The HTLV-1 Tax interactome

2.1.1 CREB/ATF factors

Tax1 was initially described as an activator of LTR-directed transcription [22]. Three imperfectly conserved 21-base-pair (bp) repeat sequences called (TxRE) located in the U₃ region of the LTR are required and sufficient to confer Tax1 responsiveness [23]. The TxRE element contains an octamer motif TGACG(T/A)(C/G)(T/A) that is flanked by a G stretch and a C stretch at the 5' and 3' sides, respectively [24]. Interestingly this octamer shares homology with the consensus cAMP-responsive element (CRE) 5'-TGACGTCA-3' [24]. Nevertheless, Tax1 exhibits poor affinity for DNA and does not bind directly to the TxRE element [25] but interacts with CRE-binding/activating transcription factors (CREB/ATF). In fact, Tax1 interacts in vitro with a number of proteins of the CREB/ATF family of transcription factors: CREB, CREM, ATF1, ATF2, ATF3, ATF4 (CREB2) and XBP1 (X-box-binding protein 1) [26-31]. These proteins share a common cluster of basic residues allowing DNA binding and a leucine zipper (b-Zip) domain involved in homo- and heterodimerization. Dimer formation modulates their DNA binding specificity and transcriptional activity [32]. Biochemical studies revealed that Tax1 promotes formation of a Tax1-CREB/ATF-TxRE ternary complex in vitro by interacting with the b-Zip domain of CREB/ATF factors. Mechanistically, Tax1 enhances the dimerization of CREB/ATF factors, increases their affinity for the viral CRE [33-36] and further stabilizes the ternary complex through direct contact of the GC-rich flanking sequences [37,38]. Tax1 then recruits co-activators (CBP/p300 and P/CAF) to facilitate transcriptional initiation (see 2.3.1). The ability of Tax1 to dimerize is required for efficient ternary complex formation and for optimal transactivation [39,40]. Interaction of Tax1-CREB/ATF with the LTR promoter DNA was further explored by chromatin immunoprecipitation (ChIP) [41]. In HTLV-1 infected human T-cells (SLB-1), Tax1 and a plethora of CREB/ATF factors as well as other b-Zip proteins bind to the LTR promoter, further confirming interaction in vivo. The fact that Tax1 interacts with ATFx adds another level of complexity since this factor represses Tax1-mediated LTR activation [42]. Tax1 is thus able to interact with positive as well as with negative CREB/ATF factors to modulate LTR promoter-directed activity.

Tax1 also binds to CREB co-activator proteins called transducers of regulated CREB activity (TORCs). In fact, Tax1 interacts with the three members of this family (TORC1, TORC2 and TORC3) [43,44] and TORCs cooperate with Tax1 to activate the LTR in a CREB and p300-dependent manner. Thus, TORCs are thought to associate with the Tax1 ternary complex and participate to transcriptional activation.

CREB/ATF members play a role in cell growth, survival and apoptosis by regulating CRE-directed gene transcription in response to environmental signals such as growth factors or stress [32,45]. Furthermore, CREB/ATF proteins also have significant impact on cancer development [45]. Depending on the cell type, Tax1 mutants deficient for CREB activation are incompetent for transformation or induction of aneuploidy [46-50]. Tax1 activates a variety of cellular genes through its interactions with CREB/ATF proteins, for example those encoding interleukin 17 or c-fos [51,52]. Conversely, Tax1 also represses expression of genes like cyclin A, p53 and c-myb by targeting CREB/ATF factors [53-55]. Transcriptomic profiling of cells expressing either a wild-type or a CREB-deficient Tax1 protein revealed several cellular genes controlled by CRE elements activated by Tax1 [50]. Among these, Sgt1 (suppressor of G2 allele of SKP1) and p97(Vcp) (valosin containing protein) have functions in spindle formation and disassembly, respectively.

Together, these reports thus demonstrate that Tax1 interacts with a series of CREB/ATF factors and modulates expression of viral and cellular genes through CRE elements. The specific contribution of each CREB/ATF member in Tax1-mediated gene transcription remains unclear.

