Plasmid 74 (2014) 45–51

Contents lists available at ScienceDirect

Plasmid journal homepage: www.elsevier.com/locate/yplas

Construction of a reporter vector for analysis of bidirectional transcriptional activity of retrovirus LTR Charlotte Arpin-André a,b,1, Sylvain Laverdure a,b,c,1, Benoit Barbeau d, Antoine Gross a,b,⇑, Jean-Michel Mesnard a,b,⇑ a

Université Montpellier 1, Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé (CPBS), France CNRS, UM5236, CPBS, F-34293 Montpellier, France c East Carolina University, Department of Microbiology and Immunology, Greenville, NC 27834, USA d Université du Québec à Montréal, Département des sciences biologiques and Centre de recherche BioMed, Montréal, Québec H2X 3X8, Canada b

a r t i c l e

i n f o

Article history: Received 22 April 2014 Accepted 16 June 2014 Available online 24 June 2014 Communicated by Philipp Berger Keywords: HIV-1 HTLV-1 Bidirectional promoter Antisense transcription Tat Tax

a b s t r a c t To study the transcriptional activity of the HIV-1 LTR, we constructed a vector containing Renilla and Firefly luciferase genes under the control of the LTR (wild-type or mutated version) and oriented in a manner that allowed them to be transcribed in opposite directions. We found that the HIV-1 LTR acted as a bidirectional promoter, which activity was controlled by NF-jB- and Sp1-binding sites in both orientations. We next analyzed with this reporter vector the bidirectional promoter activity of the HTLV-1 LTR and showed that this LTR also possessed a bidirectional transcriptional activity. Interestingly, Sp1-binding elements were also involved in the control of HTLV-1 bidirectional transcription. Moreover, both retroviral trans-activators, Tat and Tax, could preferentially activate sense transcription with no or limited effect on the extent of antisense transcription. We also cloned into this plasmid the MLV LTR and found that the LTR of a simple retrovirus also possessed bidirectional transcriptional activity. This reporter vector represents a powerful tool to analyze the bidirectional transcriptional activity of retrovirus LTRs. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Retroviruses utilize host cell transcriptional machinery to express their own genetic material integrated into the host genome. Although the 50 long terminal repeat (LTR) is known to control expression of viral genes necessary for the production of infectious viral particles, certain retroviruses are also characterized by the presence of a 30 LTR capable of generating antisense transcripts, oriented in the

⇑ Corresponding authors at: CNRS, UMR5236, Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé (CPBS), 1919 Route de Mende, 34293 Montpellier Cedex 5, France. E-mail addresses: [email protected] (A. Gross), jean-michel. [email protected] (J.-M. Mesnard). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.plasmid.2014.06.001 0147-619X/Ó 2014 Elsevier Inc. All rights reserved.

inverse direction from the transcription controlled by the 50 LTR (Barbeau and Mesnard, 2011). The human T-cell leukemia virus type 1 (HTLV-1) has been the first retrovirus from which the existence of an antisense transcript has been clearly demonstrated (Cavanagh et al., 2006; Larocca et al., 1989; Li and Green, 2007; Murata et al., 2006; Usui et al., 2008). A similar antisense transcript is also produced by viruses belonging to the HTLV family, including HTLV-2, -3, and -4 (Halin et al., 2009; Larocque et al., 2011). These antisense transcripts encode proteins (Gaudray et al., 2002; Halin et al., 2009; Larocque et al., 2011; Suemori et al., 2009), which have the potential to down-regulate sense viral transcription from the 50 LTR (Clerc et al., 2008; Gaudray et al., 2002; Halin et al., 2009; Hivin et al., 2006; Larocque et al., 2011; Lemasson et al., 2007).

