Histone acetyltransferase (HAT) activity of p300 modulates human T lymphotropic virus type 1 p30^II-mediated repression of LTR transcriptional activity

Abstract

Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated inflammatory disorders. HTLV-1 provirus has regulatory and accessory genes in four pX open reading frames. HTLV-1 pX ORF-II encodes two proteins, p13^II and p30^II, which are incompletely defined in virus replication or pathogenesis. We have demonstrated that pX ORF-II mutations block virus replication in vivo and that ORF-II encoded p30^II, a nuclear-localizing protein that binds with CREB-binding protein (CBP)/p300, represses CREB and Tax responsive element (TRE)-mediated transcription. Herein, we have identified p30^II motifs important for p300 binding and in regulating TRE-mediated transcription in the absence and presence of HTLV-1 provirus. Within amino acids 100–179 of p30^II, a region important for repression of LTR-mediated transcription, we identified a single lysine residue at amino acid 106 (K3) that significantly modulates the ability of p30^II to repress TRE-mediated transcription. Exogenous p300, in a dose-responsive manner, reverses p30^II-dependent repression of TRE-mediated transcription, in the absence or presence of the provirus, In contrast to wild type p300, p300 HAT mutants (defective in histone acetyltransferase activity) only partially rescued p30^II-mediated LTR repression. Deacetylation by histone deacetylase-1 (HDAC-1) enhanced p30^II-mediated LTR repression, while inhibition of deacetylation by trichostatin A decreases p30^II-mediated LTR repression. Collectively, our data indicate that HTLV-1 p30^II modulates viral gene expression in a cooperative manner with p300-mediated acetylation.

Introduction

Human T cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia/lymphoma (ATL), and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and many immune-mediated disorders (Khan et al., 2001; Franchini et al., 2003; Mahieux and Gessain, 2003). Molecular mechanisms used by the virus to circumvent immune elimination by the host and to facilitate lymphocyte proliferation are not fully understood. The genome of HTLV-1 encodes Gag, Pol, and Env, the common structural and enzymatic proteins characteristic of all retroviruses. Using alternative splicing and alternate initiation codons, this complex retrovirus makes several regulatory and accessory proteins encoded by four open reading frames (ORFs) of the pX region (pX ORF I to IV) between the env gene and the 3′ long terminal repeat (LTR) (Michael et al., 2004; Franchini, 1995). ORF III and IV encode the well characterized Rex and Tax proteins, respectively. Rex is a nucleolar-localizing phosphoprotein, involved in nuclear export of unspliced or singly spliced viral RNA (reviewed in Younis and Green, 2005). Tax is a nuclear-localizing phospho-protein that interacts with cellular transcription factors and activates transcription from the viral promoter and enhancer elements of various cellular genes associated with host cell proliferation (reviewed in Pise-Masison et al., 2005; Marriott and Semmes, 2005; Grassmann et al., 2005). pX ORFs I and II also produce alternatively spliced forms of mRNA that encode four accessory proteins, p12^I, p27^I, p13^II, and p30^II (Berneman et al., 1992; Ciminale et al., 1992; Koralnik et al., 1992). Less is known about the role of pX ORF I and ORF II in the replication or pathogenesis of HTLV-1. However, pX ORFs I and II mRNAs are present in infected cell lines and freshly isolated cells from HTLV-1-infected subjects (Koralnik et al., 1992) and in ATL and HAM/TSP patients (Cereseto et al., 1997). Antibodies (Dekaban et al., 2000) and cytotoxic T cells (Pique et al., 2000) against recombinant proteins or peptides of the pX ORF I and II proteins are present in HTLV-1-infected patients, and asymptomatic carriers.

p30^II is a 241 amino acid protein that contains a highly conserved bipartite nuclear localization signal (D’Agostino et al., 1999), as well as serine-and-threonine-rich regions with distant homology to transcription factors Oct-1 and -2, Pit-1, and POU-M1 (Ciminale et al., 1992). Mutations in the ACH.p30^II viral clone that insert an artificial termination codon in the p30^II mRNA, prevent the virus from obtaining typical proviral blood levels in the rabbit model of infection (Bartoe et al., 2000; Silverman et al., 2004). When provided in limiting concentrations, p30^II expression stimulates HTLV-1 LTR-driven reporter gene activity, even in the presence of Tax, whereas higher concentrations repressed LTR and CRE-driven reporter gene activity (Zhang et al., 2000). p30^II enhances the transforming potential of Myc and transcriptionally activates the human cyclin D2 promoter in a Myc-responsive E-box enhancer elements dependent manner (Awasthi et al., 2005). In addition, p30^II colocalizes with Myc-TIP60 complexes in cultured HTLV-1-infected ATL patient lymphocytes and enhances cyclin D2 promoter-driven transcription in a manner that requires TIP60 histone acetyltransferase (HAT) activity (Awasthi et al., 2005). p30^II also appears to modulate LTR-mediated transcription, in the context of the entire provirus, at the post-transcriptional level, by retaining Tax/Rex mRNA in the nucleus (Nicot et al., 2004; Younis et al., 2004, 2006). Therefore, p30^II acts as a multifunctional protein with transcriptional and post-transcriptional role in regulating viral gene expression and modulates the transcriptional control of the cell cycle.

We have demonstrated that p30^II colocalizes with p300 in the nucleus and physically interacts with CBP/p300, at the highly conserved KIX domain (Zhang et al., 2001). CBP/p300, an important coactivator of cellular and viral transcription, is available at limiting concentrations within the cell nucleus, causing an environment for competition between coactivators and transcription factors, and providing additional regulation of gene expression (Petrij et al., 1995; Suzuki et al., 1999). CBP and p300 bridge transcription factors to relevant promoters and through its intrinsic histone acetyl transferase (HAT) activity, acetylates lysine residues at the amino terminus of histones. This results in modification of chromatin structure to allow access of the transcriptional machinery. p300 also directly acetylates transcription factors such as p53 and GATA-1 and thus augment their sequence-specific DNA-binding activity (Sterner and Berger, 2000). Additionally, CBP/p300 is known to form complexes with proteins such as p300/CBP-associated factor (P/CAF), which also exhibits HAT activity (Grunstein, 1997). HTLV-1 Tax associates with the LTR through its interaction with CREB. These Tax-CREB promoter complexes act as a high-affinity binding site to recruit multifunctional cellular coactivators CBP, p300 and P/CAF to activate the expression of viral genes from the viral promoter (Lemasson et al., 2002).

Interestingly, p30^II contains six highly conserved lysine residues, four of which are part of the consensus acetylation sequence (G/SK motif) (Bannister and Miska, 2000), and thus represent potential acetylation sites for CBP/p300. Based on this, we hypothesized that the intrinsic HAT activity of CBP/ p300 is utilized to acetylate and potentially modulate the transcriptional regulatory function of p30^II. There is also evidence of a functional antagonistic relationship between transcription factors, as a result of competition for binding to common regions of CBP/p300 (Bannister and Kouzarides, 1995; Colgin and Nyborg, 1998; Kamei et al., 1996). Interestingly, p30^II disrupts CREB-Tax-CBP/p300 complexes bound to the TRE repeats (Zhang et al., 2001).