2.1.2 Serum responsive factor and members of the ternary complex factor

HTLV-1 infected T-cell lines expressing Tax1 display increased expression of AP1 (activator protein 1), a homo- or heterodimeric complex of Fos (c-Fos, FosB, Fra1 and Fra2) and Jun (c-Jun, JunB and JunD) [56,57]. Fos and Jun are under the transcriptional control of the serum responsive factor (SRF) in response to various stimuli such as cytokines, growth factors, stress signals and oncogenes. SRF binds to the SRF responsive element (SRE) located in the Fos/Jun promoters which contains two binding sites: a CarG box (CC(A/T)₆GG) and an upstream Ets box (GGA(A/T)). Once SRF occupies the CArG box, the ternary complex factor (TCF) establishes protein interaction with SRF and subsequently binds the upstream Ets site. This complex then recruits the co-activators P/CAF and CBP/p300 to activate transcription.

In reporter assays, Tax1 activates transcription of promoters under the control of SRE motifs [52,56,58] without direct binding to the DNA but through interactions with transcription factors associated with the SRF pathway. Tax1 has been shown to bind directly to SRF [59-61] and to various members of the TCF complex such as Sap1 (SRF accessory protein 1), Elk1, Spi1 (spleen focus forming virus (SFFV) proviral integration oncogene 1) and Ets1 [49,62,63]. Tax1 interaction with SRF results in increased binding of SRF to the SRE and altered site selection [64]. Once the complexes are stabilized, Tax1 recruits the co-activators CBP/p300 and P/CAF (see 2.3.1) and mediates transactivation [63].

It thus appears that Tax1 activates transcription from CREB- and SRF-responsive sites through a similar mechanism which involves its interaction with transcription factors resulting in enhanced DNA binding, altered site selection and coactivator recruitment [16].

2.1.3 Nuclear factors κB (NF-κB)

HTLV-1 infected cells display increased expression of various cytokines and cytokine receptors such as interleukin 2 (IL2) and the α-subunit of its high-affinity receptor complex (IL2Rα) [65-68]. Induction of IL2 and IL2Rα expression is mediated by Tax1 activation of the NF-κB/Rel family of transcription factors [69,70]. By modulating expression of a wide range of genes involved in apoptosis, proliferation, immune response and inflammation, NF-κB is thought playing a central role in Tax1-mediated cell transformation [16].

In mammals, the NF-κB family of transcription factors is composed of five structurally related members, RelA, RelB (p65), c-Rel, NF-κB1 (p50/p105) and NF-κB2 (p52/p100) which form various dimeric complexes that transactivate or repress target genes bearing a κB enhancer [71,72]. p105 and p100 are precursor proteins that are processed proteolytically to the mature p50 and p52 forms, respectively. These factors share a common Rel-homology domain (RHD) mediating their dimerization, DNA binding and nuclear localization. In non-activated cells, NF-κB dimers are trapped in the cytoplasm by inhibitory proteins called IκBs such as p105, p100, IκBα, IκBβ and IκBγ (C-terminal region of p105), that mask the nuclear localization signal of NF-κB factors through physical interaction [71,72]. NF-κB activation involves phosphorylation of IκB inhibitors by the IκB kinase (IKK), which triggers their ubiquitination and subsequent proteasomal degradation, resulting in nuclear translocation of NF-κB dimers [72,73].

Tax1 associates with RelA, c-Rel, p50 and p52 after their translocation in the nucleus [61,74,75] but also directly recruits RelA from the cytoplasm [76,77]. After interaction with these NF-κB factors, Tax1 increases their dimerization which is essential for binding to target promoters [61,75,78]. When the complex is bound to the promoter, Tax1 recruits the CBP/p300 and PCAF co-activators [79,80], leading to transcriptional activation

2.1.4 Other transcription factors

Tax1 has been shown to associate with CCAAT binding proteins such as NF-YB (nuclear factor YB subunit) and C/EBPβ (CCAAT/enhancer-binding protein β) [81-83]. Through its binding to NF-YB, Tax1 activates the major histocompatibility complex class II promoter [82]. Besides, C/EBPβ acts as a transcriptional repressor by preventing Tax1 binding to the LTR [83]. On the other hand, Tax1 increases binding of C/EBPβ to and activates the IL-1β promoter [81]. It is noteworthy that C/EBP factors have been implicated in regulation of cellular proliferation and differentiation but also in tumor formation and leukemia development [84].

