- Open Access
Interplay between the HTLV-2 Tax and APH-2 proteins in the regulation of the AP-1 pathway
© Marban et al.; licensee BioMed Central Ltd. 2012
- Received: 24 February 2012
- Accepted: 18 November 2012
- Published: 3 December 2012
In contrast with human T-cell leukemia virus type 1 (HTLV-1) that causes ATL (adult T-cell leukemia), HTLV-2 has not been causally linked to malignant disease. The minus strand of the HTLV genomes encode the regulatory proteins HTLV-1 bZIP factor (HBZ) for HTLV-1 and antisense protein of HTLV-2 (APH-2) for HTLV-2. Unlike the viral proteins Tax1 and Tax2, both HBZ and APH-2 are constitutively expressed in infected cells suggesting that they may play important roles in the pathogenesis of these viruses. To date, very little is known about the function of APH-2 except that it inhibits Tax2-mediated transcription of HTLV-2 genes. In the present study, we investigated the role of APH-2 in basal and Tax2B-mediated activation of the AP-1 pathway.
We demonstrate that, unlike HBZ, APH-2 stimulates basal AP-1 transcription by interacting with c-Jun and JunB through its non-conventional bZIP domain. In addition, when Tax2 and APH-2 are co-expressed, they physically interact in vivo and in vitro and APH-2 acts as an inhibitor of Tax2-mediated activation of AP-1 transcription.
This report is the first to document that HTLV-2 can modulate the AP-1 pathway. Altogether our results reveal that, in contrast with HBZ, APH-2 regulates AP-1 activity in a Tax2-dependant manner. As the AP-1 pathway is involved in numerous cellular functions susceptible to affect the life cycle of the virus, these distinct biological properties between HBZ and APH-2 may contribute to the differential pathogenic potential of HTLV-1 and HTLV-2.
Thirty years after the discovery of the first human oncogenic virus, the human T-cell leukemia virus (HTLV) family of retroviruses is now composed of four members: the well documented HTLV-1 and HTLV-2 and the recently discovered HTLV-3 and HTLV-4 [1–4]. HTLV-1 is the etiological agent of multiple disorders including adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) [5, 6]. The role of HTLV-2 in human disease is less clearly defined but infection is associated with lymphocyte proliferation and high platelet counts as well as milder neurological disorders [7–9]. However, while not being associated with ATL like disorders, HTLV-2 is still able to efficiently induce transformation of primary T-cells .
In addition to the structural and enzymatic proteins common to all retroviruses, HTLV-1 also encodes regulatory proteins such as Tax1. The HTLV-1 Tax protein is a transcriptional activator that regulates HTLV-1 gene expression but also modulates the expression of numerous cellular genes through activation of cellular transcription factors including NF-κB , CREB [12–16], SRF  and AP-1 . Activation of these major cellular signal transduction pathways plays a critical role in T-cell transformation, and therefore ATL development. Previous reports indicate that AP-1 activity is induced in ATL cells [18, 19]. Moreover, HTLV-1 Tax up-regulates the transcription of several AP-1 family members such as c-Jun, JunD, c-Fos and Fra-1 [20, 21].
AP-1 consists of a myriad of homo- or hetero- dimers that belong to the Jun, Fos, Maf and ATF subfamilies. All AP-1 family members harbour a basic leucine zipper (bZIP) motif, which consists of a DNA binding domain rich in basic amino acids adjacent to a leucine zipper structure required for protein-protein dimerization . AP-1 dimers recognize either TPA response elements (TRE) or cAMP response elements (CRE) which are present in the promoter region of many cellular genes involved in a large spectrum of biological processes including cell proliferation, apoptosis and oncogenic transformation .
Transcription from the 3’ Long Terminal Repeat (LTR) of the HTLV genomes governs the expression of antisense regulatory proteins named HTLV-1 bZIP factor (HBZ) for HTLV-1 , antisense protein of HTLV-2 (APH-2) for HTLV-2 , APH-3 and APH-4 for HTLV-3 and HTLV-4, respectively . The HBZ gene has been described as a key player in HTLV-1 pathogenesis as its expression appears to be critical for ATL development and disease severity in HAM/TSP [27–29]. HBZ contains a bZIP motif, which enables it to heterodimerize with cellular transcription factors in order to regulate viral or cellular transcription. Thus, by interacting with CREB, HBZ prevents the binding of CREB to the CRE in the HTLV-1 LTR, resulting in the inhibition of HTLV-1 gene transcription . HBZ also interacts with the transcription factor ATF3, thus preventing its ability to enhance p53 transcriptional activity, and therefore the proliferation of ATL cells . In addition, HBZ is able to inhibit the classical NF-κB pathway by binding p65 and therefore decreasing p65 DNA binding capacity, a mechanism used by the virus to escape from the host immune system . Moreover, numerous studies have also reported that HBZ interacts with AP-1 members of the Jun subfamily such as c-Jun, JunB and JunD and modulates their transcriptional activity [33, 34]. The interaction between HBZ and c-Jun as well as HBZ and JunB results in repression of c-Jun and JunB activity through degradation or sequestration into transcriptionally inactive nuclear bodies [35–38]. However, by interacting with JunD, HBZ can activate JunD-dependant transcription of cellular genes including the human telomerase reverse transcriptase [34, 39].
The role of APH-2 in the pathogenesis of HTLV-2 infection is less defined. To date, only one study reveals that APH-2 does not promote lymphocytosis . APH-2 harbours a non-conventional bZIP motif as it displays seven instead of six amino acids between the sixth and the seventh leucine. Despite the lack of a classic bZIP domain, APH-2 is still able to interact with CREB and repress Tax2-mediated transcription activation of HTLV-2 genes .
In the present report, we investigated the role of HTLV-2 proteins APH-2 and Tax2B on AP-1 activity. We demonstrated that APH-2 interacts with c-Jun and JunB through its non-canonical bZIP domain and enhances their ability to activate AP-1 transcription. Surprisingly, when APH-2 and Tax2B are co-expressed, APH-2 binds Tax2B and acts as a repressor of Tax2B-mediated activation of AP-1 transcription.