Herein, we have identified motifs within p30^II that are important in binding CBP/p300 and in regulating LTR-mediated transcription. Using N and C terminal deletion mutants of p30^II, we identified binding regions of p300 in p30^II and those important in regulating LTR-mediated transcription in the context of the provirus. We also have identified, using site-directed mutagenesis, a lysine residue at amino acid position 106 (K3) of p30^II that is critical for its repression of TRE-mediated transcription. Exogenously expressed p300 rescued p30^II-mediated repression on LTR-driven gene transcription, irrespective of the presence or absence of the provirus. p300 HAT mutants did not rescue p30^II-mediated LTR repression compared to wild type p300. The deacetylating enzyme histone deacetylase-1 (HDAC-1) enhanced p30^II-mediated HTLV-1 LTR repression. Moreover, inhibition of deacetylation by trichostatin A decreased p30^II-mediated HTLV-1 LTR repression. Our data confirm the role of p30^II as a regulator viral gene transcription, in association with p300. This is the first report that identifies the functional domains of HTLV-1 p30^II important for binding CBP/p300 and in regulating LTR-mediated transcription. Furthermore, our data indicate that p30^II opposes Tax-mediated LTR transactivation, while Tax rescues p30^II-mediated repression of LTR-driven transcription, indicating the ability of these proteins to compete with each other in modulating viral transcriptional activity.

Results

HTLV-1 p30^II accumulates with p300 in the nucleus

HTLV-1 p30^II contains a nuclear localization signal, characteristic of most proteins that function as a transcription factor. Using immunofluorescence and immunoblot methods, previous reports have shown that HTLV-1 p30^II was localized in the nucleus of transfected cells (Koralnik et al., 1993; Zhang et al., 2000). To examine the subcellular localization of p30^II, we performed confocal microscopy on HeLa-Tat cells transiently cotransfected with pMEp30^IIHA. We used Alexa Fluor 633 phalloidin to stain cytokeratin in p30^II expression cells (Fig. 1A) and Hoechst staining as a nuclear marker (Fig. 1D). Consistent with previous reports, p30^II was detected predominantly in the nucleus of our transiently transfected HeLa-Tat cells by confocal microscopy (Fig. 1B). To test whether p30^II compartmentally accumulates with p300 in the nucleus, we performed confocal microscopy of HeLa-Tat cells transiently transfected with pME p30^IIHA and pCMV-p300, and verified the subcellular localization of p30^II (Fig. 1B) and p300 (Fig. 1C). Merged images indicated that p30^II-HA accumulated with p300 in the nucleus of cells that expressed both proteins (Fig. 1E).

Fig. 1.

p30^II localizes to the nucleus and colocalizes with p300 in the nucleus of transiently transfected HeLa-Tat cells. HeLa-Tat cells were seeded in two chamber slides (Fisher Scientific) at approximately 50% confluence 18 h prior to transfection, and cotransfected with 4 µg of pME-p30II-HA and 2 µg of pCMV-p300 using Superfect (Gibco BRL). At 48 h post-transfection, media were removed and cells were fixed for 15 min using 4% paraformaldehyde. Cells were then incubated with FITC-conjugated anti-HA antibody (diluted 1:1000; Santa Cruz) and TRITC-conjugated anti-p300 antibody (diluted 1:1000; Molecular probes) overnight at 4 °C. Panel A represents the cytokeratin staining of the cells using Alexa Fluor^® 633 phalloidin, while panel D represents nuclei staining using Hoechst 33342 stain. The expression of p30II-HA (B) and p300 (C) was evaluated using Zeiss LSM 510 confocal microscope (40×) and the images were merged using Adobe photoshop (E). These results represent a minimum of triplicate experiments.

HTLV-1 p30^II physically interacts with p300 with enhanced binding with aa 1–132

HTLV-1 p30^II contains a bipartite nuclear localization sequence and regions with sequence homology to the DNA binding domain and homeodomain of transcription factors such as Oct-1 (Fig. 2A). To test if p30^II physically interacts with the cellular transcriptional coactivator p300, we transiently transfected 293T cells with pCMV-p300 and pME p30^IIHA plasmid encoding full-length p30^II, prepared whole-cell extracts, and performed coimmunoprecipitation assays. Our data indicated that p300 was immunoprecipitated as a complex with p30^II (Fig. 2B). In contrast, p300 could not be detected in the complexes precipitated using preimmune serum while p30^IIHA was detected from the cellular lysate (data not shown).

Fig. 2.

Amino acids 1–132 of HTLV-1 p30^II are critical for its physical interaction with p300. (A) Schematic diagram of the known/putative functional domains of p30^II. Schematic diagram of the carboxyl terminal and amino terminal deletion mutants of p30^II is indicated. Bipartite NLS indicates the nuclear localization sequence. Regions with sequence homology to the DNA binding domain and homeodomain of Oct-1 are indicated. The numbers on both sides of p30^II indicate length of the peptide as number of amino acids. Letters within the boxes indicate amino acids in p30^II and Oct-1. (B) Top panel represents the immunoprecipitation of p300 with p30^II and serial deletion mutants of p30^II. Physical binding between various deletion mutants of p30^II and p300 was tested by expressing pCMV-p300 and pME-p30^IIHA wild type or serial deletion mutant plasmids in 293T cells, p300 pull-down from the cellular lysates using polyclonal anti-HA antibody, followed Western immunoblot assay with anti-p300 antibody. Below each line, the binding is indicated, as density of each band measured by Gel-pro Analyzer software (middle panel). The results above are representative of three experiments. The protein expression of the carboxyl terminal and amino terminal deletion mutants of p30^II was tested using monoclonal anti-HA antibody (lower panel).

To identify the regions of p30^II that retain the ability to bind p300, we performed transient cotransfection of 293T cells with pCMV-p300 and various amino-terminal and carboxyl-terminal serial deletion mutants of p30^II, and whole-cell extracts were used for coimmunoprecipitation assays (Fig. 2A). Each of the p30^II mutant proteins was expressed at the expected molecular weight, as indicated by immunoblot analysis (Fig. 2B). The subcellular localization characteristics of these mutants were tested by confocal microscopy. Consistent with the presence or absence of the bipartite nuclear localization signal (aa 73–78 and aa 91–98), mutant constructs expressing amino acids 1–220, 1–179, 1–132, or 71–220 localized to the nucleus while mutants containing amino acids 1–71, 100–179, and 179–241 localized to the cytoplasm (Fig. 3). As seen in Fig. 2B, our binding data indicated enhanced binding of mutant representing aa 1–132 of p30^II, while mutants 1–220 and 1–179 also bound p300 more intensely compared to the wild type p30^II. Mutants representing aa 1–71 and aa 71–220 had lower binding compared to wild type p30^II, whereas mutants 100–179 and 179–241 had slightly reduced or similar p300 binding as wild type p30^II. Together, these results suggested that the nuclear localization sequence favored p300 binding, but it was not required for p30^II to bind p300. These data are consistent the known property of p300, which traffics between the cytoplasm and nucleus, but accumulates primarily in the nuclear compartment.