Tax1 forms ternary complexes in vitro with Sp1 (specificity protein 1)/Egr1 (early growth response 1) [85] and Sp1/Ets1 [62], thereby participating directly in transcriptional activation of the c-sis/PDGF-B (platelet-derived growth factor B) proto-oncogene and PTHrP (parathyroid hormone-related protein) P2 promoters, respectively. Of note, PTHrP is up-regulated during immortalization of T-lymphocytes by HTLV-1 and plays a primary role in the development of humoral hypercalcemia of malignancy that occurs in the majority of patients with ATL [86,87].

Tax1 further associates with nuclear respiratory factor 1 (NRF1) and activates the CXCR4 chemokine receptor promoter [88].

Finally, the transcriptional repressor MSX2 (msh homeobox homolog 2) impairs Tax1 mediated transactivation through direct binding [89].

2.3.1 HATs

Acetylation of lysine residues located in the N-terminal tails of histone proteins by HATs is a crucial step for activation of gene transcription. Tax1 interacts with several HATs: p300, its homologous CREB binding protein (CBP) and p300/CBP associated factor (P/CAF) [98-102]. Tax1 recruits the CBP/p300 and P/CAF once the Tax1-CREB-TxRE complex is stabilized (see 2.1.1), each of which being able to enhance Tax1-mediated transactivation of a transiently transfected LTR reporter. CBP/p300 and P/CAF bind independently on different regions of Tax1 and interaction of Tax1 with these two cofactors is required for optimal transcriptional activity from transiently transfected but also stably integrated LTR reporters [101-103]. Surprisingly, P/CAF but not CBP/p300 is able to enhance transcription from the LTR independently of its HAT activity [101,103]. Tax1 mediates recruitment of CBP/p300 on reconstituted chromatin templates and facilitates transactivation in a HAT-activity dependent manner [104,105]. CBP/p300 presence at the LTR template correlates with histone H3 and H4 acetylation as well as increased binding of basal transcription factors and RNA polymerase II. ChIP analyses with HTLV-1 infected T cell lines indicate that Tax1, CBP/p300 and acetylated histone H3 and H4 are indeed associated with the LTR promoter [41,105].

There is a long lasting debate about how Tax1 recruits CBP/p300 at the Tax1-CREB/ATF-TxRE complex. Phosphorylation of CREB at serine 133 by protein kinases A or C is required for CBP/p300 recruitment via physical interaction with the KIX domain [106-108]. It has long been suggested that Tax1 bypasses the requirement for CREB phosphorylation to recruit coactivators [98,100]. Nevertheless, recent reports indicate that Tax1 rather cooperates with phosphorylated CREB (pCREB) to induce transactivation [109,110]. High levels of pCREB are detected in Tax1 expressing cells and in HTLV-1-infected human T-lymphocytes [110]. Tax1 and pCREB interact simultaneously at two distinct binding sites on the KIX domain forming a very stable complex with the viral CRE [110,111]. Both CREB phosphorylation and Tax1 binding are needed for efficient interaction of full-length CBP to pCREB and subsequent transcriptional activation [112].

Finally, Tax1 is able to repress the activity of some transcription factors by competitive usage of CBP, p300 and P/CAF. As mentionned above, stable complex formation between Tax1, a transcription factor (e.g. CREB or SRF) and CBP/p300 contributes to transcriptional activation. On the contrary, when Tax1 has poor affinity for a transcription factor (e.g. p53, MyoD or STAT2), it interferes with co-activator recruitment and prevents their activition [113-116]. Although controversial, this mechanism termed trans-repression could partipate to p53 inactivation in Tax1 expressing cells and HTLV-1 infected lymphocytes (for a review see [117]).