Taken together, our results reveal that both APH-2 and Tax2 act as transcription factors that subtly regulate AP-1 transcription. These findings strongly suggest that APH-2 and Tax2 are involved in the regulation of many biological processes involving AP-1 and therefore indirectly help the virus to replicate and/or counteract the host’s immune system.
APH-2 stimulates the transcriptional activity of c-Jun, JunB and JunD
Previous studies have shown that HBZ affects AP-1 transcription by modulating the transcriptional activity of all members of the Jun subfamily. On the one hand, HBZ represses transcription mediated by c-Jun and JunB while it activates JunD-dependant transcription [34, 35, 37, 38]. To examine whether APH-2-mediated activation of AP-1 transcription results in the stimulation of Jun activity, we performed luciferase assays. Cells were co-transfected with the AP-1-Luc reporter construct, c-Jun, JunB or JunD as well as APH-2 expression vectors (Figure 1C, 1D and 1E, respectively, upper panels). Western blot analysis demonstrated that FLAG-APH-2 does not affect the levels of overexpressed c-Jun-Myc, FLAG-JunB and FLAG-JunD (Figure 1C, 1D and 1E, respectively, lower panels). As expected, in the absence of APH-2 expression, c-Jun (Figure 1C, column 2), JunB (Figure 1D, column 2) and JunD (Figure 1E, column 2) activate AP-1 transcription. Interestingly, co-expression of APH-2 further enhances c-Jun (Figure 1C, columns 3–5), JunB (Figure 1D, columns 3–5) and JunD-mediated (Figure 1E, columns 3–5) transactivation.
These results collectively reveal that APH-2 is a co-activator of c-Jun, JunB and JunD.
APH-2 interacts with c-Jun and JunB but not JunD
To further characterize the interaction between APH-2 and c-Jun/JunB, we tested whether APH-2 also associates with endogenous c-Jun and JunB. We, therefore, co-immunoprecipitated endogenous c-Jun and JunB from nuclear extracts of FLAG-APH-2 transfected cells. As shown in Figure 2D (column 3) and Figure 2E (column 3), FLAG-APH-2 was specifically detected in the c-Jun and JunB immunoprecipitates, respectively. Taken together, these results demonstrate that APH-2 dimerizes with endogenous c-Jun and JunB.
The non-conventional bZIP domain of APH-2 is critical for binding c-Jun and JunB and stimulating their transcriptional activation
The leucine zipper motif of a conventional bZIP domain is a protein-protein interaction domain consisting of amphipathic α-helices that dimerize either as homodimers or heterodimers to form a coiled-coil. Despite the lack of a conventional bZIP domain, APH-2 is still able to interact with CREB and repress Tax2-dependant activation of HTLV-2 gene transcription .
Finally, to test whether the absence of the non-conventional bZIP domain could abolish the ability of APH-2 to activate c-Jun and JunB-mediated transactivation, we carried out luciferase assays. 293T were transfected with the AP-1-Luc reporter construct, c-Jun-Myc or FLAG-JunB as well as APH-2ΔbZIP expression vectors (Figure 3C and 3D, respectively, upper panels). The expression levels of the transfected proteins were verified by Western blot (Figure 3C and 3D, lower panels). As expected, APH-2ΔbZIP was unable to stimulate the transcriptional activity of c-Jun and JunB (Figure 3C, columns 3–5 and Figure 3D, columns 3–5, respectively). Similar experiments conducted with JunD show that even though APH-2ΔbZIP did not interact with JunD, the non-conventional bZIP domain is required for APH-2-mediated stimulation of JunD transactivation (Additional file 2A and 2B).
Altogether, these results demonstrate that APH-2 binds c-Jun and JunB via its non-conventional bZIP domain. Moreover, this domain is crucial for APH-2 ability to stimulate c-Jun and JunB-dependent AP-1 transcription.
APH-2 interacts with Tax2B in vitro and in vivoand represses the ability of Tax2B to stimulate AP-1 transcription
Taken together, our data show that both HTLV-2 proteins Tax2B and APH-2 are individually able to activate AP-1 transcription. In order to monitor AP-1 activity when Tax2B and APH-2 are co-expressed, we conducted luciferase assays on 293T cells co-transfected with the AP1-Luc reporter construct and Tax2B-His and/or FLAG-APH-2 expression vectors (Figure 4B, upper panel). The expression levels of Tax2B-His and FLAG-APH-2 were confirmed by Western blot analysis (Figure 4B, lower panel). We speculated that Tax2B and APH-2 effects on AP-1 activity are either additive or synergistic. Unexpectedly, our data demonstrate that APH-2 suppressed the activation of AP-1 transcription by Tax2B, indicating that APH-2 acts as a repressor of Tax2B-mediated transactivation (Figure 4B, columns 3–5).
We next investigated whether Tax2B and APH-2 interact in vitro. To address this issue, we performed GST pull-down assays with GST-APH-2 incubated with purified Tax2B-His. As illustrated in Figure 4C, GST-APH-2 binds Tax2B-His but not GST, indicating that APH-2 interacts directly with Tax2B (Figure 4C, columns 2 and 3).
To test whether APH-2 and Tax2B also interact in vivo, cells were transfected with Tax2B-His and/or FLAG-APH-2 and cellular lysates were subjected to co-immunoprecipitation assays with anti-His antibodies (Figure 4D). Our data demonstrate that FLAG-APH-2 was detected in the immunoprecipitates from cells co-transfected with FLAG-APH-2 and Tax2B-His (Figure 4D, WB anti-FLAG, column 6) but not FLAG-APH-2 or Tax2B-His alone (Figure 4D, WB anti-FLAG, columns 4 and 5).