Fig. 3.

Subcellular localization of p30^II serial deletion mutants. p30^II in transiently transfected HeLa-Tat cells. HeLa-Tat cells were seeded in two chamber slides (Fisher Scientific) at approximately 50% confluence 18 h prior to transfection, and cotransfected with 4 µg of each pME-p30^II-HA serial deletion mutant using Superfect (Gibco BRL). At 48 h post-transfection, media were removed and cells were fixed for 15 min using 4% paraformaldehyde. Cells were then incubated with FITC conjugated anti-HA antibody (diluted 1:1000; Santa Cruz) overnight at 4 °C. Cytokeratin staining of the cells was performed using anti-Phalloidin. The amino acid domains present in each mutant are indicated. The expression of p30^II-HA was evaluated using Zeiss LSM 510 confocal microscope (40×). The results represent a minimum of triplicate experiments.

HTLV-1 p30^II modulates HTLV-1 LTR-driven transcription

To test the effect of p30^II on LTR-mediated transcription, we transiently transfected increasing concentrations of pME p30^IIHA plasmid with constant amounts of our LTR-luc reporter plasmid and tested for luciferase reporter activity. We found that lower concentrations of the p30^II plasmid (below 0.6 µg) consistently activated the HTLV-1 LTR reporter gene activity, but increased amounts (above 0.6 µg) of the plasmid repressed LTR reporter gene activity (Fig. 4A). Consistent with our previous report (Zhang et al., 2000) and recent findings of Awasthi et al. (2005), these data confirm that, at low concentrations, p30^II has the potential to enhance transcription, while at higher doses p30^II represses transcription.

Fig. 4.

HTLV-1 p30^II differentially modulates LTR-mediated transcription in the absence or the presence of the provirus. (A) In the absence of the provirus, p30^II enhances LTR-mediated transcription at low concentrations (up to 0.6 µg) and represses LTR-mediated transcription at higher doses (above 0.6 µg). (B) In the presence of full-length provirus, p30^II represses LTR-mediated transcription in a dose responsive fashion. 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, increasing amounts of pME-p30^II HA, and ±0.4 µg of ACH.30 proviral plasmid and the luciferase activity was measured. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student’s t test. *Indicates P < 0.05.

We then tested the ability of p30^II to regulate LTR-mediated transcription in the presence of the HTLV-1 provirus. By transiently transfecting pME-p30^IIHA at various doses with ACH.30, provirus deleted for p30^II expression, we have found that p30^II represses LTR-mediated transcription, in a dose responsive fashion (Fig. 4B). Interestingly, at concentrations that activated LTR-mediated transcription in the absence of ACH.30 (below 0.6 µg), we found that p30^II indeed represses LTR-mediated transcription if the provirus is present.

p300 rescues p30^II-dependent repression of LTR-driven transcription

HTLV-1 Tax-mediated transactivation is dependent, in part, on the coadaptors CBP/p300. Tax binds p300 at the KIX domain similar to p30^II. Our previous reports suggested that p30^II and Tax serve apposing roles in the regulation of LTR-mediated transcription. Based on the hypothesis that p30^II represses LTR-driven reporter gene activities via competition for limited basal quantities of CBP/p300, we tested if overexpressing p300 rescues p30^II-mediated repression of LTR-driven reporter gene expression. By transiently transfecting increasing concentrations of pCMV-p300 with constant amounts of pME-p30^IIHA plasmid, our data indicated that p300 expression rescues the p30^II-dependent repression of LTR-luciferase reporter gene activity (Fig. 5A).

Fig. 5.

p300 expression reverses the p30^II-dependent repression of LTR-driven transcription, in the absence or the presence of the provirus. (A) In the absence of the provirus, p300 expression reverses the p30^II-dependent repression of LTR-driven transcription, in a dose-responsive manner. (B) p300 reverses the p30^II-dependent repression of LTR-driven transcription, in the presence of the provirus, in a dose-responsive manner. 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, 1.2 µg pME-p30II-HA, increasing quantities of pCMV-p300, and ±0.4 µg of ACH.30 proviral plasmid and the luciferase activity was measured. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student’s t test. *Indicates P<0.05.

To test if the p30^II-mediated repression of LTR-mediated transcription in the presence of the HTLV-1 provirus, is influenced by p300, we transiently transfected increasing amounts of pCMV-p300 with constant amounts of pME-p30^IIHA and ACH.30, a provirus that lacks wild type p30^II expression (Robek et al., 1998). Interestingly, even in the presence of the provirus, increasing doses of p300 reversed the ability of p30^II to repress the LTR-mediated transcription, in a dose responsive fashion (Fig. 5B). Taken together, these data indicated that p300 has the ability to modulate the transcriptional regulatory function of p30^II on the LTR.

HTLV-1 p30^II competes with Tax in LTR-driven transcription

Since p30^II and Tax bind CBP/p300 at the KIX domain and since the transcriptional regulatory functions of p30^II and Tax on the LTR are influenced by p300, we hypothesized that Tax and p30^II might competitively influence the transcriptional regulatory function of each other on the HTLV-1 LTR. To test the effect of p30^II on Tax-mediated LTR transactivation, we transiently cotransfected increasing concentrations of pME p30^IIHA plasmid with constant concentration of LTR-luc reporter plasmid, as well as pCMV-Tax plasmid and tested the luc reporter activity. We found that p30^II consistently repressed the Tax transactivation of the HTLV-1 LTR reporter gene activity, in a dose-dependent manner (Fig. 6A). Conversely, to test the effect of HTLV-1 Tax on p30^II-mediated repression of the LTR transcriptional activity, we transiently cotransfected increasing concentrations of pCMV-Tax plasmid along with constant concentration of LTR-luc reporter plasmid as well as pME p30^IIHA plasmid and tested the luc reporter activity. We found that Tax consistently rescued the p30^II-mediated repression of the LTR reporter gene activity, in a dose-dependent manner (Fig. 6B).

Fig. 6.

HTLV-1 p30II and Tax appear to compete with each other in modulating the LTR-mediated transcription. (A) p30II represses Tax-mediated LTR transactivation in a dose-dependent manner. (B) Tax rescues p30II-mediated repression of LTR-driven transcription in a dose-dependent fashion. 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid along with either 0.4 µg of pCMV-Tax plasmid and increasing quantities of pME-p30II HA (Fig. 6A) or 1.2 µg of pME-p30II HA plasmid and increasing quantities of pCMV-Tax (Fig. 6B). pME 18S (Fig. 6A) or a plasmid containing CMV promoter (Fig. 6B) was used as carrier DNA to equalize DNA concentrations for each transfection. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student’s t test. *Indicates P < 0.05.

