Group I p21-activated kinases facilitate Tax-mediated transcriptional activation of the human T-cell leukemia virus type 1 long terminal repeats
© Chan et al.; licensee BioMed Central Ltd. 2013
Received: 28 August 2012
Accepted: 23 April 2013
Published: 26 April 2013
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and tropical spastic paraparesis. HTLV-1 encodes transactivator protein Tax that interacts with various cellular factors to modulate transcription and other biological functions. Additional cellular mediators of Tax-mediated transcriptional activation of HTLV-1 long terminal repeats (LTR) remain to be identified and characterized.
In this study, we investigated the regulatory role of group I p21-activated kinases (Paks) in Tax-induced LTR activation. Both wild-type and kinase-dead mutants of Pak3 were capable of potentiating the activity of Tax to activate LTR transcription. The effect of Paks on the LTR was attributed to the N-terminal regulatory domain and required the action of CREB, CREB-regulating transcriptional coactivators (CRTCs) and p300/CREB-binding protein. Paks physically associated with Tax and CRTCs. Paks were recruited to the LTR in the presence of Tax. siRNAs against either Pak1 or Pak3 prevented the interaction of Tax with CRTC1 and the recruitment of Tax to the LTR. These siRNAs also inhibited LTR-dependent transcription in HTLV-1-transformed MT4 cells and in cells transfected with an infectious clone of HTLV-1.
Group I Paks augment Tax-mediated transcriptional activation of HTLV-1 LTR in a kinase-independent manner.
KeywordsHTLV-1 Tax p21-activated kinases CREB CREB-regulating transcriptional activators
Human T-cell leukemia virus type 1 (HTLV-1) infects more than 20 million people worldwide and causes adult T-cell leukemia (ATL) or tropical spastic paraparesis (TSP) in a small subset of infected individuals. While ATL is a highly lethal malignancy of T lymphocytes, TSP is a disabling neuroinflammatory disorder of the central nervous system . Although the development of ATL and TSP is rare and slow, high proviral load is the major risk factor for disease progression in carriers of HTLV-1 . Understanding the mechanism by which HTLV-1 drives proviral expression might provide new strategies for prevention and control of HTLV-1-associated diseases.
The master regulator of HTLV-1 proviral expression is viral transactivator Tax which potently activates transcription from the long terminal repeats (LTR). For this activation, Tax forms a dimer to complex with dimeric CREB and the three imperfectly conserved Tax-responsive elements (TREs) in the LTR [3–5]. Tax also engages transcriptional coactivators such as p300/CREB-binding protein (CBP) and CREB-regulating transcriptional coactivators (CRTCs), also known as transducers of regulated CREB activity (TORCs) [5–10]. In addition, phosphorylation of Tax, CREB and CRTCs has also been implicated in LTR activation [10–14]. Because the activation of the LTR by Tax is a tightly regulated process accomplished through multiple mechanisms [4, 5, 15–17], we hypothesized that additional Tax-binding cellular proteins might also be involved in its execution and regulation. Particularly, it will be of great interest to see whether any Tax-binding protein kinases would play a role in this process.
p21-activated kinase 3 (Pak3) is one of the 32 Tax-binding proteins identified in a mass spectrometric analysis of proteins precipitated from HTLV-1-transformed C8166 cells using an anti-Tax antibody . However, the roles of Pak3 in Tax-induced LTR activation have not yet been characterized. Paks are a family of serine-threonine kinases that regulate gene transcription, cell cycle progression, cytoskeletal organization and cell migration in response to small GTPases Cdc42 and Rac1 . Pak signaling is commonly activated in cancer cells [20–22]. Paks are also implicated in the replication and spread of viruses such as human immunodeficiency virus type 1 (HIV-1) . Currently there are six members in the Pak family that can be divided into group I (Pak1, Pak2 and Pak3) and group II (Pak4, Pak5 and Pak6) based on structural and biochemical properties. They have highly conserved Cdc42/Rac-interactive binding (CRIB) domain and kinase domain, but differ in tissue distribution and the N-terminal regulatory domain. Particularly, CRIB and kinase domains in Pak1, Pak2 and Pak3 share more than 95% identity . It is noteworthy that Paks can fulfill some of their functions, such as the adaptor role in signal transduction, the inhibition of cell cycle progression as well as the induction of lamellipodia and membrane ruffling, in a kinase-independent manner [24–27].