As previously described, Tax2B is mainly distributed in the cytoplasm but can also be found in punctate nuclear structures whereas APH-2 displays a predominant nuclear localization [25, 41]. To test whether the expression of Tax2B was able to alter APH-2 localization, we carried out immunofluorescence experiments. Interestingly, Tax2B expression was able to relocate APH-2 to the nuclear periphery (Additional file 3A).
Altogether, these results suggest that Tax2B-His and FLAG-APH-2 form a stable protein complex in vitro and in vivo.
APH-2 and c-Jun/JunB interaction is independent of Tax2B
As a control, we tested whether c-Jun was able to affect Tax2B localization. The results obtained from our immunofluorescence experiments show that c-Jun was able to delocalize Tax2B from the cytoplasm to the nucleus (Additional file 3B).
Similar co-immunoprecipitation experiments were performed with nuclear extracts from cells transfected with APH-2-His and FLAG-JunB either alone or together with increasing amounts of Tax2B-His and immunoprecipitated with FLAG antibodies (Figure 5B). As expected, Tax2B was also part of the APH-2/JunB complex (Figure 5B, columns 3–4) but did not affect the interaction between APH-2 and JunB (Figure 5B, columns 6–8). Conversely, additional co-immunoprecipitations reveal that c-Jun and JunB did not have an effect on the interaction between APH-2 and Tax2B (Additional file 4A and 4B, respectively).
Overall our results strongly suggest that APH-2, Tax2B and c-Jun/JunB can form a ternary complex. Similarly, Tax2A also binds APH-2 but this interaction does not affect the association between APH-2 and c-Jun/JunB (Additional file 5A and 5B, respectively).
The interaction between APH-2 and Tax2B neither involves the non-canonical bZIP domain nor the LXXLL domain of APH-2
A recent study reported that the LXXLL motif of APH-2 is important for CREB binding and repression of Tax function on viral genes . We therefore tested whether this motif was also involved in Tax2B binding and repression of Tax2B function on AP-1 transcription. To this aim, we generated a mutant of APH-2 that lacks the LXXLL motif and named it APH-2ΔLXXLL (Figure 6A). Co-immunoprecipitations were carried out with cellular extracts overexpressing FLAG-APH-2ΔLXXLL and/or Tax2B-His and immunoprecipitated with His antibodies (Figure 6C). Results revealed that FLAG antibodies were able to detect FLAG-APH-2ΔLXXLL in nuclear extracts overexpressing both proteins, thus suggesting that Tax2B can still interact with the APH-2ΔLXXLL mutant (Figure 6C, WB anti-FLAG, column 6).
Furthermore, we carried out luciferase assays to test the effects of these APH-2 mutants on Tax2B-mediated AP-1 transcription (Figure 6D, upper panel). 293T cells were transfected with the AP-1-Luc reporter construct, Tax2B and APH-2 full-length, ΔbZIP or ΔLXXLL. The expression levels of Tax2B-His and FLAG-APH-2 constructs were confirmed by Western blot analysis (Figure 6D, lower panel). Interestingly, even if the non-canonical bZIP domain of APH-2 is not required for its interaction with Tax2B, it appears crucial for Tax2B function on AP-1 transcription as APH-2-mediated repression of Tax2B function is completely inhibited when this domain is deleted (Figure 6D, column 4). Unlike ΔbZIP, the ΔLXXLL mutant was not able to abolish the ability of APH-2 to repress Tax2B-mediated transactivation of AP-1 (Figure 6D, column 5).
Thus far, we have demonstrated that the interaction between APH-2 and Tax2B does not involve the two main domains of APH-2: the bZIP domain (103–136) and the LXXLL domain (178–186). In order to further study the interaction between APH-2 and Tax2B, we constructed a FLAG-tagged mutant of APH-2 lacking the C-terminal part of the protein: APH-2 (1–102) (Figure 6A). We then performed co-immunoprecipitations with cellular extracts from 293T cells overexpressing Tax2B and FLAG-APH-2 full-length or N-terminal (1–102) in combination with Tax2B-His (Figure 6E). Our data reveal that in contrast with the full-length APH-2 (Figure 6E, WB anti-His, column 4), the N-terminal part of APH-2 (1–102) is unable to interact with Tax2B-His (Figure 6E, WB anti-His, column 6). Overall, these results suggest that, by default, APH-2 interacts with Tax2B through its C-terminal part (137–177).
APH-2 and Tax2B finely regulate the activity of the collagenase promoter
These findings demonstrate that APH-2 acts synergistically with c-Jun and JunB to activate transcription of the collagenase promoter. Taken together our data suggest that the collagenase promoter well illustrates how APH-2 and Tax2B regulate AP-1 activity.
HTLV-1 and HTLV-2 differ both in their specific epidemiologic and pathogenic properties. In contrast to HTLV-1 that causes ATL and HAM/TSP, HTLV-2 has not been clearly linked to any disease but has been associated with lymphocyte proliferation and high platelet counts as well as rare cases of chronic neuromyelopathy [7–9]. This suggests that HTLV-2 fails to promote a critical stage of leukemogenesis and neurologic disease development.
The distinct clinical manifestations of HTLV-1 and HTLV-2 can be attributed in part to distinct biological functions of Tax1 and Tax2 . For instance, the transforming potential of Tax1 is higher than that of Tax2 mainly due to the fact that, unlike Tax1, Tax2 cannot activate the non-canonical NF-κB pathway because of its inability to interact with NF-κB2/p100 [48, 49].
Although Tax plays a pivotal role in HTLV-associated transformation of T-cells, the tax gene is frequently genetically and epigenetically inactivated in ATL cells, suggesting that Tax is not required for the maintenance of the leukemic stage in ATL [50, 51]. Recent studies showed that transcription from the 3’ LTR of the HTLV genomes governs the expression of antisense regulatory proteins [24–26]. HBZ, the antisense protein encoded by HTLV-1, is a bZIP factor that is consistently expressed in ATL cells and plays a key role in the malignant proliferation in ATL . HBZ interacts with numerous cellular transcription factors of the bZIP family and modulates their transcriptional activity. Notably, by interacting with c-Jun, JunB and JunD, HBZ highly influences AP-1 transcription [33, 34].