2.3.2 HDACs

Among three HDACs (-1, -2 and -3) interacting with the viral LTR in HTLV infected cell lines [118], Tax1 binds directly to HDAC1. HDAC1 overexpression represses Tax1-mediated transactivation owing to its HDAC activity [119]. Nevertheless, the presence of Tax1 and HDAC1 on the viral promoter is mutually exclusive [118,120]. HDAC1 binds to the non-activated LTR and is released from the promoter through physical interaction with Tax1 allowing recruitment of co-activators and transcription initiation. Tax1 is also able to tether HDAC1 to the tyrosine phosphatase SHP1 promoter and selectively down-regulate gene expression [121].

HDACs form multiprotein complexes together with DNA-histone binding proteins such as SMRT (silencing mediator for retinoid and thyroid receptor) and MBD2 (methyl-CpG-binding domain 2) that both interact with Tax1 and are involved in Tax1 transcriptional activities [122,123]. It thus seems that Tax1, through direct association with HDACs and HDAC-containing complexes is able to selectively activate or repress viral and cellular genes expression.

2.3.3 HMTs and HDMs

Mono-, di- and tri-methylation of histone H3 at lysine 9 (H3K9) play a crucial role in structural modification of chromatin. Tax1 associates with two enzymes involved in regulation of H3K9 methylation: SUV39H1 (suppressor of variegation 3–9 homologue 1), a HMT and JMJD2A (Jumonji containing domain 2A), a HDM [124,125]. Methylated H3K9 is a hallmark of transcriptionally inactive chromatin whereas demethylation rather promotes transcriptional activation [126]. SUV39H1 interacts with Tax1 and represses Tax1-mediated transactivation of the LTR [124]. JMJD2A is highly expressed in HTLV-1 infected cell lines but its role on Tax1-mediated transcription is currently unknown [125].

Methylation of histone H3 at arginine residues is another important regulatory mechanism of transcriptionnal regulation. Tax1 associates with coactivator-associated arginine methyltransferase (CARM1), which preferentially induces methylation at residues R2, R17 and R26 of histone H3 [127]. CARM1 is recruited by Tax1 to the LTR and increases Tax1-mediated transactivation of the LTR. Consistently, silencing of CARM1 impairs Tax1 transcriptional activation, R2-, R17- and R26-methylated histone H3 proteins being present on the LTR promoter in HTLV-1 infected cells.

Tax1 thus interacts with different histone methyltranferases and demethylases to modulate histone methylation and regulate gene expression.

2.3.4 The SWI/SNF complex

The SWI/SNF (Switch/Sucrose Non Fermentable) complex utilizes the energy of ATP hydrolysis to remodel chromatin structures, thereby allowing transcription factors to gain access to DNA during initiation and elongation steps of transcription [128,129]. Tax1 interacts with different components of SWI/SNF: BRG1, BAFs 53, 57 and 155 [130]. Overexpression and silencing of BRG1 increments and impedes Tax1 transactivation of the LTR, respectively [130]. It was first suggested that Tax1 targets BRG1/BRM downstream of RNA polymerase II in order to prevent stalling of transcription. This model was apparently contradicted by the capacity of Tax1 to efficiently activate transcription from chromosomally integrated LTR and NF-κB promoter in a BRG1/BRM deficient cell line [131]. Nevertheless, this observation does not exclude that factors of the SWI/SNF complex cooperate with Tax1 to promote gene transcription. Consistent with this idea, Tax1 cooperates with SWI/SNF complex and RNA polymerase II to promote nucleosome eviction during transactivation [132]. Histone eviction increases accessibility of DNA to transcription factors and requires activity of SWI/SNF and RNA polymerase II [128,133]. Of note, Tax1 may also impact indirectly on SWI/SNF function [134] by interaction with DNA topoisomerase I [135].

Tax1 is thus able to target SWI/SNF complex components to promote nucleosome displacement and participate to transcriptional activation.