HTLV-1 p30^II domains important for its repression of LTR-driven transcription

To identify the regions of p30^II required for repression of LTR-driven transcription, 293T cells were transiently cotransfected with a series of carboxyl-terminal and amino-terminal truncation mutants of p30^II and our LTR-luciferase reporter plasmid, and then tested for their ability to reduce the LTR-luciferase activity. The luciferase activity elicited by LTR-luciferase construct, in the presence of the full-length p30^II was compared to luciferase activity in the presence of each of the serial deletion mutants (Fig. 7A). Luciferase activity elicited by the serially deleted mutants 1–220, 1–179, 1–132, and 1–71 were higher than that by the full-length p30^II, while in the presence of mutants 179–241, 100–179, and 71–220, the luciferase activity was less than that in the presence of the wild type p30^II. Together, these data indicated that the region between 71 and 220 is able to repress the LTR-luc reporter to a greater degree than wild type p30^II, suggesting that these amino acid sequences are important for the repression of LTR-driven transcription by p30^II. Interestingly, mutants representing aa 1–220, aa 1–179, and aa 1–132 abolished the ability of p30^II to repress LTR-driven reporter activity, which suggests the carboxyl region of the protein contains domain(s) that modify the transcriptional repressive function of p30^II. The mutant representing aa 1–71, while lacking the bipartite nuclear localization domain, retained some repressive activity, indicate that nuclear accumulation is not a strict requirement for this activity.

Fig. 7.

Amino acids 100–179 of p30II are critical for its repression of LTR-driven transcription, in the absence or the presence of the provirus. (A) In the absence of the provirus, amino acids 100–179 of p30II are critical for its repression of LTR-driven transcription. (B) In the presence of the provirus, amino acids 100–179 of p30II are critical for its repression of LTR-driven transcription. 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, 1.2 µg pME-p30II-HA wild-type or serial deletion mutants, and ±0.4 µg of ACH.30 proviral plasmid and the luciferase activity was measured. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student’s t test. *Indicates P < 0.05.

Based on our serial deletion mutant data, we cannot rule out the possibility of a change in the physical conformation or other properties of the mutant proteins (e.g., altered post translation modifications), causing the observed differences in their transcriptional activity. These data, however, suggest that the amino acid sequences from 71–220 of p30^II are important for its function as a repressor of LTR-mediated transcription. The mutant containing amino acid sequence 100–179 was also able to repress LTR-mediated transcription at levels similar to that of 71–220. The differences in activity between these mutant were not statistically significant (P<0.15), and therefore, the amino acids 71–179 appear to be the structural motif within p30^II, important for its repression of LTR-mediated reporter activity.

To determine which of our serial deletion mutants of p30^II retained the ability to repress LTR-mediated transcription in the presence of the provirus, we performed transient transfection of 293T cells with ACH.30 proviral clone, LTR-luc, and the carboxyl-terminal and amino-terminal truncation mutants of p30^II and measured the luciferase activity (Fig. 7B). While most mutants retained repressive activity, those that lacked the carboxyl end of the p30^II (1–220 and 1–179) were more limited in their ability to repress LTR reporter gene activity in the presence of the provirus and mutant 100–179 retained the greatest amount of repressive ability. Taken together, these data indicate that the amino acid sequence 100–179 of p30^II is critical in repression of LTR-mediated transcription in the presence or absence of the HTLV-1 provirus.

Lysine 106 (K3) of HTLV-1 p30^II influences repression of LTR-driven transcription

HTLV-1 p30^II contains six lysine residues (K1–K6) (Table 1). Interestingly, five of these are within the domain comprised of amino acids 100–179. Importantly, four (K2–K5) of the lysine residues within this domain are part of a consensus acetylation sequence (G/SK motif) (Bannister and Miska, 2000). To determine the role of these lysine residues in p30^II-mediated repression of LTR-driven transcription, 293T cells were transiently cotransfected with arginine substitution mutants for each lysine residue of p30^II and tested for their ability to repress LTR-luciferase reporter gene activity (Fig. 8A). Luciferase activity elicited by the arginine substitution mutant K3 (amino acid 106) was higher than that by the full-length p30^II and each of the other lysine mutants (K2, K4, and K5) and the pME empty plasmid. These data indicated that the lysine at amino acid position 106 of p30^II is important for its ability to repress LTR-luc reporter gene activity.

Table 1.

Lysine residues in p30^II are conserved among various HTLV-1 isolates

Lysine residue and amino acid position within p30II	Percentage conservationa
K1 (50)	87.5
K2 (103)	66.7
K3 (106)	87.5
K4 (123)	87.5
K5 (143)	91.7
K6 (152)	91.7

^ap30^II amino acid sequences were compared using data available from public databases and compared using commercial software (Vector NTI, AlignX).

Fig. 8.

Lysine 106 (K3) of p30II is critical for its repression of LTR-driven transcription in the absence or the presence of the provirus. (A) In the absence of the provirus, lysine 106 (K3) of p30II is critical for its repression of LTR-driven transcription. (B) In the presence of the provirus, lysine 106 (K3) of p30II is critical for its repression of LTR-driven transcription. 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, 1.2 µg pME-p30II-HA wild-type or site directed mutants, and ±0.4 µg of ACH.30 proviral plasmid and the luciferase activity was measured. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student’s t test. *Indicates P < 0.05.

To determine the role of these lysine residues in p30^II-mediated repression of LTR-driven transcription, in the presence of the provirus, we performed transient transfection of 293T cells with ACH.30, LTR-luc, and each of the arginine substitution mutants of p30^II and measured luciferase activity (Fig. 8B). Similar to our data using only the LTR reporter gene, the arginine substitution mutant K3 (amino acid 106) significantly altered, but did not eliminate the ability of wild type p30^II to mediated repression. Taken together, these data indicate that the lysine residue at amino acid position 106 of p30^II is important for its function as a repressor of LTR-mediated transcription.

Histone acetyltransferase (HAT) activity of p300 modulates p30^II-dependent repression of LTR-driven transcription

Our findings that the lysine residue at amino acid position 106 (K3) of p30^II was important for repressing TRE-mediated transcription, suggested a potential role of post translation modification of the viral protein, perhaps through acetylation, in p30^II-mediated LTR repression. To test the role of p300 HAT domain on p30^II-dependent repression of LTR-driven transcription, we transiently cotransfected pME-p30^IIHA and an LTR-luciferase reporter plasmid, along with either wild type or p300 HAT mutant plasmids. In contrast to the wild type p300, p300 HAT mutants only partially rescued the p30^II-dependent repression of LTR-luciferase reporter gene activity (Fig. 9A).