In the current study, we reveal that the antisense protein of HTLV-2 (APH-2) also regulates AP-1 activity. However, we postulate that HBZ and APH-2 display opposite effects on AP-1 basal transcription. While HBZ inhibits AP-1-mediated transcription, APH-2 acts as an activator of AP-1 basal activity. Moreover, whereas HBZ has been described as an inhibitor of c-Jun and JunB-mediated transcription [33, 38], we demonstrated that APH-2 enhances the transcriptional activity of c-Jun and JunB on AP-1 binding sites. As AP-1 controls numerous biological processes critical in virus replication such as oncogenic transformation, cell proliferation, differentiation and apoptosis, we hypothesize that this divergence in AP-1 transcription regulation might explain, in part, the differences between HTLV-1 and HTLV-2 pathogenesis. A recent study already excluded the involvement of APH-2 in lymphocyte proliferation , but further investigation is needed to establish the role of APH-2 in other AP-1-associated biological functions.
We focused our studies on elucidating the molecular mechanisms involved in the regulation of AP-1 activity by APH-2 and demonstrated that APH-2 physically interacted with c-Jun and JunB through its non-conventional bZIP domain. HBZ harbours a bZIP domain that is involved in numerous biological functions and especially in its interaction with c-Jun and JunB . Interestingly, despite the lack of a consensus bZIP domain, APH-2 was still able to interact with c-Jun and JunB and therefore still share some similarities with HBZ. Using a reporter assay, we established that this non-canonical bZIP domain is critical for APH-2 to enhance c-Jun and JunB transcriptional activity on AP-1 transcription. These findings confirm that the physical interaction between APH-2 and c-Jun/JunB is essential for APH-2 to regulate c-Jun and JunB-mediated AP-1 transcription.
Interestingly, as previously described for HBZ , APH-2 cooperates with JunD through its non-canonical bZIP domain to stimulate the AP-1 activity. However, our results show that APH-2 and JunD are not able to form a stable complex in vivo. We postulate that APH-2 and JunD might involve a different mechanism of action than APH-2 and c-Jun/JunB to stimulate AP-1 transcription. Further investigation is needed to better understand how APH-2 indirectly stimulates JunD transcriptional activity on AP-1 transcription.
The development of ATL in HTLV-1 infected patients has been associated with the deregulation of cellular gene transcription. Among all HTLV-1 proteins, Tax1 is known to play a critical role in ATL development by disrupting major cellular signal transduction pathways such as NF-κB and AP-1 [18, 47]. In the present study, we reveal that HTLV-2 Tax enhances AP-1 transcription. However, when Tax2B and APH-2 are co-expressed, they interact directly and APH-2 impairs the ability of Tax2B to activate AP-1 transcription. These functional effects could be explained by the fact that Tax2B can relocate APH-2 to the nuclear periphery, a mechanism that could prevent APH-2 from activating AP-1 transcription. Interestingly, similar functional effects have been described on the HTLV-2 promoter , but further experiments are required to investigate whether Tax2B and APH-2 are using a common molecular mechanism to regulate HTLV-2 and AP-1 transcription.
Here, we report that APH-2, Tax2B and Jun can form a ternary complex, as there is no competition between Tax2B and c-Jun/JunB for APH-2 binding. Indeed, unlike c-Jun/JunB, APH-2 and Tax2B, interaction does not involve the non-canonical bZIP domain of APH-2. However, this domain, and consequently the interaction between APH-2 and c-Jun/JunB, is crucial to repress Tax function on AP-1 transcription.
According to a recent study, the LXXLL motif of APH-2 is involved in its interaction with CREB and in the repression of Tax function on viral genes . Surprisingly, we demonstrated that this motif is not essential for APH-2 repressive function on Tax2B-mediated AP-1 transcription and is not involved in Tax2B binding.
The human collagenase promoter is an extensively studied AP-1 responsive promoter [43, 44]. Here we used the collagenase promoter as an example to illustrate AP-1 transcription regulation by the two HTLV-2 proteins APH-2 and Tax2B. We established that, according to our model, APH-2 stimulates c-Jun and JunB-mediated transactivation. Moreover, APH-2 or Tax2B expressed individually highly enhance AP-1 transcription whereas co-expression of both viral proteins results in a suppression of Tax2B-mediated transactivation by APH-2. We also speculate that the molecular mechanism by which APH-2 and Tax2B regulate the collagenase transcription could be common to numerous AP-1 target genes.
A recent study reported that both APH-2 and Tax2 mRNA expression are correlated with HTLV-2 proviral loads (PVL). However, whereas APH 2 was expressed in vivo in the majority of HTLV-2 carriers, Tax expression was not detected among HTLV-2 carriers with low PVL . With the intention of correlating this study with our findings, we speculate that in HTLV-2 carriers with low PVL, only APH-2 is expressed and consequently, AP-1 activity is stimulated. However, in HTLV-2 carriers with high PVL, APH-2 and Tax2 are co-expressed and AP-1 activity is down-regulated, suggesting that APH-2 might balance the transactivation activity of Tax2B on HTLV-2 and AP-1 transcription in order to silence the virus and allow it to escape host immune responses.
The present study is the first to demonstrate that HTLV-2 deregulates AP-1 activity. Moreover, together with a previous report , we confirm that APH-2 and Tax2B act as viral transcription factors that subtly regulate HTLV-2 and AP-1 transcription to possibly help the virus to replicate and counteract host immune responses. Moreover, in accordance with previous studies [33, 34, 38], we report that APH-2 and HBZ display similar repressive effects on AP-1-mediated transcription in the presence of Tax but have opposite effects in its absence. These findings highlight the fact that APH-2 and HBZ have distinct biological properties that may contribute to the differential pathogenic potential of HTLV-1 and HTLV-2.