In this study, we investigated the regulatory roles of group I Paks in Tax-induced activation of HTLV-1 LTR in transfected and infected cells. We found that all three Paks of group I can facilitate Tax-induced LTR activation. This activity of Paks was kinase-independent and was mediated through the N-terminal regulatory domain. Tax interacted with Paks and facilitated their recruitment to the viral promoter. Our work reveals new cellular mediators of HTLV-1 transcription and a new kinase-independent function of group I Paks in transcriptional regulation.
Augmentation of Tax-induced activation of HTLV-1 LTR by group I Paks
Pak3 is known to interact with Tax in HTLV-1-transformed cells . Within group I Paks, Pak1 and Pak2 are strikingly homologous to Pak3, sharing 80% and 69% identical amino acid residues [19, 20]. While all three Paks in group I are activated by Cdc42/Rac to mediate similar biological effects, they are differentially expressed in different tissues and might have non-redundant functions by targeting different substrates. Particularly, Pak1 is abundantly expressed in brain, muscle and spleen. Pak2 is ubiquitous and Pak3 are primarily found in the brain . Consistent with the previous finding on Pak3-Tax interaction in C8166 cells , we detected Pak1, Pak2 and Pak3 mRNA and protein in Jurkat cells and several lines of HTLV-1-transformed T cells, although the levels of Pak2 were relatively low (data not shown). With this in mind, we set out to explore whether Pak1, Pak2 and Pak3 might affect Tax-induced activation of HTLV-1 LTR.
We next repeated the experiments in Jurkat cells and obtained similar results indicating the ability of Pak1 and Pak3 to further augment LTR activation by Tax (Figure 1D, bars 4–6 compared to bar 3, and bars 10–13 compared to bar 9). In addition, when we depleted endogenous Pak1 or Pak3 in Jurkat cells with a pre-validated siRNA, the activation of the LTR by Tax was compromised (Additional file 1: Figure S1A, bars 5 and 6 compared to bar 4). Consistent with this, Tax-mediated LTR activation was attenuated in HTLV-1-transformed MT2 and MT4 cells in which Pak1 or Pak3 was depleted by siRNA (Figure 1E, bars 8–10 compared to bar 5; Additional file 1: Figure S1B, bar 4 compared to bar 3). As a positive control, we also verified the suppression of LTR activation in Tax-depleted MT2 cells transfected with siTax-A or siTax-B (Figure 1E, bars 6 and 7 compared to bar 5). Since antibodies that can reproducibly detect endogenous Pak1 and Pak3 proteins in MT2 cells were not available, the silencing effect of siRNAs used was verified by RT-PCR analysis of Pak1 and Pak3 transcripts. Representative examples of RT-PCR results were presented in Additional file 2: Figure S2. Two independent siRNAs targeting Pak1 (siPak1A and siPak1B) were found to have a substantial suppressive effect on Pak1 mRNA expression in HeLa and MT4 cells (Additional file 2: Figure S2A, lanes 2 and 3 compared to lane 1; Additional file 2: Figure S2B, lanes 2 and 3 compared to lane 1). They also suppressed Pak3 mRNA expression to a lesser extent, but did not influence the expression of β-globin transcript (Additional file 2: Figure S2A, lanes 5 and 6 compared to lane 4; Additional file 2: Figure S2B, lane 5 compared to lane 4). Likewise, diminution of the steady-state levels of Pak3 mRNA was most pronounced in cells transfected with siPak3A and siPak3B (Additional file 2: Figure S2). The cross suppression of Pak1 and Pak3 by siPak3 and siPak1 respectively was due to the high homology between Pak1 and Pak3. Because the expression levels of Pak2 was low in MT2 and MT4 cells, RNAi depletion experiments were not performed for Pak2. Nevertheless, our results demonstrated that compromising group I Paks attenuated Tax-induced activation of HTLV-1 LTR.
