition of template plasmid DNA augment ATPase activity (Fig. 4A). These results demonstrate that purified RTA complexes contain a strong DNA-dependent ATPase activity that appears to be derived from the SWI/SNF complex.
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To investigate the functional role of the interaction between the SWI/SNF complex and RTA, we cotransfected expression vectors containing RTA and Brg1 cDNA together with the RTA promoter (Rp) luciferase reporter vector into SW13 adrenal carcinoma cells, which lack a functional Brg1 gene. In the absence of a functional Brg1, RTA induced basal Rp promoter activity (Fig. 4C). In contrast, coexpression of Brg1 strongly enhanced RTA activity, resulting in over eightfold induction of Rp promoter activity (Fig. 4C). This result suggests that the efficient activation of Rp promoter activity by RTA requires a functional SWI/SNF complex.
TRAP/Mediator complex is essential for RTA-mediated transcription in vitro. To establish the functional consequences of an interaction of RTA with the TRAP/Mediator complex, we first asked if TRAP could function as a coactivator for RTA-mediated transcription in an in vitro transcription reaction with nuclear extracts. We have shown previously that TRAP25 antibody efficiently depletes the majority of TRAP/Mediator complex from the nuclear extract, leaving RNA polymerase II and other general transcription factors unaffected (4). Thus, we tested the intact nuclear extracts and TRAP/Mediator-depleted nuclear extracts for the ability of TRAP/Mediator to support transcriptional activation induced by Gal4-RTA, which contains the C-terminal activation domain of RTA (16, 28). In addition, the G5HML plasmid has five copies of a Gal4 binding site upstream of a hybrid core promoter and permits measurement of the transcriptional activity of the Gal4-RTA fusion (4).
In the intact nuclear extracts, Gal4-RTA protein activated in vitro transcription of G5HML up to 46-fold (Fig. 5A). By striking contrast, Mediator-depleted extracts had only a basal level of Gal4-RTA transcriptional activation (Fig. 5A, lane 3). The reconstitution of purified TRAP/Mediator complex into Mediator-depleted nuclear extracts completely restored Gal4-RTA transcription activity (Fig. 5A, lane 4). These results demonstrate that the reduction of Gal4-RTA transcription activity in TRAP/Mediator-depleted nuclear extracts is due solely to the absence of TRAP/Mediator rather than other coimmunoprecipitated factors and that the TRAP/Mediator complex plays an important role in RTA-mediated transcriptional activation.
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The resulting blots showed that while RTA interacted with TRAP230, TRAP95, TRAP80, MED6, TRAP25, and SRB7 subunits after treatment with the crosslinking agent, TRAP230 exhibited a saturated level of binding activity to GST-RTA(C581-691) even at the lowest concentration of DSP tested (Fig. 5B, lane 5). In contrast, the amounts of other TRAP subunits binding to GST-RTA(C581-691) gradually increased as the DSP concentration was increased (Fig. 5B). None of the TRAP subunits tested was shown to bind to GST protein under the same conditions, indicating the specificity of the RTA interaction with TRAP subunits (Fig. 5B). This also suggests that TRAP230 has a higher affinity for RTA protein than other TRAP subunits.
To further characterize this, GST, GST-RTA(C598-691), and GST-RTA(C598-691 DD-ILQ) fusion proteins were mixed with [35S]methionine-labeled TRAP subunits, TRAP230, TRAP100, TRAP80, and PCQAP, followed by autoradiography. In addition to full-length TRAP230, three fragments of TRAP230, TRAP230N, TRAP230C1, and TRAP230C2, were included in this assay. The results showed that the carboxyl activation domain of RTA interacted efficiently with TRAP230, TRAP230C1, and TRAP230C2 but not with TRAP230N, TRAP100, TRAP80, and PCQAP (Fig. 5C). In contrast, the RTA(DD-ILQ) mutant did not interact with full-length TRAP230 and its C-terminal fragments under the same conditions (Fig. 5C). This result shows an activation domain-dependent interaction of RTA with the TRAP230 subunit.
Recruitment of cellular transcriptional cofactors into RTA-dependent promoters.
RTA activates expression from the promoters of several KSHV genes, including its own promoter (Rp), ORF57 (Mp), polyadenylated nuclear RNA (PANp), vOX2 (vOX2p), and vIRF1 (vIRFp), but RTA cannot activate expression of KSHV DNA polymerase (POLp) and glycoprotein B (gBp). To investigate whether RTA recruits cellular transcriptional cofactors into the RTA-dependent promoters, we performed chromatin immunoprecipitation experiments. For this assay, we used a KSHV-infected BCBL1 cell line (TRExBCBL1) in which Myc epitope-tagged wild-type RTA or the RTA AD mutant gene was integrated into the chromosomal DNA under the control of a tetracycline-inducible promoter. Treatment of these cells with doxycycline strongly activated RTA and RTA AD expression (data not shown).