46

C. Arpin-André et al. / Plasmid 74 (2014) 45–51

The existence of antisense transcription in HIV-1 has also been demonstrated in infected cell lines and cells derived from HIV-1-infected patients (Bukrinsky et al., 1990; Landry et al., 2007; Michael et al., 1994; VanheeBrossollet et al., 1995). By RACE analyses, we have identified several transcription initiation sites near the 50 border of the 30 LTR (Landry et al., 2007). This multiplicity of initiation sites has also been described for HTLV-1 and might be a consequence of the absence of a TATA box at close distance (Carninci et al., 2006; Cavanagh et al., 2006; Landry et al., 2007). Like HTLVs, HIV-1 antisense transcript is involved in the synthesis of a viral antisense protein (Briquet and Vaquero, 2002; Clerc et al., 2011; Laverdure et al., 2012; Torresilla et al., 2013). A certain number of reports have focussed on the regulation of the expression of this gene and have thereby aimed to characterize the promoter region of the HIV-1 antisense transcript. Experiments on deletion and point mutants further revealed the importance of NF-jB-binding sites for the regulation of antisense promoter (Bentley et al., 2004; KobayashiIshihara et al., 2012; Ludwig et al., 2006; Michael et al., 1994). Other experiments have concluded that an unexplored Sp1-binding site was important for antisense transcription (Peeters et al., 1996). Interestingly, Sp1-binding sites have also been described to be involved in the control of HTLV-1 antisense transcription (Gazon et al., 2012; Yoshida et al., 2008). Antisense transcription has also been detected in feline immunodeficiency virus (Briquet et al., 2001) and murine leukemia virus-infected cells (Rasmussen et al., 2010). In the latter case, antisense transcripts initiated on the negative strand in the U3 region of the proviral 50 LTR. As both LTRs of a retroviral provirus are identical, altogether these data thereby suggest that retroviral LTRs can act as bidirectional promoters. Indeed, different data have shown that transcriptional regulatory regions in eukaryotes have the capacity to simultaneously initiate transcription in opposite directions. Thus, a substantial fraction of human genes are controlled by bidirectional promoters, which are defined as DNA regions flanked by the transcription start sites of two genes in opposite directions (Adachi and Lieber, 2002; Trinklein et al., 2004). Therefore, we have constructed bidirectional expression vectors, containing two different reporter genes (encoding Renilla and Firefly luciferases) under the control of the LTR sequence and oriented in opposite directions. By this approach, we demonstrate for the first time that HIV-1 and HTLV-1 LTRs possess bidirectional transcription activities both being controlled by Sp1-binding elements and that the viral trans-activators Tat and Tax are key-players for the controlled balance between sense and antisense transcription. We also show that the LTR of the simple retrovirus MLV possesses bidirectional transcriptional activity.

2. Materials and methods 2.1. Plasmids The HIV-1 bidirectional reporter vector, pAsLuc(Fire)HIV-Luc(Reni), was constructed by PCR amplifying a region

containing an antisense Firefly luciferase gene under the control of the 30 LTR from pNL4.3AsLucE- (Laverdure et al., 2012) and by cloning this sequence into the XmaI/ NdeI-digested pRL-null vector (Promega). The HTLV-1 reporter vector was constructed by replacing the HIV-1 LTR of pAsLuc(Fire)-HIV-Luc(Reni) by the HTLV-1 LTR, which was PCR-amplified from previously described K3030 asLuc (containing the wt LTR) or K30-30 asLucDvCRE (containing an LTR mutated in all three vCREs) (Landry et al., 2009). pAsLuc(Fire)-MLV-Luc(Reni) was constructed by replacing the HIV-1 LTR by the MLV LTR. Site-directed mutagenesis was performed as already described (Clerc et al., 2009) and the constructs were sequenced to ensure that no unintended mutations were introduced during PCR amplification. The Sp1-, Tat- and Tax-expression vectors have already been published (Basbous et al., 2003; Gazon et al., 2012; Laverdure et al., 2012). 2.2. Cotransfections and luciferase assays CEM and NIH-3T3 cells were transiently cotransfected according to the previously published protocol (Clerc et al., 2009). NIH-3T3 and CEM cells were transfected with 0.5 lg or 2 lg of bidirectional expression vector and 1 or 5 lg of b-galactosidase-containing reference vector, respectively. The total amount of DNA in each transfection was kept constant through the addition of appropriate quantities of empty plasmids. Luciferase activity was measured in a centro XS3 LB 960 microplate luminometer (Berthold Technologies) and normalized by b-galactosidase activity. Negative controls correspond to background signals obtained from cells transfected without bidirectional expression vectors. The Student’s t-test (two tailed) was used to assess significance of differences between two groups and P values were considered significant when ⁄ P < 0.05 and highly significant when ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001. 3. Results 3.1. pAsLuc(Fire)-HIV-Luc(Reni) reporter vector We generated a bidirectional expression vector, pAsLuc(Fire)-HIV-Luc(Reni), which contained two different reporter genes (encoding Renilla and Firefly luciferases) under the control of the HIV-1 LTR and oriented in opposite directions (Fig. 1). A region containing an antisense Firefly luciferase gene under the control of the HIV-1 30 LTR was PCR amplified from pNL4.3AsLucE- (Laverdure et al., 2012) and ligated into the pRL-null vector so that the gene would be transcribed in the opposite direction of the Renilla reporter gene. To avoid any interference from antisense transcription on Renilla luciferase expression (Landry et al., 2007), the SV40 late polyadenylation signal was cloned downstream of the Firefly luciferase gene. Because the transcriptional activity of an integrated proviral LTR can be influenced by transcriptional interference due to the nearby presence of either chromatin remodeling factors or active host transcriptional start sites (Han et al., 2008; Melamed et al., 2013), HIV-1 LTR activity was analyzed after transient transfection. CEM cells were transfected with constructs