Cells and plasmids
The 293T cells (obtained from the ATCC) were cultured under standard tissue culture conditions. The pAP1-Luc and pRL-TK-Renilla plasmids are commercially available at Stratagene and Promega, respectively. The pCAGGS-Tax2B-His was previously described . The pFLAG-JunB and pFLAG-JunD were generous gifts from Dr. Rong Li (Stowers Institute for Medical Research, Kansas City, USA). The p-c-Jun-HA, pCollagenase-Luc and p-c-Jun-Myc were kindly provided by Dr. Anna Maria Musti (University of Würzburg, Germany), Dr. Sagar Ghosh (University of Texas, San Antonio, USA) and Dr. Kunitada Shimotohno (Chiba Institute of Technology, Japan), respectively. The pGFP-APH-2 was obtained by cloning the APH-2 cDNA into the pEGFP (Invitrogen) using standard techniques. The pFLAG-APH-2 and pFLAG-HBZ were generated by cloning the APH-2 or HBZ cDNAs as a HindIII/EcoRI fragment obtained from pcDNA-APH-2-Myc-His or pcDNA-HBZ-Myc-His (kindly provided by Dr. Jean-Michel Mesnard, Université de Montpellier, France) into pFLAG-CMV (Sigma-Aldrich). The pFLAG-APH-2ΔbZIP and pAPH-2ΔbZIP-His mutants were generated by site-directed mutagenesis (Phusion® Site-Directed Mutagenesis kit, Thermo Scientific) using pFLAG-APH-2 or pcDNA-APH-2-Myc-His as a template and the following primers: 5’-TATACACTCCAACTGCTGATGCCTTTC-3’ and 5’-GAGGAACTATTTGAGGCAATTATTCAG-3’. The pFLAG-APH-2ΔLXXLL and pFLAG-APH-2 (1–102) mutants were also constructed by site-directed mutagenesis (Phusion® Site-Directed Mutagenesis kit, Thermo Scientific) using pFLAG-APH-2 as a template and the primers: 5’-TAAGAATTCATCGATAGATCTGATATCGGT-3’ and 5’- CTTCTGCAGCAAATCCCCATGGTT-3’ for pFLAG-APH-2ΔLXXLL and 5’-TAAGAATTCATCGATAGATCTGATATCGGT-3’ and 5’- TATACACTCCAACTGCTGATGCCTTTC-3’ for pFLAG-APH-2 (1–102). To obtain the GST-APH-2 construct, we generated an APH-2 PCR product using the pcDNA-APH-2-Myc-His as a template. The APH-2 PCR product was then digested with BamHI/EcoRI and cloned into the pGEX-2 T (GE Healthcare).
293T cells were transiently transfected with the pAP1-Luc or pcollagenase reporter plasmids, different combinations of expression vectors and the pRL-TK-Renilla vector as an internal control using Lipofectamine™ 2000 (Invitrogen). DNA amounts were normalized across samples using the respective empty vectors. Cells were harvested 48 hours post-transfection and processed for luciferase assays (Dual Luciferase® Reporter Assay System, Promega) or Western blot.
293T cells were transiently transfected with different combinations of expression vectors using Lipofectamine™ 2000 (Invitrogen). DNA amounts were normalized across samples using the respective empty vectors. Protein extracts were prepared 48 hours after transfection (NE-PER® Nuclear and Cytoplasmic Extraction Reagents, Thermo Scientific) and 400ug of protein extracts were immunoprecipitated with 5ug of anti-FLAG® M2 (Sigma-Aldrich, F3165), anti-Myc (Invitrogen, R950), anti-6xHis (Clontech, 631212), anti-c-Jun (Abcam ab31419), anti-JunB (Abcam ab31421) or rabbit IgG (Millipore) overnight at 4°C. Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) were then added to the samples and incubated for 2 hours at 4°C. Beads were washed 3 times with IPLS buffer (50 mM Tris–HCl pH7.5, 120 mM NaCl, 0.5 mM EDTA, 0.5% NP-40), 3 times with IPHS buffer (50 mM Tris–HCl pH7.5, 400 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) and twice with IPLS buffer. Samples were then subjected to Western blot.
Cell lysates were subjected to SDS-PAGE and analyzed by Western blot using standard procedures. Membranes were probed using the SNAP i.d. system (Millipore). The antibodies used for Western blot are as follows: anti-FLAG® M2 (Sigma-Aldrich, F3165), anti-α-tubulin (Abcam, Ab7291), anti-Myc (Invitrogen, R950), anti-6xHis (Clontech, 631212) and anti-HA (Invitrogen, 32–6700).
Purified GST or GST-APH-2 fusion proteins were immobilized on Glutathione Sepharose™ 4 Fast Flow beads (GE Healthcare) and incubated with purified Tax2B-His overnight at 4°C. After extensive washing with GST Wash Buffer (0.5% Triton® X-100 in PBS supplemented with protease inhibitors), bound proteins were eluted using GST Elution Buffer (50 mM Tris–HCl pH8.0, 10 mM reduced glutathione) and separated by Western blot. Tax2B-His was detected with anti-6xHis antibodies (Clontech, 631212).
COS-7 cells were plated onto chamber slides and co-transfected with the indicated expression vectors using FuGENE® HD (Promega). One day post-transfection, cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.2% Tween/PBS for 10 minutes at room temperature and incubated with a blocking reagent (TSA kit, Molecular Probes) for 1 hour at room temperature. Tax2B was detected with an anti-Tax2B antibody (Fusion Antibodies) followed by anti-mouse IgG-HRP and Alexa Fluor® 594 tyramide staining (Molecular Probes). c-Jun-Myc was detected using an anti-Myc-HRP antibody (Invitrogen, R951) followed by Alexa Fluor® 488 tyramide staining (Molecular Probes). DAPI (Sigma-Aldrich) was used to stain the nuclei. Slides were mounted using the ProLong® Gold Antifade reagent (Invitrogen). Images were acquired with a Zeiss Axio Imager microscope.