Fig. 9.

p300 HAT mutants only partially rescue p30^II-dependent repression of LTR-driven transcription in the absence of the provirus (A) or the presence of the provirus (B). 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, 1.2 µg pME-p30II HA, 2.4 µg of either the wild type p300 plasmid or one of the p300 HAT mutant plasmids, M3 or M7, and ±0.4 µg of ACH.30 proviral plasmid and the luciferase activity was measured. A plasmid containing CMV promoter was used as carrier DNA to equalize DNA concentrations for each transfection. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed, comparing to the values obtained in the presence of p30II and CMV empty plasmid, using Student’s t test. *Indicates P < 0.05.

We then tested the role of histone acetyltransferase activity mediated by p300 plasmids on p30^II-dependent repression of LTR-driven transcription, in the presence of the HTLV-1 provirus. We transiently cotransfected pME-p30^IIHA, LTR-luc reporter plasmid, and ACH.30, a provirus deleted for p30^II expression, along with either wild type or HAT mutants of p300, and tested the luciferase reporter activity. Interestingly, p300 HAT mutant plasmids failed to rescue p30^II-dependent repression of LTR-luciferase reporter gene activity, compared to the wild type p300 plasmid, in the presence of the HTLV-1 provirus as well (Fig. 9B). These data confirm that the HTLV-1 LTR transcriptional regulatory effect of p30^II is influenced by the HAT activity of p300.

Deacetylation by HDAC-1 enhances p30^II-dependent repression of HTLV-1 LTR-driven transcription

Histone acetyl transferases and histone deacetylases (HDACs) form multiprotein complexes with transcriptional factors and are known to regulate their transcriptional activity (Ego et al., 2002). In addition, HDAC-1 is known to physically and functionally interact with Tax and repress the LTR transactivation function of Tax (Ego et al., 2002). To test if deacetylation by HDAC-1 has a similar role on p30^II-dependent repression of LTR-mediated transcription, we transiently cotransfected increasing concentrations of HDAC-1 plasmid with constant concentration of pME-p30^IIHA plasmid and LTR-luciferase reporter plasmid and tested the luciferase reporter activity. Our data indicated that HDAC-1 expression enhanced p30^II-dependent repression of LTR-luciferase reporter gene activity, in a dose-dependent manner (Fig. 10A).

Fig. 10.

Addition of HDAC-1 enhances p30^II-dependent repression of HTLV-1 LTR-driven transcription in the absence of the provirus (A) or the presence of the provirus (B). 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, 1.2 µg pME-p30II HA, 0.0, 0.5, 1.0, or 1.5 µg HDAC-1 plasmid, and ±0.4 µg of ACH.30 proviral plasmid and the luciferase activity was measured. A plasmid containing the same promoter as the HDAC-1 plasmid was used as carrier DNA to equalize DNA concentrations for each transfection. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed, comparing to the values obtained in the presence of p30^II and no HDAC-1 plasmid, using Student’s t test. *Indicates P < 0.05.

We then tested the role of deacetylation on p30^II-dependent repression of LTR-mediated transcription, in the presence of the HTLV-1 provirus, by transiently cotransfecting increasing concentrations of the HDAC-1 plasmid with constant concentration of pME-p30^IIHA plasmid, LTR-luciferase reporter plasmid, and ACH.30, a provirus deleted for p30^II expression. Our data indicated that addition of HDAC-1 enhanced p30^II-dependent repression of LTR-luciferase reporter gene activity, in a dose-dependent manner, in the presence of the provirus (Fig. 10B). These data further support our findings that the HTLV-1 LTR transcriptional modulatory function of p30^II is dependent on deacetylation by HDAC-1.

Acetylation by trichostatin A (TSA) decreases p30^II-dependent repression of HTLV-1 LTR-driven transcription

Reversible modification of the core histones is crucial in the regulation of gene expression. While histone acetylation is typically associated with nucleosomal decondensation necessary for transcriptional activation, histone deacetylation is associated with nucleosomal condensation and subsequent transcriptional repression (Ego et al., 2002). Repression of Tax transactivation of HTLV-1 LTR by HDAC-1 can be restored when treated with an HDAC inhibitor, namely, trichostatin A (Ego et al., 2002). Similarly, we tested the effect of inhibition of deacetylation on p30^II-dependent repression of LTR-mediated transcription. To determine the most effective concentration of TSA, we transiently cotransfected 293T cells with 1.0 µg of HDAC-1 plasmid, 1.2 µg of pME-p30^IIHA plasmid, and 0.3 µg of LTR-luciferase reporter plasmid, and added either 0.0, 100, 200, or 400 nM TSA at 24 h post-transfection (Fig. 11A). The most effective dose of TSA was determined in a dose titration experiment to be 100 nM. Higher concentrations of TSA likely were not as effective due to cell toxicity. We then tested the luciferase reporter activity at 48 h post-transfection and compared the difference in luciferase activity in the presence of various concentrations of TSA and found that 100 nM TSA had the highest effect in decreasing p30^II-dependent LTR repression (data not shown). Next, to determine the effect of TSA at different concentrations of HDAC-1, we transiently cotransfected 293T cells with increasing concentrations of a HDAC-1 plasmid with constant concentration of pME-p30^IIHA plasmid and LTR-luciferase reporter plasmid, and added 100 nM TSA at 24 h post-transfection. We then tested the luciferase reporter activity at 48 h post-transfection and compared the difference in luciferase activity in the presence or absence of TSA. Our data demonstrated that TSA, an inhibitor of deacetylation, decreases p30^II-dependent repression of LTR-luciferase reporter gene activity, at various doses of HDAC-1, in the absence of the provirus (Fig. 11B).

Fig. 11.

Addition of 100 nM Trichostatin A (TSA) counteracts enhancement of p30^II-dependent repression of HTLV-1 LTR-driven transcription by HDAC-1. (A) To determine effective concentration of TSA, 293T cells were cotransfected with 1.0 µg of HDAC-1 plasmid, 1.2 µg of pME-p30^IIHA plasmid, and 0.3 µg of LTR-luciferase reporter plasmid, with either 0.0, 100, 200, or 400 nM TSA added to culture media at 24 h post-transfection and the luciferase activity measured. (B and C) Addition of TSA counteracts the enhancement of p30^II-dependent repression of HTLV-1 LTR-driven transcription by HDAC-1 in the absence (B) or the presence of the provirus (C). 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, 1.2 µg pME-p30II HA, 0.0, 0.5, 1.0 or 1.5 µg HDAC-1 plasmid, and ±0.4 µg of ACH.30 proviral plasmid. A plasmid containing the same promoter as the HDAC-1 plasmid was used as carrier DNA to equalize DNA concentrations for each transfection. At 24 h post-transfection, 100 nM TSA (Sigma) was added to the culture medium, when specified, and the luciferase activity was measured. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed, comparing between values obtained in the presence or absence of TSA, at the same concentration of HDAC-1, using Student’s t test. *Indicates P<0.05.