C. Arpin-André et al. / Plasmid 74 (2014) 45–51

47

Fig. 1. Analysis of bidirectional transcription activity of the HIV-1 LTR in transfected CEM cells. (A) Schematic representation of the U3, R, and U5 regions of the HIV-1 LTR. Sp1- and NF-jB-binding sites are shown. The solid and dotted arrows show sense- and antisense-transcription initiation sites, respectively. Although antisense transcription initiates at multiple positions, only the proximal initiation site is shown. (B) Antisense and sense transcriptional activities of the HIV-1 LTR. Two days after transfection of CEM cells with wt or mutated pAsLuc(Fire)-HIV-Luc(Reni), cells were lysed and luciferase activities were analyzed. Results show mean luciferase activity values of three independent samples ± S.D. Results of Student’s t-test are indicated on the graph.

containing either the wild type (wt) or mutant versions of the HIV-1 LTR. As shown in Fig. 1, the wt HIV-1 LTR effectively possesses bidirectional promoter activity. We next examined the contribution of the NF-jB and Sp1 sites (see Fig. 1A), known to be involved in the regulation of HIV-1 transcription from the 50 LTR (Burnett et al., 2009). Both sense and antisense promoter activities respectively dropped 7- and 22-fold following mutations of the two NF-jB-binding sites (Fig. 1B). Likewise, up to 14- and 6-fold less sense and antisense activities were measured respectively when all the three Sp1 sites (Sp1A, B, and C, Fig. 1A) were mutated. As a fourth Sp1 site (Sp1D, Fig. 1A) has been described to abolish HIV-1 antisense transcription (Peeters et al., 1996), we equally mutated this site in our bidirectional promoter, which showed a more modest 2-fold decrease in antisense promoter activity (Fig. 1B). In conclusion, our results show that the HIV-1 LTR functions bidirectionally and that the pAsLuc(Fire)-HIV-Luc(Reni) plasmid is a functional vector to study its bidirectional transcriptional activity. We also demonstrate that the NF-jB and Sp1 sites known to control HIV-1 sense transcription are importantly involved in the regulation of antisense transcription as well. 3.2. Analysis of the bidirectional activity of the HTLV-1 LTR To confirm the efficiency of the reporter plasmid, we next analyzed the promoter activity of an LTR from another complex retrovirus, HTLV-1. The HTLV-1 LTR contains three key enhancer elements, referred to as viral cyclic AMP-responsive elements (vCRE) (Fig. 2A). New reporter vectors were constructed by replacing the HIV-1 LTR of pAsLuc(Fire)-HIV-Luc(Reni) by the HTLV-1 LTR mutated or not in all three vCREs. The bidirectional promoter activity of these vectors was analyzed in transfected CEM cells.

Significant antisense promoter activity was measured from this construct (Fig. 2B) while sense activity was low. Three Sp1 sites, here termed A, B, and C (Fig. 3A), have previously been identified upstream of the antisense transcription initiation sites (Yoshida et al., 2008). Although the mutation of the first two Sp1 sites did not significantly impair antisense promoter activity, the third one was described to be critical for antisense transcription (Gazon et al., 2012; Yoshida et al., 2008). We therefore constructed mutants targeting these Sp1-binding sites and analyzed bidirectional promoter activity from resulting reporter vectors. As shown in Fig. 3B, upon transfection in CEM cells, a decrease in antisense promoter activity was observed for all mutants (DSp1AB, DSp1C, and DSp1ABC), demonstrating that the three sites were important for antisense activity. On the other hand, no significant effect was observed on sense promoter activity. To further examine the role of Sp1 in HTLV-1 bidirectional transcription, an Sp1expression vector was cotransfected with pAsLuc(Fire)HTLV-Luc(Reni) in CEM cells. Fig. 3C shows that Sp1 induced antisense and sense transcription up to 4-fold and 5.5-fold, respectively. These results suggested that Sp1 was not only involved in antisense transcription but also in sense transcription. A fourth Sp1 site (Sp1D, Fig. 3A) has been described to be the major cis-acting element for Sp1-induced sense transcription (Livengood and Nyborg, 2004). To evaluate its role in HTLV-1 bidirectional transcription, this specific site was inactivated, as previously described (Livengood and Nyborg, 2004). When the activity of this mutant was compared with the wt reporter vector, a low but significant decrease (1.4-fold) was detected for both sense and antisense promoter activities (Fig. 3D). Taken together, our results show that our reporter vector is an efficient tool to study bidirectional transcriptional activity of complex retrovirus LTRs.