2.6.1 Tristetraprolin (TTP)

TTP belongs to a family of adenine/uridine-rich element (ARE)-binding proteins that contain tandem CCCH zinc finger RNA-binding domains [145]. TTP is therefore an important player in posttranscriptional regulation of mRNA containing ARE elements. Indeed, TTP delivers ARE-containing mRNAs in discrete cytoplasmic regions, called RNA granules, involved in regulation of translation or decay of these transcripts [146]. The repertoire of ARE-containing genes includes Tumor Necrosis Factor α (TNFα) and Granulocyte Macrophage-Colony-Stimulating Factor (GM-CSF) [145] involved in cell signaling, metabolism, cell proliferation, immune response, death, differentiation and morphogenesis [147].

Tax1 interacts with TTP and redirects TTP from the cytoplasm to the nuclear compartment as well as in a region surrounding the nucleus [148]. Through its interactions with TTP, Tax1 stabilizes TNFα mRNA and indirectly increases TNFα protein expression. This observation is of importance for the cell transformation process induced by HTLV-1, because TNFα overexpression plays a central role in pathogenesis.

2.6.2 Int6 and TRBP

Tax also binds Int6 (Integration site 6) and TRBP (TAR binding protein) that regulate translation and RNA interference, respectively. In fact, Int6 is a subunit of translation initiation factor eIF3, which regulates mRNA binding to the ribosome [149] while TRBP (TAR binding protein) is a componant of RISC (RNA-induced silencing complex) that mediates RNA interference [150]. Currently, the role of these interactions remains unclear.

3.2.1 MEKK1

MEKK1 primarily regulates JNK and ERK1/2 but also contributes to the NF-κB pathway [176,177]. Tax1 binds to the amino terminus of MEKK1 and stimulates MEKK1 kinase activity [157]. As a result, Tax1 expression increases IKKβ activity, leading to phosphorylation and degradation of IκBα. Dominant negative mutants of both IKKβ and MEKK1 prevent Tax1 activation of the NFκB pathway but, intriguingly, silencing of MEKK1 does not affect Tax1-induced NF-κB activation [160].

3.2.2 TAK1

TAK1 is involved in JNK, TGF-β and NF-κB dependent signaling pathways [178]. TAK1 acts in concert with TAK1 binding proteins (TABs) which link TAK1 to the upstream activating TNF receptor associated factor (TRAFs) proteins. TAK1 phosphorylates IKKβ and MKK6, thereby activating NF-κB and JNK [179].

TAK1 is constitutively activated in Tax1-expressing cells and in HTLV-1 infected lymphocytes [158,160,180]. Tax1 activates TAK1 through complexation with TAK1 and TAB2 and connects TAK1 onto the IKK complex thereby stimulating IKK activity [180,181]. Consistently, overexpression of TAK1 or TAB2 increases Tax1 transactivation of a NF-κB reporter [180,181]. However, RNA interference of TAK1 suppresses activation of JNK and p38 but not NF-κB. Constitutive activation of TAK1 is thus not absolutely required for NF-κB activation [160,180]. TAK1 rather participates to JNK signaling, which is constitutively activated in Tax1-expressing cells, in Tax1-transformed murine fibroblasts and in human lymphocytes transformed with HTLV-1 [182-185].

3.4.1 Rho GTPases and the cytoskeleton proteins

Tax1 complexes with several members of the small GTPase Rho family such as RhoA, Rac, Gap1m and Cdc42 [130]. Rho GTPases are activated in response to external stimuli (e.g. growth factor, stress, cytokines) and exert a wide range of biochemical functions like cytoskeleton organization, regulation of enzymatic activities as well as gene expression [188]. Notably, Tax1 binds to proteins involved in cytoskeleton structure and dynamics: α-internexin, cytokeratin, actin, gelsolin, annexin and γ-tubulin [130,189-191]. Through these interactions, Tax1 might thus connect Rho GTPases to their targets and affect cytoskeleton organization. Consistent with this idea, Tax1 localizes around the microtubule organization center (MTOC) and in the cell-cell contact region [192]. Thereby, Tax1 provides an intracellular signal that synergizes with ICAM1 engagement to cause the T-cell microtubule polarization and formation of the virological synapse. Through the formation of complexes with both Rho GTPases and their targets, Tax1 could thus favor HTLV-1 cell-to-cell transmission.