We then tested the role of inhibition of deacetylation on p30^II-dependent repression of LTR-mediated transcription, in the presence of the HTLV-1 provirus, by transiently cotransfecting increasing concentrations of HDAC-1 plasmid with constant concentration of pME-p30^IIHA plasmid, LTR-luciferase reporter plasmid, and ACH.30, a provirus deleted for p30^II expression and by adding 100 nM TSA at 24 h post-transfection. We then tested the luciferase reporter activity at 48 h post-transfection and compared the difference in luciferase activity in the presence or absence of TSA. Interestingly, addition of TSA decreased p30^II-dependent repression of LTR-luciferase reporter gene activity, at various concentrations of HDAC-1, in the presence of the provirus as well (Fig. 11C). These data further implicated that the HTLV-1 LTR transcriptional regulatory effect of p30^II is dependent on deacetylation by HDAC-1 and inhibition of deacetylation by TSA.

P/CAF does not rescue p30^II-dependent repression of HTLV-1 LTR-driven transcription

P/CAF is an important coactivator that mediates transcription through HAT activity (Sterner and Berger, 2000). P/CAF acetylates various nonhistone transcription-related proteins, such as the chromatin proteins HMG17 and HMG I(Y), activators p53, MyoD, human immunodeficiency virus (HIV) Tat, and general transcription factors TFIIE and TFIIF (Sterner and Berger, 2000). In addition, P/CAF is recruited to the Tax responsive element sites on the HTLV-1 LTR, through direct interaction with Tax and enhance Tax-mediated HTLV-1 transcription, in a HAT-independent manner (Jiang et al., 1999). We therefore tested if P/CAF has a role on p30^II-dependent repression of HTLV-1 LTR-driven transcription. We transiently cotransfected increasing concentrations of P/CAF plasmid with constant concentration of pME-p30^IIHA plasmid and LTR-luc reporter plasmid and tested the luciferase reporter activity. Our data indicated that, unlike p300, P/CAF does not rescue p30^II-dependent repression of HTLV-1 LTR-driven transcription (Fig. 12). In addition, to test if the HAT activity of P/CAF influences p30^II-dependent repression of HTLV-1 LTR-driven transcription, we transiently cotransfected pME-p30^IIHA plasmid and LTR-luc reporter plasmid along with either wild type P/CAF plasmid or P/CAF HAT mutant plasmid, and tested the luc reporter activity. We found that the P/CAF HAT mutant did not significantly alter p30^II-dependent repression of HTLV-1 LTR-driven transcription, compared to wild type P/CAF (Fig. 12). These data confirm that the HTLV-1 LTR transcriptional regulatory effect of p30^II is p300-dependent and not P/CAF-dependent.

Fig. 12.

Expression of P/CAF does not rescue p30II-dependent repression of HTLV-1 LTR-driven transcription. 293T cells were transiently cotransfected with 0.3 µg of pLTR-luciferase reporter plasmid, 1.2 µg pME-p30II HA and 0.0, 0.6, 1.2, or 2.4 µg wild type P/CAF, or 2.4 µg P/CAF HAT mutant plasmid, and the luciferase activity was measured. A plasmid containing the same promoter as the P/CAF plasmids (CMV promoter) was used as carrier DNA to equalize DNA concentrations for each transfection. Results represent the mean of optimized luciferase activity in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed, comparing to the values obtained in the presence of p30II alone, using Student’s t test. Statistical significance was also performed, to compare between pCAF wild type and HAT mutant, using Student’s t test. *Indicates P<0.05.

Discussion

Our data are the first to identify functional motifs of p30^II that are critical in binding p300 and in repressing LTR-mediated transcription. Identification of these motifs is important for defining the molecular mechanism of p30^II-mediated repression of LTR-driven transcription. We found that the region important for binding p300 is amino acid sequence 1–132, while the motif that plays a major role in repressing LTR-mediated transcription is amino acid sequence 100–179, which contains the entire DNA binding region, part of the homeodomain with homology to Oct-1 and POU family of transcription factors as well as 5 lysine residues, four of which are part of a G/SK consensus acetylation sequence (Bannister and Miska, 2000) representing potential acetylation sites for CBP/p300. The lysine residue at amino acid position 106 (K3) of HTLV-1 p30^II was important for p30^II-mediated repression of LTR-driven transcription in the presence or absence of the provirus. Our data using p300 HAT mutants demonstrated that the histone acetyl transferase domain of p300 influences HTLV-1 LTR-mediated transcription by p30^II. These data were further supported by experiments that indicated that p30^II LTR repression was enhanced on deacetylation by HDAC-1 and inhibited by TSA, an inhibitor of HDAC-1.

The coactivators CREB binding protein (CBP) and p300 mediate transcriptional control of various cellular and viral DNA binding transcription factors. Although these coactivators have divergent functions, they are similar in nucleotide sequence, are evolutionarily conserved, and are commonly referred to as CBP/p300 (Blobel, 2000; Goodman and Smolik, 2000). CBP and p300 are highly related and share many functional properties; however, there is evidence that these factors are not interchangeable (Vo and Goodman, 2001). Several cellular and viral proteins that interact with either CBP or p300 have been identified, including steroid and retinoid hormone receptors, CREB, c-Jun, c-Myb, Sap-1a, c-Fos, MyoD, p53, Stat-1/2, NF-κB, pp90rsk, TATA-binding protein, TFIIB (Avantaggiati et al., 1997; Goodman and Smolik, 2000; Janknecht and Hunter, 1996a, 1996b), HTLV-1 Tax, adenovirus E1A, Kaposi’s sarcoma-associated herpes virus viral interferon regulatory factor protein, and simian virus 40 large T antigen (Arany et al., 1995; Ariumi et al., 2000; Avantaggiati et al., 1996; Colgin and Nyborg, 1998; Kashanchi et al., 1998; Kwok et al., 1996; Suzuki et al., 1999).

CBP and p300 bridge transcription factors to relevant promoters, have intrinsic histone acetyltransferase (HAT) activity, and form complexes with proteins such as CBP/p300 binding protein-associated factor, which also exhibits HAT activity (Grunstein, 1997). Recently, there is increasing knowledge of the mechanism and functional significance of the interactions between many viral proteins and CBP/p300. In the case of adenovirus oncoprotein E1A, interaction with CBP/ p300 is critical for regulation of transcription, suppression of differentiation, and immortalization of cells in culture (Arany et al., 1995; Avantaggiati et al., 1996; Lundblad et al., 1995). Simian virus T antigen regulates the expression of a group of cellular genes by modifying the HAT activity of CBP/p300 or by bridging the gap between DNA binding transcription factors and components of the general transcription machinery (Avantaggiati et al., 1997; Goodman and Smolik, 2000). p30^II has recently been demonstrate to increase the transforming potential of Myc and transcriptionally activate the human cyclin D2 promoter (Awasthi et al., 2005). The enhanced c-Myc transforming activity by p30^II was dependent upon the transcriptional coactivators, transforming transcriptional activator protein/p434 and TIP60, and required TIP60 histone acetyltransferase (HAT) activity. Similar to our findings, Awasthi et al. (2005) demonstrated that aa 99 to 154 within p30^II were required for transcriptional regulation by TIP60. Therefore, identifying the molecular mechanism and functional significance of HAT-dependent transcription will be important to understanding of the role of p30^II in lymphocyte transformation by HTLV-1 and replication of the virus in vivo.