This work was supported by the National Virus Reference Laboratory (NVRL), University College Dublin, Ireland. We are grateful to Dr. Anna Maria Musti, Dr. Jean-Michel Mesnard, Dr. Rong Li, Dr. Sagar Ghosh and Dr. Kunitada Shimotohno for kindly providing plasmids. We thank Pr. Olivier Rohr and Dr. Virginie Gautier for helpful discussions during the course of this work.
- Calattini S, Chevalier SA, Duprez R, Bassot S, Froment A, Mahieux R, Gessain A: Discovery of a new human T-cell lymphotropic virus (HTLV-3) in Central Africa. Retrovirology. 2005, 2: 30-10.1186/1742-4690-2-30.PubMed CentralView ArticlePubMedGoogle Scholar
- Kalyanaraman VS, Sarngadharan MG, Robert-Guroff M, Miyoshi I, Golde D, Gallo RC: A new subtype of human T-cell leukemia virus (HTLV-II) associated with a T-cell variant of hairy cell leukemia. Science. 1982, 218: 571-573. 10.1126/science.6981847.View ArticlePubMedGoogle Scholar
- Poiesz BJ, Ruscetti FW, Reitz MS, Kalyanaraman VS, Gallo RC: Isolation of a new type C retrovirus (HTLV) in primary uncultured cells of a patient with Sezary T-cell leukaemia. Nature. 1981, 294: 268-271. 10.1038/294268a0.View ArticlePubMedGoogle Scholar
- Wolfe ND, Heneine W, Carr JK, Garcia AD, Shanmugam V, Tamoufe U, Torimiro JN, Prosser AT, Lebreton M, Mpoudi-Ngole E, et al: Emergence of unique primate T-lymphotropic viruses among central African bushmeat hunters. Proc Natl Acad Sci U S A. 2005, 102: 7994-7999. 10.1073/pnas.0501734102.PubMed CentralView ArticlePubMedGoogle Scholar
- Uchiyama T, Yodoi J, Sagawa K, Takatsuki K, Uchino H: Adult T-cell leukemia: clinical and hematologic features of 16 cases. Blood. 1977, 50: 481-492.PubMedGoogle Scholar
- Iwasaki Y: Pathology of chronic myelopathy associated with HTLV-I infection (HAM/TSP). J Neurol Sci. 1990, 96: 103-123. 10.1016/0022-510X(90)90060-Z.View ArticlePubMedGoogle Scholar
- Bartman MT, Kaidarova Z, Hirschkorn D, Sacher RA, Fridey J, Garratty G, Gibble J, Smith JW, Newman B, Yeo AE, Murphy EL: Long-term increases in lymphocytes and platelets in human T-lymphotropic virus type II infection. Blood. 2008, 112: 3995-4002. 10.1182/blood-2008-05-155960.PubMed CentralView ArticlePubMedGoogle Scholar
- Hall WW, Ishak R, Zhu SW, Novoa P, Eiraku N, Takahashi H, Ferreira Mda C, Azevedo V, Ishak MO, Ferreira Oda C, et al: Human T lymphotropic virus type II (HTLV-II): epidemiology, molecular properties, and clinical features of infection. J Acquir Immune Defic Syndr Hum Retrovirol. 1996, 13 (Suppl 1): S204-S214.View ArticlePubMedGoogle Scholar
- Roucoux DF, Murphy EL: The epidemiology and disease outcomes of human T-lymphotropic virus type II. AIDS Rev. 2004, 6: 144-154.PubMedGoogle Scholar
- Ross TM, Narayan M, Fang ZY, Minella AC, Green PL: Human T-cell leukemia virus type 2 tax mutants that selectively abrogate NFkappaB or CREB/ATF activation fail to transform primary human T cells. J Virol. 2000, 74: 2655-2662. 10.1128/JVI.74.6.2655-2662.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Sun SC, Ballard DW: Persistent activation of NF-kappaB by the tax transforming protein of HTLV-1: hijacking cellular IkappaB kinases. Oncogene. 1999, 18: 6948-6958. 10.1038/sj.onc.1203220.View ArticlePubMedGoogle Scholar
- Zhao LJ, Giam CZ: Interaction of the human T-cell lymphotrophic virus type I (HTLV-I) transcriptional activator Tax with cellular factors that bind specifically to the 21-base-pair repeats in the HTLV-I enhancer. Proc Natl Acad Sci U S A. 1991, 88: 11445-11449. 10.1073/pnas.88.24.11445.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhao LJ, Giam CZ: Human T-cell lymphotropic virus type I (HTLV-I) transcriptional activator, Tax, enhances CREB binding to HTLV-I 21-base-pair repeats by protein-protein interaction. Proc Natl Acad Sci U S A. 1992, 89: 7070-7074. 10.1073/pnas.89.15.7070.PubMed CentralView ArticlePubMedGoogle Scholar
- Suzuki T, Fujisawa JI, Toita M, Yoshida M: The trans-activator tax of human T-cell leukemia virus type 1 (HTLV-1) interacts with cAMP-responsive element (CRE) binding and CRE modulator proteins that bind to the 21-base-pair enhancer of HTLV-1. Proc Natl Acad Sci U S A. 1993, 90: 610-614. 10.1073/pnas.90.2.610.PubMed CentralView ArticlePubMedGoogle Scholar
- Goren I, Semmes OJ, Jeang KT, Moelling K: The amino terminus of Tax is required for interaction with the cyclic AMP response element binding protein. J Virol. 1995, 69: 5806-5811.PubMed CentralPubMedGoogle Scholar
- Paca-Uccaralertkun S, Zhao LJ, Adya N, Cross JV, Cullen BR, Boros IM, Giam CZ: In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator. Tax. Mol Cell Biol. 1994, 14: 456-462.View ArticlePubMedGoogle Scholar
- Fujii M, Tsuchiya H, Chuhjo T, Akizawa T, Seiki M: Interaction of HTLV-1 Tax1 with p67SRF causes the aberrant induction of cellular immediate early genes through CArG boxes. Genes Dev. 1992, 6: 2066-2076. 10.1101/gad.6.11.2066.View ArticlePubMedGoogle Scholar