KSHV-infected TRExBCBL1 cells with or without doxycycline treatment were subjected to the chromatin immunoprecipitation assay. RTA-dependent viral promoters (Rp, Mp, PANp, vOX2p, and vIRFp) and RTA-independent viral promoters (POLp and gBp) were tested for RTA binding and the recruitment of cellular transcription cofactors. Twenty-four hours after doxycycline treatment, wild-type RTA was readily detected on the RTA-dependent promoters (Rp, Mp, PANp, vOX2p, and vIRFp) but not on the RTA-independent promoters (POLp and gBp) (Fig. 6A). Additionally, the recruitment of TBP, CBP, Brg1, BAF170, TRAP220, and TRAP100 as well as acetylated histone was strongly detected on the RTA-dependent promoters (Rp, Mp, PANp, vOX2p, and vIRFp), whereas it was not detected on the RTA-independent promoters (POLp and gBp) (Fig. 6A). In the absence of RTA expression, however, none of the cellular transcription cofactors were recruited to viral promoters, indicating that RTA is necessary for the recruitment of cellular transcription cofactors (Fig. 6A). This finding was further supported by the results from TRExBCBL1 cells expressing the RTA AD mutant. Because of the presence of the amino-terminal DNA binding region, RTA AD was detected on the Rp promoter (Fig. 6A, bottom two panels). However, due to the lack of the carboxyl activation domain on the RTA AD mutant, none of these cellular transcriptional cofactors were recruited onto the Rp promoter (Fig. 6A, bottom two panels). Finally, a reChip assay further confirmed that RTA interacted with and recruited CBP, Brg1, and TRAP220 onto the Rp promoter (Fig. 6B).
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Recruitment of TRAP/Mediator and SWI/SNF complexes is required for RTA-mediated KSHV lytic reactivation.
RTA has been shown to be sufficient to induce a complete cycle of KSHV lytic reactivation. To assay the induction of KSHV gene expression by wild-type RTA and its mutants, we treated TRExBCBL1-cDNA5, TRExBCBL1-RTA, TRExBCBL1-RTA AD, and TRExBCBL1-RTA(DD-ILQ) cells with 1 µg of doxycycline per ml. As shown in Fig. 3, RTA(DD-ILQ), which contains a deletion of amino acids 612 to 621, does not interact with TRAP/Mediator and SWI/SNF complexes and lacks transcriptional activation. Despite its complete loss of transcriptional activation activity, the RTA AD mutant still contains intact DNA binding activity and thus functions as a dominant negative mutant that suppresses KSHV lytic reactivation (28).
For the RNase protection assay, we chose to analyze three genes (RTA, ORF57, and vIRF1) having RTA-dependent expression. Also, a probe for the gene encoding ribosomal protein L32, which serves as a housekeeping gene, was included as a control. Total RNA was harvested at 0, 6, and 12 h after doxycycline (1 µg/ml) treatment and analyzed by the RNase protection assay. Within a short period of doxycycline treatment, rapid, robust expression of the RTA, ORF57, and vIRF1 genes was detected in TRExBCBL1-RTA cells, and the expression of these genes was further increased at 12 h of treatment (Fig. 7A). By striking contrast, the expression of RTA, ORF57, and vIRF1 was undetectable in TRExBCBL1-cDNA5, TRExBCBL1-RTA AD, and TRExBCBL1-RTA(DD-ILQ) cells under the same conditions (Fig. 7A). Similar levels of L32 transcript were detected in all these cells (Fig. 7A).
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AD, and TRExBCBL1-RTA(DD-ILQ) cells. The reactivation assay used a polyclonal antibody that recognizes the K8.1 envelope glycoprotein expressed with late kinetics. This protein is specific to lytically infected cells, since it is expressed only on cells that are committed to viral reactivation. Approximately 1.7% of TRExBCBL1-cDNA5 cells showed spontaneous surface expression of K8.1 before doxycycline stimulation and did not increase K8.1 surface expression after 3 days of doxycycline stimulation (Fig. 7B). In contrast, TRExBCBL1-RTA cells showed a dramatic increase in K8.1 surface expression: more than 74% of these cells underwent lytic reactivation after 3 days of stimulation (Fig. 7B). However, both TRExBCBL1-RTA(DD-ILQ) and TRExBCBCL1-RTA AD cells showed little or almost no increase in K8.1 surface expression after 3 days of doxycycline stimulation (Fig. 7B). In particular, TRExBCBCL1-RTA AD cells showed extremely low K8.1 expression with or without stimulation, which was likely due to the dominant negative activity of the RTA AD mutant (Fig. 7B). Wild-type RTA, RTA(DD-ILQ), and RTA AD were expressed at equivalent levels in KSHV-infected TRExBCBL1 cells (Fig. 7C). In addition, expression of RTA(DD-AA) and RTA(ILQ-AAA) also showed little or no increase in K8.1 surface expression under the same conditions (data not shown). Thus, these results show a strong correlation between the ability of RTA to activate viral lytic reactivation and the ability to recruit SWI/SNF and TRAP/Mediator complexes. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using affinity purification and mass spectrometry analysis, we found that KSHV RTA recruits numerous cellular transcription cofactors, including CBP, SWI/SNF complex, and TRAP/Mediator complex. While most but not all subunits of SWI/SNF and TRAP/Mediator complexes were isolated from our analysis, additional coimmunoprecipitation and immunoblot assays unambiguously demonstrated that RTA interacts with these complexes. Furthermore, our chromatin immunoprecipitation assay showed that RTA also recruits a p160 family member, NcoA-1/steroid receptor coactivator-1 (SRC-1), to its own promoter (unpublished results). Transcriptional activation by numerous cellular factors, specifically nuclear receptors, also requires multiple cellular cofactors that act in both a sequential and combinatorial manner to reorganize chromatin templates and to modify and recruit basal transcription factors and RNA polymerase II (20, 32, 33). These cofactors include the p160 family, SWI/SNF complex, CBP/p300/PCAF, TRIP/DRIP/ARC complex, and TRAP/SMCC/Mediator complex. This suggests that RTA resembles cellular nuclear receptors in the recruitment of cellular transcription cofactors and in the initiation of gene expression. Thus, our findings indicate that the molecular strategies that underlie RTA-mediated KSHV reactivation require the actions of a large number of transcriptional cofactors, which reorganize viral promoter chromatin structure and further recruit basal transcriptional factors to initiate well-controlled viral gene expression.