Previously, using deletion mutants of CBP/p300 in GST pull-down assays, we localized the binding site of CBP/p300 for p30^II to a highly conserved KIX region. Interestingly, KIX domain is the binding site of CBP/p300 for HTLV-1 Tax protein as well. In addition, p30^II was able to disrupt CREB-Tax-CBP/ p300 complexes bound to the viral 21-bp TRE repeats (Zhang et al., 2001). These results indicate that p30^II might act in contrast to the role of HTLV-1 Tax. The role of CBP and p300 coactivators in HTLV-1 gene expression has been the focus of many previous reports. HTLV-1 Tax, a transactivator of LTR-mediated transcription, is critical in the activation of the HTLV-1 viral genes through its interaction with the p300 and CBP coactivators (Bex and Gaynor, 1998).

Although CBP and p300 mediate the activities of various transcription factors, many earlier reports prove that it is present in the cell at limiting concentrations. Even small reductions in the concentrations of these coactivators are damaging in many instances, such as in the human Rubinstein–Taybi syndrome where loss of a single CBP allele causes developmental defects (Petrij et al., 1995). In experiments where transcription factors are exogenously expressed, it is possible that the capacity of the endogenous CBP/p300 is overridden and the effects due to competition may be more obvious. Many such studies have demonstrated the competition between different molecules for CBP/p300. Even though some of these involve two mutually antagonistic transcription factors with the same shared binding site on CBP/p300, competition does not necessarily require having the same binding site. In circumstances where CBP/ p300 levels are limiting, there could be selective preference of one over the other, causing an exclusion of one of the proteins. In our study, we found that HTLV-1 p30^II and Tax appear to compete with each other in modulating the transcriptional activity from the LTR. Since HTLV-1 p30^II and Tax interact with CBP/p300 through the same KIX domain, we believe that this is through competitive CBP/p300 binding between p30^II and Tax. Analogously, the binding of Tax and c-Myb to KIX domain of CBP was found to be mutually exclusive and Tax expression was shown to interfere with transcriptional activity of c-Myb (Colgin and Nyborg, 1998).

An environment of coactivator competition between transcription factors provides an additional layer of regulated gene expression. The relative amounts of Tax and p30^II may be critical in determining which protein binds to CBP/p300 and the consequent regulation of LTR-mediated transcription of viral genes from the HTLV-1 viral promoter. When its levels are low, p30^II also enhances transcription from the viral promoter. Under such circumstances, p30^II and Tax might act synergistically. However, at higher concentrations, p30^II may antagonize Tax transactivation of the HTLV-1 promoter. There are similar reports on positive and negative effects by the same protein in other viruses, such as herpes simplex virus type 1 (HSV-1), in which the interaction between cellular and viral transcription factors plays a critical role in the regulation of the immediate-early (IE) gene promoter. VP16, a potent transcription factor from HSV-1, binds the host cell protein HCF, which facilitates the stable complex formation of the viral protein with Oct-1 (Wilson et al., 1997). The IE gene promoter contains an Oct-1-like motif (TAATGARAT) that is important for IE gene expression, with both positive and negative effects, depending on the context of these cellular transcription factors and VP16 (Thomas et al., 1998).

CBP and p300 bridge transcription factors to relevant promoters and through its intrinsic histone acetyl transferase (HAT) activity acetylates lysine residues at the amino terminus of histones, which results in modification of chromatin structure to allow access of the transcriptional machinery. CBP/p300 is known to form complexes with proteins such as CBP/p300 binding protein-associated factor, which also exhibits HAT activity (Grunstein, 1997). p300 also directly acetylate transcription factors such as p53, GATA-1, and c-myb and thus augment their sequence-specific DNA-binding activity (Sterner and Berger, 2000). Simian virus T antigen regulates the expression of a group of cellular genes by modifying the HAT activity of CBP/p300 or by bridging the gap between DNA binding transcription factors and components of the general transcription machinery (Avantaggiati et al., 1997; Goodman and Smolik, 2000). c-myb oncoprotein of the avian retrovirus group is known to be acetylated by CBP/p300 at its multiple lysine residues (Colgin and Nyborg, 1998).

p30^II contains six highly conserved lysine residues, 5 of which are within the amino acid region 100–179, four of which are part of the consensus acetylation sequence (G/SK motif) (Bannister and Miska, 2000) and thus represent potential acetylation sites for CBP/p300 (Table. 1). We found that the lysine residue at amino acid position 106 (K3) of HTLV-1 p30^II is critical for its repression of LTR-driven transcription in the presence or absence of the provirus. We tested the expression level of all the arginine substitution mutants for each of the lysines (K2–K5) by Western immunoblot and found that the expression of all the mutants was similar to that of the wild type p30^II (data not shown). Based on this, it is possible that the intrinsic HAT activity of CBP/p300 is utilized to acetylate and potentially modulates the transcriptional regulatory function of p30^II. Our ongoing studies seek to determine if p30^II is directly acetylated or post-translationally modified in other ways (e.g., phosphorylation) and if these potential modifications regulate its function.

Recently, it was reported that p30^II modulates LTR-mediated transcription, in the context of the entire provirus, by a post-transcriptional mechanism (Nicot et al., 2004; Younis et al., 2004). Previous studies from our laboratory demonstrated that HTLV-1 p30^II directly interacts with CBP/p300 to modulate gene transcription (Zhang et al., 2000, 2001). Data presented in this report further confirm the role of CBP/p300 in the modulation of LTR-mediated transcription by p30^II, in the context of the provirus. In the presence of increasing concentrations of p300, there was a dose-dependent rescue of LTR-driven gene transcription, irrespective of the presence or absence of the provirus. Although, we cannot rule out the possibility of a post-transcriptional mechanism by HTLV-1 p30^II, this report presents convincing evidence for a p300-dependent transcriptional regulatory function. Therefore, HTLV-1 p30^II appears to be a multifunctional protein with transcriptional and post-transcriptional role in regulating gene expression.

As of yet, there is no much information regarding the relative levels of various HTLV-1 proteins during various stages of the infection. It is possible that differences in expression levels of various viral proteins, leading to differences in transcriptional regulation as described above might be the mechanism by which HTLV-1 infection/disease progresses though various stages (Princler et al., 2003). This is likely to be in synergy with differential regulation of cellular gene regulation by Tax and/or p30^II and thus changing the immune responses in accordance with different stages of progression of HTLV-1 infection and disease. Our current data are supported by recent findings from our laboratory where an infectious HTLV-1 molecular clone failed to maintain viral loads in vivo when p30^II expression was abolished (Silverman et al., 2004). Taken together, our data indicate that HTLV-1, a complex retrovirus associated with lymphoproliferative disorders, uses accessory genes to promote cell-to-cell transmission of the virus, clonal expansion of infected cells, and maintenance of proviral loads in vivo.