In addition to HTLV-1 LTR, Tax can also activate NFκB-dependent transcription . For example, Tax activates cellular HIAP promoter through NFκB . Since Pak1 was also implicated in the activation of NFκB , we asked whether Pak1 or Pak3 might influence Tax-induced activation of NFκB. When we co-expressed Tax with Pak1 or Pak3, the activation of HIAP promoter by Tax was not further enhanced (Figure 1F, bars 3–5 compared bar 2, and bars 8–10 compared to bar 7). Additionally, when Pak1 or Pak3 was depleted from HeLa cells, Tax-induced activation of HIAP promoter was unaffected (data not shown). Hence, Pak1 and Pak3 had no influence on Tax-induced activation of NFκB. Collectively, our gain-of-function and loss-of-function experiments consistently supported the notion that group I Paks facilitate Tax-induced LTR activation specifically.
Pak3 augments Tax-induced activation of HTLV-1 LTR in a kinase-independent manner
To shed light on the functional domain of Tax that interacts with Pak3, we also examined the effect of Pak3 on various point mutants of Tax. Pak3 was found to augment all Tax mutants that are able to activate HTLV-1 LTR [33, 34], including S258A, S150A, and C23S (Additional file 3: Figure S3). Thus, as in the case of many other Tax-binding proteins , we were unable to define a discrete domain of Tax that interacts with Pak3. Plausibly, the interaction is conformation-dependent and requires the full protein of Tax.
CREB, CRTCs and p300/CBP are required for Tax-augmenting effect of Pak3
Tax and CRTC1 associate with Paks
Tax interacts with Pak1 (Additional file 4: Figure S4B) and with CRTC1 individually . In addition, Tax, Pak1 and CRTC1 can all be localized to the nucleus [8, 39, 40]. However, it was still unclear whether all three proteins might be found in the same protein complex. To investigate this, we expressed all three proteins in HEK293T cells and precipitated V5-tagged CRTC1 using an α-V5 antibody (Additional file 4: Figure S4C). The α-V5 precipitates contained both Pak1 and Tax (Additional file 4: Figure S4C, lanes 6 and 9). This was consistent with the formation of a protein complex that contains CRTC1, Pak1 and Tax. We also noted the association of Pak1 with CRTC1 in the absence of Tax (Additional file 4: Figure S4C, lane 5). Similar results compatible with the formation of CRTC1-Pak3 and CRTC1-Pak3-Tax protein complexes were also obtained (Figure 5E, lanes 9, 10 and 15). Hence, Pak1 and Pak3 associate with Tax and CRTC1.
Paks are recruited to HTLV-1 LTR
Paks are required for optimal Tax activity
We next assessed LTR recruitment of Tax in Pak1/3-depleted cells. As anticipated, suppression of Pak1/3 expression in HeLa cells by siPak1/3 also compromised their recruitment to the LTR (Figure 7B, middle panels). In addition, LTR recruitment of Tax was also suppressed in siPak1/3-transfected cells (Figure 7B, upper panel, lanes 3 and 4 compared to lane 2, and lane 7 compared to lane 6). These results suggest that group 1 Paks are also necessary for Tax recruitment to HTLV-1 LTR.
Paks are required for optimal HTLV-1 transcription
On the other hand, we also measured the expression of Pak1 and Tax transcripts in HTLV-1-transformed and Tax-expressing MT4 cells transfected with siPaks (Figure 8B). Again, silencing of Pak1 or Pak3 correlated with down-regulation of Tax expression (Figure 8B, bars 3–8), but did not apparently affect cell proliferation (Figure 8C). Taken together, these results provided further support to the notion that group I Paks are facilitators of Tax-activated HTLV-1 transcription in both acutely and chronically infected cells.