- Iwai K, Mori N, Oie M, Yamamoto N, Fujii M: Human T-cell leukemia virus type 1 tax protein activates transcription through AP-1 site by inducing DNA binding activity in T cells. Virology. 2001, 279: 38-46. 10.1006/viro.2000.0669.View ArticlePubMedGoogle Scholar
- Mori N, Fujii M, Iwai K, Ikeda S, Yamasaki Y, Hata T, Yamada Y, Tanaka Y, Tomonaga M, Yamamoto N: Constitutive activation of transcription factor AP-1 in primary adult T-cell leukemia cells. Blood. 2000, 95: 3915-3921.PubMedGoogle Scholar
- Fujii M, Sassone-Corsi P, Verma IM: c-fos promoter trans-activation by the tax1 protein of human T-cell leukemia virus type I. Proc Natl Acad Sci U S A. 1988, 85: 8526-8530. 10.1073/pnas.85.22.8526.PubMed CentralView ArticlePubMedGoogle Scholar
- Fujii M, Tsuchiya H, Seiki M: HTLV-1 Tax has distinct but overlapping domains for transcriptional activation and for enhancer specificity. Oncogene. 1991, 6: 2349-2352.PubMedGoogle Scholar
- Landschulz WH, Johnson PF, McKnight SL: The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988, 240: 1759-1764. 10.1126/science.3289117.View ArticlePubMedGoogle Scholar
- Shaulian E, Karin M: AP-1 in cell proliferation and survival. Oncogene. 2001, 20: 2390-2400. 10.1038/sj.onc.1204383.View ArticlePubMedGoogle Scholar
- Gaudray G, Gachon F, Basbous J, Biard-Piechaczyk M, Devaux C, Mesnard JM: The complementary strand of the human T-cell leukemia virus type 1 RNA genome encodes a bZIP transcription factor that down-regulates viral transcription. J Virol. 2002, 76: 12813-12822. 10.1128/JVI.76.24.12813-12822.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Halin M, Douceron E, Clerc I, Journo C, Ko NL, Landry S, Murphy EL, Gessain A, Lemasson I, Mesnard JM, et al: Human T-cell leukemia virus type 2 produces a spliced antisense transcript encoding a protein that lacks a classic bZIP domain but still inhibits Tax2-mediated transcription. Blood. 2009, 114: 2427-2438. 10.1182/blood-2008-09-179879.PubMed CentralView ArticlePubMedGoogle Scholar
- Larocque E, Halin M, Landry S, Marriott SJ, Switzer WM, Barbeau B: Human T-Cell Lymphotropic Virus Type 3 (HTLV-3)- and HTLV-4-Derived Antisense Transcripts Encode Proteins with Similar Tax-Inhibiting Functions but Distinct Subcellular Localization. J Virol. 2011, 85: 12673-12685. 10.1128/JVI.05296-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Satou Y, Yasunaga J, Yoshida M, Matsuoka M: HTLV-I basic leucine zipper factor gene mRNA supports proliferation of adult T cell leukemia cells. Proc Natl Acad Sci U S A. 2006, 103: 720-725. 10.1073/pnas.0507631103.PubMed CentralView ArticlePubMedGoogle Scholar
- Saito M, Matsuzaki T, Satou Y, Yasunaga J, Saito K, Arimura K, Matsuoka M, Ohara Y: In vivo expression of the HBZ gene of HTLV-1 correlates with proviral load, inflammatory markers and disease severity in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Retrovirology. 2009, 6: 19-10.1186/1742-4690-6-19.PubMed CentralView ArticlePubMedGoogle Scholar
- Arnold J, Zimmerman B, Li M, Lairmore MD, Green PL: Human T-cell leukemia virus type-1 antisense-encoded gene, Hbz, promotes T-lymphocyte proliferation. Blood. 2008, 112: 3788-3797. 10.1182/blood-2008-04-154286.PubMed CentralView ArticlePubMedGoogle Scholar
- Lemasson I, Lewis MR, Polakowski N, Hivin P, Cavanagh MH, Thebault S, Barbeau B, Nyborg JK, Mesnard JM: Human T-cell leukemia virus type 1 (HTLV-1) bZIP protein interacts with the cellular transcription factor CREB to inhibit HTLV-1 transcription. J Virol. 2007, 81: 1543-1553. 10.1128/JVI.00480-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Hagiya K, Yasunaga J, Satou Y, Ohshima K, Matsuoka M: ATF3, an HTLV-1 bZip factor binding protein, promotes proliferation of adult T-cell leukemia cells. Retrovirology. 2011, 8: 19-10.1186/1742-4690-8-19.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhao T, Yasunaga J, Satou Y, Nakao M, Takahashi M, Fujii M, Matsuoka M: Human T-cell leukemia virus type 1 bZIP factor selectively suppresses the classical pathway of NF-kappaB. Blood. 2009, 113: 2755-2764. 10.1182/blood-2008-06-161729.View ArticlePubMedGoogle Scholar
- Basbous J, Arpin C, Gaudray G, Piechaczyk M, Devaux C, Mesnard JM: The HBZ factor of human T-cell leukemia virus type I dimerizes with transcription factors JunB and c-Jun and modulates their transcriptional activity. J Biol Chem. 2003, 278: 43620-43627. 10.1074/jbc.M307275200.View ArticlePubMedGoogle Scholar
- Thebault S, Basbous J, Hivin P, Devaux C, Mesnard JM: HBZ interacts with JunD and stimulates its transcriptional activity. FEBS Lett. 2004, 562: 165-170. 10.1016/S0014-5793(04)00225-X.View ArticlePubMedGoogle Scholar