Materials and methods

Cell lines

293T cells were obtained from G. Franchini (National Cancer Institute, NIH, Bethesda) and HeLa-Tat cells were obtained from AIDS Research and Reference Reagent Program, National Institute of Health. 293T and HeLa-Tat cells were grown in modified Dulbecco’s eagle medium containing 10% fetal bovine serum and 1% streptomycin and penicillin at 37 °C. Cells were split and cultured in six-well plates, 10 cm dishes, or chamber slides to 50–60% confluence 16–18 h before transfection.

Plasmids

The pTRE-Luc plasmid and pRSV-B-Gal have been described previously (Zhang et al., 2000, 2001). pME-p30^IIHA wild-type and serial deletion mutant plasmids were created by cloning the p30^II sequence from HTLV-1 molecular clone, ACH with downstream influenza hemagglutinin (HA1) tag, into pME-18S plasmid (G. Franchini, National Cancer Institute) between 5′ EcoRI and 3′ NotI sites. Site-directed mutants were made by substituting lysines in pMEp30^IIHAwith arginine. Fidelity of the plasmids was confirmed by Sanger sequencing and p30^II HA protein expression was confirmed by Western blot using monoclonal anti-HA antibody (Covance, Berkeley, CA). ACH.30 plasmid has a 24 bp insertion that causes an artificial termination codon in p30^II (Robek et al., 1998). pCMV-Tax expresses the HTLV-1 Tax protein and has been described previously (Newbound et al., 1996). pCMV-p300 expresses the full-length p300 protein from a CMV I/E promoter (Upstate USA, Inc., Charlottesville, VA). The HDAC-1 plasmid encodes for HDAC-1 (S. Schreiber, Harvard University, Cambridge, MA). Wild type and HAT mutants of P/CAF have been described previously (Jiang et al., 1999).

Cell transfection and reporter gene assay

For each transfection, 0.3 µg of pTRE-Luc reporter plasmid was cotransfected with pME-p30^IIHA wild type or serial deletion/arginine substitution mutant plasmids and 0.4 µg ACH.30 plasmid when specified, using Lipofectamine Plus (Invitrogen, Carlsbad, CA). When pCMV-p300 plasmid was used, it was also cotransfected. As an internal control for transfection efficiency, 0.1 µg of pRSV-(Gal (Invitrogen) was also used in each transfection. pME 18S was used as carrier DNA to equalize DNA concentrations for each transfection. Transfected cells were lysed with 400 (l lysis buffer (Promega, Madison, WI) per well for 25 min. Twenty microliters of each lysate was used to test luciferase reporter gene activity using an Enhanced (Luciferase Assay kit (Promega). To normalize transfection efficiency, cells were washed with PBS, stained with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) (Sigma, St. Louis, MO) and counted (Gal expressing cells using 20× objective of light microscope. Cotransfection of pME-p30IIHA had no effect on the X-Gal staining (data not shown). Results were expressed as mean of optimized luciferase activity (luciferase activity/percentage cells stained positive for β-Gal expression) in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student’s t test, P<0.05.

Western immunoblot assay

Transiently transfected cells were lysed in RIPA buffer containing PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Cell lysates were prepared by centrifugation at 14,000 rpm (Beckman, Fullerton, CA) for 20 min at 4 °C. Equal amounts of proteins were mixed with Laemmli buffer (62.5 mM Tris; pH 6.8), 2% SDS, 10% glycerol, 0.2% bromophenol blue, and 100 mM dithiothreitol (DTT). After boiling for 5 min, samples were electrophoresed through 6 or 10% polyacrylamide gels. The fractionated proteins were transferred to nitrocellulose membranes (Amersham Pharmacia Biotechnology, Piscataway, NJ) at 100 V for 1 h at 4 °C. Membranes were then blocked in Tris-buffered saline containing 5% nonfat milk and 0.1% Tween 20. Proteins were detected with primary monoclonal anti-hemagglutinin (HA) antibody (Covance) followed by an anti-mouse (Cell Signaling Technology, Beverly, MA) immunoglobulin G (IgG)-horseradish peroxidase-conjugated goat antibody. Blots were developed using an enhanced chemiluminescence detection system (Cell Signaling Technology).

Coimmunoprecipitation of p30II with p300

Sixty percent confluent 293T cells was cotransfected by pME-p30^IIHA, pME-p30^IIHA serial deletion, or arginine substitution mutants and pCMV-p300 using Superfect (Gibco BRL, Gaithersburg MD). After 48 h, the transfected cells were washed with PBS and resuspended in 400 µl of lysis buffer containing 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 µg of leupeptin per ml, and 1 µg of aprotinin per ml. Cell suspensions were incubated on ice for 20 min and then lysed by homogenization. The lysates were centrifuged at 14,000 rpm for 20 min at 4 °C and incubated overnight at 4 °C with 100 ng of a polyclonal HA antibody (Covance). After adding 40 µl of 100% of protein Aagarose slurry, the mixture was incubated at 4 °C for 1 h. The immunoprecipitated complexes were washed twice with 10 volumes of lysis buffer and three times with PBS buffer. The components of the complexes were resolved on 6% SDS-polyacrylamide gels and detected by Western immunoblot assay using anti-p300 antibody (Santa Cruz biotechnology, Santa Cruz, CA). The density of each band was measured and compared to wild type p30^II using a commercial software package (Gel-pro Analyzer software; Media Cybernatic Inc., San Diego, CA). Normal rabbit IgG was used as a negative control in immunoprecipitation assays and equal amounts of input cell extracts were processed to determine the expression of p30^II by Western immunoblot assay.

Localization of p30^II and colocalization with CBP/p300 by confocal microscopy

To detect cellular colocalization of p30^II and p300 by immunofluorescence, HeLa-Tat cells were seeded in two chamber slides (Fisher Scientific) at approximately 50% confluence 18 h prior to transfection. Transfection with 4 µg of pME-p30^II-HA wild-type or pME-p30^IIHA serial deletion mutants alone or with 2 µg of pCMV-p300 was performed using Superfect (Gibco BRL). At 48 h post-transfection, media were removed and cells were fixed for 15 min using 4% paraformaldehyde. Cells were then incubated with FITC (Fluorescin isothyocyanate) conjugated anti-HA antibody alone (diluted 1:1000; Santa Cruz) or with TRITC (Texas red isothyocyanate) conjugated anti-p300 antibody (diluted 1:1000; Molecular probes) in antibody dilution buffer containing 0.01 M sodium phosphate, 0.5 M NaCl, 0.5% Triton-X 100, and 2% bovine serum albumin overnight at 4 °C. Actin filaments were stained with Alexa Fluor 633 phalloidin and nuclei were visualized using Hoechst 33342, trihydrochloride, and trihydrate-FluoroPure grade (Invitrogen/Molecular Probes, Carlsbad, CA). The expression of p30II-HA and p300 was evaluated using Zeiss LSM 510 confocal microscope.