In this study, we provided the first evidence for a facilitator function of group I Paks in Tax-induced activation of HTLV-1 LTR. This kinase-independent function of Pak1, Pak2 and Pak3 was characterized in the context of Tax and HTLV-1 LTR (Figures 1 and 2). CREB, CRTCs, p300/CBP and the N-terminal regulatory domain of Paks are indispensable for this function (Figures 3, 4, 5 and 6). Paks associate with Tax and CRTCs and are recruited to HTLV-1 LTR by Tax (Figures 5, 6 and Additional file 4: Figure S4). Compromising Paks by RNAi resulted in a suppression of the interaction of Tax with CRTC1 and the recruitment of Tax to the LTR (Figure 7), plausibly leading to an inhibition of HTLV-1 transcription in cells infected with an HTLV-1 clone and in HTLV-1-transformed T cells (Figures 1, 8, Additional file 1: Figures S1 and Additional file 2: Figures S2). Collectively, our findings reveal new mechanistic details of Tax-induced transcriptional activation of HTLV-1 LTR, in which Tax interacts with and recruits group I Paks to the TRE enhancers to facilitate transcription.
Group I Paks are Cdc42/Rac-regulated kinases that regulate transcription, cell cycle, cell motility and other aspects of cell physiology [19, 20]. Some functions of Paks do not require their kinase activity [24–27]. For example, Pak1 induces lamellipodia formation and membrane ruffling through its N-terminal regulatory domain in a kinase-independent fashion [24, 25]. Inhibition of cell cycle progression by the CRIB domain of Pak1 is also independent of its kinase activity . In addition, Pak1 serves a kinase-independent scaffolding role in Akt signaling . However, it is still surprising that group I Paks augment Tax transcriptional activity on HTLV-1 LTR in a kinase-independent manner. Exactly how these Paks facilitate Tax in LTR activation remains to be elucidated. The diminution of CRTC1- and LTR-bound Tax in Pak1/3-compromised cells (Figure 7) was in support of an adaptor function of group I Paks in Tax-induced activation of the LTR. On the other hand, Pak1 was previously shown to stimulate transcription when tethered to the promoter through Gal4 DNA-binding domain (Gal4BD) . One alternative mechanism by which Pak1 modulates transcription is through inactivation of transcriptional corepressor CtBP , which inhibits the function of CBP . Pak1 also interacts with and phosphorylates histone H3 . We found that the M5 mutant of Pak3 containing the N-terminal regulatory domain alone could sufficiently interact with Tax, promote Tax recruitment to the LTR, and augment Tax-induced LTR activation (Figures 3, 5 and 6). Although neither CRIB nor kinase activity was required for the augmentation of Tax activity (Figures 2 and 3), we cannot completely rule out the involvement of the kinase domain since the Tax-augmenting activity of M1 lacking the kinase domain was weakened compared to the wild type (Figure 3). Our experiments suggested that the augmentation of Tax activity by group I Paks could not bypass CREB, CRTCs or p300/CBP (Figure 4). However, our analysis could not distinguish the effect of the dominant inhibitors of CREB, CRTC1 and p300/CBP on Tax or on Paks. Particularly, the incomplete inhibitory effect of CRTC1M1 (Figure 4B) suggested that compromising CRTC1 alone is insufficient to abrogate the activity of Tax and Pak3. It will be of interest to determine whether CRTC2 and CRTC3 could account for the remaining activity of Tax and Pak3 in the presence of CRTC1M1. Exactly how Paks affect the activity of CREB, CRTCs and p300/CBP remains to be elucidated. In this regard, we showed that depletion of Pak1 suppressed the recruitment of Tax to the LTR (Figure 7B). Because Tax is required for the recruitment and activation of CREB, CRTCs and p300/CBP [5–9], the activity of these transcriptional regulators should also be compromised in the absence of Paks. At least three additional lines of experiments are required to derive mechanistic insight on the transcriptional regulatory function of group I Paks. First, the dispensability of the kinase domain for the augmentation of Tax activity should be determined. Second, the M5 mutant recruited to the promoter through Gal4BD should be assessed for transcriptional activity. Finally, possible involvement of CtBP and histone H3 phosphorylation in Pak-augmented activation of HTLV-1 LTR by Tax should be investigated.