Since Rho GTPases modulate a wide range of signaling networks (SRF, JNK, p38 and NF-kB) [188], complex formation with Tax1 is also likely to modulate transcription.

3.4.2 Heterotrimeric Gβ subunit

Heterotrimeric G proteins are the molecular switches that turn on intracellular signaling cascades in response to activation of G protein coupled receptor (GPCR). After binding of an agonist, the activated GPCR induces an exchange of GDP to GTP on the Gα subunit and facilitates the dissociation of GTP-bound Gβγ and Gα subunits [193]. Through its interaction with Gβ, Tax1 affects SDF-1 dependent activation of the CXCR4 GPCR chemokine receptor. Tax1 enhances response to SDF-1 resulting in MAPK pathway over-activation and increased cell chemotaxis. The HTLV-1 associated pathologies (ATL, HAM/TSP and dermatitis) are characterized by invasion and accumulation of infected T-cells in organs such as lymph nodes, central nervous system or dermis [194]. These results thus provide a rationale for the mechanisms of cell migration observed in HTLV-1 associated pathologies.

4.3.1 RanBP1 and Tax1BP2

The presence of two centrosomes at mitosis is crucial for formation of bipolar mitotic spindles and correct chromosome segregation. Multipolar mitosis which happens when more than two centrosomes emerge in one cell is a possible cause of aneuploidy in solid tumors and leukemias [245]. Supernumerary centrosomes are observed in approximately 30% of ATL cells [246-248]. Tax1 colocalizes with the centrosome during mitosis and causes centrosome amplification through physical interaction with Ran/Ran Binding protein 1 (RanBP1) and Tax1BP2 [249,250]. RanBP1 is involved in the Ran GTP cycle that controls microtubule nucleation and/or stabilization and centrosome cohesion during mitosis [251,252]. Centrosome fragmentation requires direct Tax1/RanBP1 interaction and Tax1's ability to transactivate NF-κB. Tax1BP2 is thought to act as an intrinsic block to centrosome overreplication [253]. Consistently, overexpression of Tax1BP2 abolishes Tax1-induced centrosome amplification. On the other hand, a Tax1 mutant unable to bind to Tax1BP2 is impaired in centrosome overreplication [250].

4.3.2 Mad1 and cdc20

In eukaryotes, the mitotic spindle assembly checkpoint (SAC) monitors the fidelity of chromosome segregation [254]. SAC functioning requires complex formation between Mad1-2-3 and Bub1-2-3 proteins that arrest mitosis in response to microtubule damage [255]. At the molecular level, SAC activation involves formation of inhibitory complexes between Mad2 and/or Mad3/BubR1 and Cdc20, preventing Cdc20 from activating the anaphase promoting complex/cyclosome (APC/C). APC/C is active during mitosis where it mediates ubiquitination and degradation of an inhibitory chaperone of separase called securin. Once liberated from its inhibitor, separase triggers anaphase by hydrolysing cohesin leading to subsequent separation of sister chromatin. Furthermore, APC/C regulates the degradation of mitotic cyclin, activates CDK1 and, ultimately, promotes mitotic exit [256].

Through physical interactions with Mad1 and Cdc20, Tax1 subverts activation of SAC and APC/C. Tax1 inhibits Mad1 homodimerization, a process that is required for formation of a inhibitory complex between Mad2 and Cdc20 [257-259]. Consistently, ATL cells exhibit a defect in the mitotic spindle assembly checkpoint [257]. On the other hand, Tax1 associates with and activates Cdc20-associated APC/C leading to unscheduled degradation of securin and cyclin B1, a delay or failure in mitotic entry and progression, and faulty chromosome transmission [260,261]. Tax1-induced premature activation of APC/C provokes permanent G1 arrest and senescence [262,263]. Finally, coexpression of Tax1 and securin enhances chromosomal instability and favors cell transformation in vitro and in vivo [264].