Previous reports have demonstrated that numerous cellular proteins, including Oct-1, RBP J, and the novel cellular protein MGC2663, interact with RTA and that this interaction is mediated through the amino-terminal DNA binding domain and the central leucine zipper motif of RTA. Despite low sequence homology at the carboxyl transcriptional activation domain among RTA homologs of gamma-2 herpesviruses, this region has been shown to play a critical role in transcription activation of KSHV genes and thereby induction of viral reactivation. Furthermore, we have demonstrated that mutations of two glutamic acids at residues 612 and 613 and the ILQ sequence at residues 619 to 621 of RTA cripple the interaction of RTA with SWI/SNF and TRAP/Mediator complexes and that the loss of interaction with cellular transcription cofactors results in a drastic reduction in RTA-mediated transcriptional activity and thereby KSHV reactivation. This indicates that a specific interaction of RTA with the SWI/SNF and TRAP/Mediator complexes plays an important role in RTA-mediated transcription.
Of note, while the loss of interaction with these cellular cofactors results in a drastic reduction in RTA-mediated gene expression, a minor level, less than 3%, of RTA activity still remained in viral reactivation. This low level of RTA activity may be derived from other cellular factors, including Oct-1, Stat3, novel cellular protein MGC2663, RBP J, or other proteins not yet characterized. Finally, we also found that, similar to KSHV RTA, the herpesvirus saimiri and gammaherpesvirus 68 RTA homologs interact with CBP, SWI/SNF, and TRAP/Mediator, suggesting that an interaction with these cellular cofactors is a general mechanism for gamma-2 herpesvirus reactivation (unpublished results). Further study of the synergistic contribution of these cellular cofactors to viral gene expression will provide the detailed molecular mechanism of gamma-2 herpesvirus replication and assist in the development of antiviral therapeutic agents.
Upon external signals, such as chemical treatment, RTA gene expression is initiated, and subsequently RTA recruits a number of transcription coactivator complexes (such as CBP, SWI/SNF complex, TRAP/mediator complex) and basic transcription machinery onto its own promoter and other RTA-dependent promoters. A recent report (39) showed that a complete preinitiation complex, including phosphorylated RNA polymerase II, is assembled at the human antitrypsin promoter long before the recruitment of transcription cofactors, suggesting that chromatin reconfiguration is a defining step of the initiation process, acting after the assembly of the RNA polymerase II machinery. In contrast, chromatin remodeling prior to preinitiation complex assembly is required at the beta interferon gene promoter (1). Further study is necessary to define the specific order of transcription factor assembly on the RTA promoter.
An important step in the herpesvirus life cycle is the switch from latency to lytic replication, and this switch should be well controlled at the molecular and cellular level. In this study, we have demonstrated that KSHV RTA recruits CBP and SWI/SNF chromatin remodeling complex and TRAP/Mediator transcriptional regulatory complex into viral promoters through interactions with a short acidic sequence in its carboxyl-terminal region. The recruitment strongly correlates with and presumably is essential for RTA-dependent viral gene expression and thereby viral lytic reactivation. Thus, these results not only have important implications for understanding the molecular basis of KSHV RTA action but also suggest a basis for the synergistic contributions of cellular transcription cofactors to gamma-2 herpesvirus reactivation.
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ACKNOWLEDGMENTS |
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This work was partly supported by U.S. Public Health Service grants CA82057, CA91819, AI38131, and RR00168 and ACS grant RPG0010201. J. Jung is a Leukemia & Lymphoma Society Scholar.
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FOOTNOTES |
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