Emerging evidence implicates group I Paks in supporting the replication of various human viruses . Thus, association and activation of Pak2 by viral Nef protein were thought to play a role in HIV-1 replication . In addition, an RNAi screen identified Pak1 and Pak3 to be important host factors that support HIV-1 infection in HeLa cells and T lymphocytes . On the other hand, hepatitis B virus oncoprotein HBx was recently shown to activate Pak1 to promote cell survival . Our findings that group I Paks facilitate HTLV-1 transcription are generally consistent with the notion that these Paks play an important role in the life cycle of human viruses of different families. Small-molecule inhibitors of Paks are thought to be attractive therapeutic agents for different types of cancer and viruses . Because the facilitator function of Paks on Tax-induced LTR activation is kinase-independent, kinase inhibitors of Paks might not be useful in anti-HTLV-1 therapy. However, peptide mimetics that can inhibit the interaction between Paks and Tax would still hold the promise for the design and development of new molecularly targeted anti-HTLV-1 and anti-ATL therapeutics.
Group I Paks are a critical regulatory point in cell signaling on which diverse upstream signals converge [18, 19]. It remains to be understood how these signals might affect the interaction between Tax and Paks. Tax appears to be able to promote the recruitment of Paks to HTLV-I LTR (Figure 6). The mechanisms of this recruitment and the cellular factors that regulate this process warrant further investigations. Paks are frequently upregulated in cancer cells and virus-infected cells [19, 23]. In line with this, Pak1 and Pak3 were found to be expressed in HTLV-1-transformed leukemic cells and compromising their expression led to inhibition of HTLV-1 transcription (Figures 1, 8, Additional file 1: Figure S1 and Additional file 2: Figure S2). To understand whether group I Paks might indeed be induced by HTLV-1 infection, expression profiles of Pak mRNA and protein in HTLV-1-infected individuals and ATL patients should be determined systematically. Pak1, Pak2 and Pak3 are strikingly homologous and serve redundant and non-redundant functions in cells [18, 19]. We found that all three were equally active in facilitating Tax-induced LTR activation (Figure 1). Comparing their relative abundance in HTLV-1-infected T cells in patients will shed light on whether they are differentially expressed and which ones might be more important in HTLV-1 transcription.
The activation of CREB signaling by Tax not only mediates LTR activation, but is also required for full-blown oncogenic transformation [15, 49]. In addition to the LTR, Tax also activates a wide array of cellular genes through CREB to effect cell proliferation and survival [47, 48]. We noted in our study that Pak1 and Pak3 associated with CRTC1 even in the absence of Tax (Figures 5 and Additional file 4: Figure S4), raising the possibility that group I Paks might serve a general facilitator function in cellular CREB-dependent transcription. That is to say, group I Paks could act as cofactors in CREB-induced cellular transformation. In HTLV-1-infected cells, Tax might further enhance the activity of Paks to facilitate CREB-dependent transcription by recruiting them to CRE-containing promoters. In this regard, full characterization of the role of group I Paks in the activation of cellular CREB-regulated genes both in the absence and in the presence of Tax will enable us to have a complete picture of how Tax, group I Paks and CREB cooperate to mediate transcriptional activation and oncogenic transformation.
We demonstrate that group I Paks interact with HTLV-1 Tax and are recruited to the LTR to serve a kinase-independent facilitator function in Tax-induced activation of LTR transcription.
Cell culture and transfection
HeLa and HEK293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Jurkat and HTLV-1-transformed T cells MT2 and MT4 were maintained in RPMI medium supplemented with 10% fetal bovine serum. HeLa and HEK293T cells were transfected using GeneJuice transfection reagent (Novagen). Jurkat, MT2 and MT4 cells were transfected using Lipofectamine 2000 (Invitrogen).