- Barbeau B, Mesnard JM: Does the HBZ gene represent a new potential target for the treatment of adult T-cell leukemia?. Int Rev Immunol. 2007, 26: 283-304. 10.1080/08830180701690843.View ArticlePubMedGoogle Scholar
- Clerc I, Hivin P, Rubbo PA, Lemasson I, Barbeau B, Mesnard JM: Propensity for HBZ-SP1 isoform of HTLV-I to inhibit c-Jun activity correlates with sequestration of c-Jun into nuclear bodies rather than inhibition of its DNA-binding activity. Virology. 2009, 391: 195-202. 10.1016/j.virol.2009.06.027.View ArticlePubMedGoogle Scholar
- Hivin P, Basbous J, Raymond F, Henaff D, Arpin-Andre C, Robert-Hebmann V, Barbeau B, Mesnard JM: The HBZ-SP1 isoform of human T-cell leukemia virus type I represses JunB activity by sequestration into nuclear bodies. Retrovirology. 2007, 4: 14-10.1186/1742-4690-4-14.PubMed CentralView ArticlePubMedGoogle Scholar
- Matsumoto J, Ohshima T, Isono O, Shimotohno K: HTLV-1 HBZ suppresses AP-1 activity by impairing both the DNA-binding ability and the stability of c-Jun protein. Oncogene. 2005, 24: 1001-1010. 10.1038/sj.onc.1208297.View ArticlePubMedGoogle Scholar
- Kuhlmann AS, Villaudy J, Gazzolo L, Castellazzi M, Mesnard JM, Duc Dodon M: HTLV-1 HBZ cooperates with JunD to enhance transcription of the human telomerase reverse transcriptase gene (hTERT). Retrovirology. 2007, 4: 92-10.1186/1742-4690-4-92.PubMed CentralView ArticlePubMedGoogle Scholar
- Douceron E, Kaidarova Z, Miyazato P, Matsuoka M, Murphy EL, Mahieux R: HTLV-2 APH-2 expression is correlated with proviral load but APH-2 does not promote lymphocytosis. J Infect Dis. 2012, 205: 82-86. 10.1093/infdis/jir708.PubMed CentralView ArticlePubMedGoogle Scholar
- Bertazzoni U, Turci M, Avesani F, Di Gennaro G, Bidoia C, Romanelli MG: Intracellular localization and cellular factors interaction of HTLV-1 and HTLV-2 Tax proteins: similarities and functional differences. Viruses. 2011, 3: 541-560. 10.3390/v3050541.PubMed CentralView ArticlePubMedGoogle Scholar
- Yin H, Kannian P, Dissinger N, Haines R, Niewiesk S, Green PL: HTLV-2 APH-2 is dispensable for in vitro immortalization, but functions to repress early viral replication in vivo. J Virol. 2012, 86: 8412-8421. 10.1128/JVI.00717-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, Karin M: Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987, 49: 729-739. 10.1016/0092-8674(87)90611-8.View ArticlePubMedGoogle Scholar
- Lee W, Mitchell P, Tjian R: Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements. Cell. 1987, 49: 741-752. 10.1016/0092-8674(87)90612-X.View ArticlePubMedGoogle Scholar
- Angel P, Karin M: Specific members of the Jun protein family regulate collagenase expression in response to various extracellular stimuli. Matrix Suppl. 1992, 1: 156-164.PubMedGoogle Scholar
- Westermarck J, Lohi J, Keski-Oja J, Kahari VM: Okadaic acid-elicited transcriptional activation of collagenase gene expression in HT-1080 fibrosarcoma cells is mediated by JunB. Cell Growth Differ. 1994, 5: 1205-1213.PubMedGoogle Scholar
- Higuchi M, Fujii M: Distinct functions of HTLV-1 Tax1 from HTLV-2 Tax2 contribute key roles to viral pathogenesis. Retrovirology. 2009, 6: 117-10.1186/1742-4690-6-117.PubMed CentralView ArticlePubMedGoogle Scholar
- Higuchi M, Tsubata C, Kondo R, Yoshida S, Takahashi M, Oie M, Tanaka Y, Mahieux R, Matsuoka M, Fujii M: Cooperation of NF-kappaB2/p100 activation and the PDZ domain binding motif signal in human T-cell leukemia virus type 1 (HTLV-1) Tax1 but not HTLV-2 Tax2 is crucial for interleukin-2-independent growth transformation of a T-cell line. J Virol. 2007, 81: 1900-11907.View ArticleGoogle Scholar
- Shoji T, Higuchi M, Kondo R, Takahashi M, Oie M, Tanaka Y, Aoyagi Y, Fujii M: Identification of a novel motif responsible for the distinctive transforming activity of human T-cell leukemia virus (HTLV) type 1 Tax1 protein from HTLV-2 Tax2. Retrovirology. 2009, 6: 83-10.1186/1742-4690-6-83.PubMed CentralView ArticlePubMedGoogle Scholar
- Furukawa Y, Kubota R, Tara M, Izumo S, Osame M: Existence of escape mutant in HTLV-I tax during the development of adult T-cell leukemia. Blood. 2001, 97: 987-993. 10.1182/blood.V97.4.987.View ArticlePubMedGoogle Scholar
- Takeda S, Maeda M, Morikawa S, Taniguchi Y, Yasunaga J, Nosaka K, Tanaka Y, Matsuoka M: Genetic and epigenetic inactivation of tax gene in adult T-cell leukemia cells. Int J Cancer. 2004, 109: 559-567. 10.1002/ijc.20007.View ArticlePubMedGoogle Scholar
- Matsuoka M, Green PL: The HBZ gene, a key player in HTLV-1 pathogenesis. Retrovirology. 2009, 6: 71-10.1186/1742-4690-6-71.PubMed CentralView ArticlePubMedGoogle Scholar
- Sheehy N, Lillis L, Watters K, Lewis M, Gautier V, Hall W: Functional analysis of human T lymphotropic virus type 2 Tax proteins. Retrovirology. 2006, 3: 20-10.1186/1742-4690-3-20.PubMed CentralView ArticlePubMedGoogle Scholar
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