RNA knockdown experiments were carried out as described [51, 52]. HeLa, Jurkat, MT2 and MT4 cells were transfected with 100 nM siRNA using Lipofectamine 2000 (Invitrogen). siRNA sequences are as follows:
siPak1A: 5′-GCUCUGUCAA GCUAACUGAdT dT-3′
siPak1B: 5′-GCUCUGGAGU UCUUGCAUUdT dT-3′
siPak3A: 5′-CAACCCAAGA AGGAAUUAAdT dT-3′
siPak3B: 5′-CCAGAUCACU CCUGAGCAAdT dT-3′
siTax-A: 5′-GGCCUCAUAC AGUACUCUUdT dT-3′
siTax-B: 5′-GGCAGAUGAC AAUGACCAUdT dT-3′
siGFP: 5′-GAAGCAGCAC GACUUCUUCdT dT-3′
Reporter plasmid pLTR-Luc and expression plasmids for Tax, A-CREB, CRTC1, CRTC1M1 and Pak1 were described elsewhere [4, 8, 21, 36, 37]. pLTR-Luc contains the full LTR of HTLV-1. Reporter plasmid pTRE-Luc was constructed by inserting into pGL3-basic (Promega) three copies of TREs amplified from HTLV-1 LTR using primers 5′-CCCAAGCTTG GTCAGGGCCC AGACTAAGGC TC-3′ and 5′-CCCAAGCTTT GGATGGCGGC CTCAGGTAAG G-3′. Reporter plasmid pHIAP-Luc was a gift from R. Grassmann . Expression plasmid for E1A-12S was provided by J. Lundblad . HTLV-1 molecular clone pX1MT was provided by D. Derse . Pak2 expression construct was provided by W. Hahn  and Pak2 was subcloned using primers 5′-GGGGTACCAT CATGTCTGAT AACGGAGAAC TGGAAG-3′ and 5′-ATAAGAATGC GGCCGCTTAA CGGTTACTCT TCATTGCTTC TTT-3′. Pak3 was derived from I.M.A.G.E. cDNA clone 4798769 and was amplified using primers 5′-GCGGATCCAG ATGTCTGACG GTCTGGATA ATG-3′ and 5′-CCGCTCGAGT TAGCGGCTGC TGTTCTTAAT TGCTTCC-3′. R67C, K297L, A365E, R419X and T421E mutants of Pak3 were constructed by PCR method using the following sets of sense (s) and antisense (as) primers (the mutated nucleotides are underlined):
R67C-s: 5′-CCAATAAGAA GAAAGAGAAA GAGTGCCCAG AGATCTCTCT TCC-3′
R67C-as: 5′-GGAAGAGAGA TCTCTGGGCA CTCTTTCTCT TTCTTCTTAT TGG-3′
K297L-s: 5′-GAGGTGGCCA TACTGCAGAT GAACCTTCAA CAGC-3′
K297L-as: 5′-GCTGTTGAAG GTTCATCTGC AGTATGGCCACC TC-3′
A365E-s: 5′-CCTGTATGGA TGAAGGACAG ATAGAAGCTG TCTG-3′
A365E-as: 5′-CAGACAGCTT CTATCTGTCC TTCATCCATA CAGG-3′
R419X-s: 5′-CTGAGCAAAG TAAATGAAGC ACTATGGTGG GAAC-3′
R419X-as: 5′-GTTCCCACCA TAGTGCTTCA TTTACTTTGC TCAG-3′
T421E-s: 5′-CTGAGCAAAG TAAACGAAGC GAGATGGTGG GAAC-3′
T421E-as: 5′-GTTCCCACCA TCTCGCTTCG TTTACTTTGC TCAG-3′
Truncated mutant M4 of Pak3 was made by PCR using primers 5′-GCGGATCCAG ATGCGCCCAG AGATCTCTCT TCCTTCA-3′ and 5′-CCGCTCGAGT TACTTCTTCT GTTCCAATTT TGTTATGTTG G-3′. Truncated mutants M1, M2, M3 and M5 of Pak3 were generated by PCR using the common sense primer 5′-GCGGATCCAG ATGTCTGACG GTCTGGATAA TG-3′ and the following anti-sense primers:
M1-as: 5′-CCGCTCGAGT TATGCTGGTG AAGCAATGGA TTCAACCAC-3′
M2-as: 5′-CCGCTCGAGT TAATGGGCTG CTATGTATCC ATGTGCACT-3′
M3-as: 5′-CCGCTCGAGT TATGGGTTCT TCTTCTGTTC CAATTTTGTT AT-3′
M5-as: 5′-CCGCTCGAGT TAGATCTCTG GGCGCTCTTT CT-3′
Luciferase reporter assays
RNA was extracted using Trizol reagent (Invitrogen). RNA (2 μg) was digested with DNase I (Ambion) at 37°C for 30 min. cDNA synthesis was performed using oligo (dT). Semi-quantitative RT-PCR was performed as previously described [37, 51]. Primer sets were as follows:
Tax-s: 5′-TCTCACACGG CCTCATACAG-3′
Tax-as: 5′-ATATTTGGGG CTCATGGTCA-3′
Gag-s: 5′-CTTTGCTCCT CCCTCGTG-3′
Gag-a: 5′-TTGCTGGTAT TCTCGCCTTA-3′
Env-s: 5′-TGGCGGAGGC TATTATTCAG-3′
XII-s: 5′-CGGATACCCA GTCTACGTGT TTG-3′
XII-as: 5′-GGGAGTCGAG GGATAAGGAA CT-3′
Pak1-s: 5′-TGAGAGCCTT GTACCTCATT GCCA-3′
Pak1-as: 5′-TCCTTAGCTG CAGCAATCAG TGGA-3′
Pak3-s: 5′-ACAACCGGGA TTCTTCAGCA CTCA-3′
Pak3-as: 5′- AGTAATCGTG CCCATTGCTC TGGA-3′
globin-s: 5′-AGCGTACTCC AAAGATTCAG GTT-3′
globin-as: 5′-TACATGTCTC GATCCCACTT AACTAT-3′
Real-time RT-PCR was performed as previously described [37, 50]. Relative expression levels were quantified by normalizing to the corresponding β-globin values using the comparative threshold cycle method where:
Fold difference = 2–(ΔCTof gene of interest -ΔCTof β-globin) = 2–ΔΔCT
Primer sets for quantitative RT-PCR were as follows:
Tax-s: 5′-TACTACAGTC CTCCTCCT-3′
Tax-as: 5′-CCCTCATTTC TACTCTCAC-3′
Pak1-s: 5′-GACATCCAAC AGCCAGAA-3′
Pak1-as: 5′-ACACAGCCTT CACATTCAA-3′
Gag-s: 5′-CTTTGCTCCT CCCTCGTG-3′
Gag-as: 5′-TTGCTGGTAT TCTCGCCTTA-3′
Env-s: 5′-TGGCGGAGGC TATTATTCAG-3′
Env-as: 5′-TTGAGGCGTG ACACTTCTTG-3′
Co-immunoprecipitation was carried out as described [52, 54]. HEK293T cells were lysed with lysis buffer (20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 0.1% NP-40, and 0.5 mM EDTA) supplemented with protease inhibitors (Roche). Cell debris was removed by centrifugation at 14,000 rpm at 4°C. Cell lysate was incubated with primary antibodies at 4°C overnight. The immunocomplex was incubated with 30 μl protein A-agarose (Invitrogen), washed three times with lysis buffer, and then resuspended with SDS-PAGE loading buffer (60 mM Tris-Cl, 2% SDS, 6% glycerol, 1% β-mercaptoethanol, and 0.002% bromophenol blue).
Chromatin immunoprecipitation (ChIP) was performed as previously described [37, 55]. HeLa cells were cross-linked by 1% formaldehyde for 10 min at room temperature. The DNA–protein complex was immunoprecipitated, and the genomic DNA was purified by phenol-chloroform extraction. Promoter sequence spanning the three 21-bp TREs in HTLV-1 LTR was PCR-amplified using primers 5′-GGCTTAGAGC CTCCCAGTG-3′ and 5′-CTCCTGAACT GTCTCCACGC-3′.
Cell proliferation assay
Cell proliferation assay was performed using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium (MTT) method . MT4 cells (5 × 104) were treated with 5 mg/ml MTT solution (10 μl) and OD550 was measured with a microplate reader (Spectra Max 340, Molecular Devices). Cell proliferation was presented as a percentage of the control.
We thank D. Derse, R. Grassmann, W. Hahn, K.T. Jeang, J. Lundblad and C. Vinson for gifts of plasmid and members of Jin laboratory for critical reading of manuscript. This work was supported by S.K. Yee Medical Research Fund (2011) and Hong Kong Research Grants Council (HKU7683/05M, HKU7661/08M, HKU7674/12M and HKU1/CRF/11G).
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