48

C. Arpin-André et al. / Plasmid 74 (2014) 45–51

Fig. 2. Analysis of bidirectional transcription activity of the HTLV-1 LTR. (A) Schematic representation of the U3, R, and U5 regions of the HTLV-1 LTR. The three vCREs are also indicated. The solid and dotted arrows respectively show sense- and antisense-transcription initiation sites (only the proximal initiation site is shown for antisense transcription). (B) Antisense and sense transcriptional activities of the HTLV-1 LTR. Transfected CEM cells were lysed and luciferase activities were measured. Results show mean luciferase activity values of three independent samples ± S.D. Results of Student’s t-test are indicated on the graph.

3.3. The retroviral trans-activators preferentially activate sense transcription We next compared both human retrovirus LTRs to the bidirectional promoter activity of a simple retrovirus, the Moloney Murine Leukemia Virus (MLV). After cloning of its LTR into our vector, this construct termed pAsLuc(Fire)-MLV-Luc(Reni) was transfected in NIH-3T3 cells. As shown in Fig. 4A, sense transcription activity was 68fold higher for MLV than for HIV-1 while both viruses presented equivalent antisense promoter activity. This result showed that the MLV LTR basal transcriptional activity is strongly oriented in the sense direction. Interestingly, MLV encodes no transcriptional trans-activator. It was therefore interesting to determine whether HIV-1 and HTLV-1 trans-activators Tat and Tax were able to promote sense activity to the detriment of antisense transcription. In CEM cells transfected with pAsLuc(Fire)-HIV-Luc(Reni), sense activity was increased 24-fold in the presence of a Tat-expressing plasmid, while in contrast Tat led to a weak reduction (0.6-fold) of antisense transcription (Fig. 4B). On the other hand, Tax was able to trans-activate both sense and antisense transcriptions in cells transfected with pAsLuc(Fire)-HTLV-Luc(Reni) (Fig. 4B). However, while Tax enhanced sense transcription activity by 130-fold, the antisense transcription stimulation was ten times lower. In conclusion, both viral trans-activators Tat and Tax preferentially activated sense transcription with no or limited effect on the extent of antisense transcription.

4. Discussion A substantial fraction of human genes occurs as divergently oriented pairs being controlled by a shared region

with bidirectional promoter activity (Adachi and Lieber, 2002; Trinklein et al., 2004). In this paper, we show for the first time that a vector containing Renilla and Firefly luciferase genes under the control of the HIV-1 or HTLV-1 LTR is capable of expressing genes in a opposite directions from a bidirectional promoter activity. We also demonstrate that Sp1-binding elements are critical for bidirectional promoter activity of both LTRs. Sp1 is an ubiquitously expressed transcription factor that binds to GC-rich boxes and recruits the transcription factor IID (TFIID) to stimulate transcription initiation (Wierstra, 2008). Efficient recruitment of TFIID provides the ability of Sp1 to induce the transcription of TATA-less genes. It is noteworthy that HIV-1 and HTLV-1 antisense transcripts initiate in the 30 LTR at multiple positions, precisely due to the absence of TATA boxes at a close distance (Cavanagh et al., 2006; Landry et al., 2007). Interestingly, most cellular bidirectional promoters are TATA less and are GC rich (Trinklein et al., 2004). Our results also show the involvement of the two NF-jB-binding sites in the regulation of HIV-1 bidirectional promoter activity in CEM cells. The involvement of NF-jB in the regulation of antisense transcription activity has previously been described by others in different T-cell lines including SupT1 and Molt-4 cells (Kobayashi-Ishihara et al., 2012; Michael et al., 1994). We also used these vectors to study the effects of the viral trans-activators, Tat and Tax, on the LTR bidirectional transcription activity. Controversial data have been published so far on the effects of both Tat (KobayashiIshihara et al., 2012; Landry et al., 2007; Laverdure et al., 2012; Michael et al., 1994; Peeters et al., 1996) and Tax (Belrose et al., 2011; Landry et al., 2009; Larocca et al., 1989; Yoshida et al., 2008) on the antisense transcription activity. We found that Tax stimulated both sense and antisense transcriptional activities from the HTLV-1 LTR.

C. Arpin-André et al. / Plasmid 74 (2014) 45–51

49

Fig. 3. Sp1 sites are involved in the regulation of HTLV-1 antisense transcription. (A) Schematic representation showing the different Sp1-binding sites within the HTLV-1 LTR. (B) Analysis of the Sp1-binding sites A, B, and C. CEM cells transfected with wt or mutated pAsLuc(Fire)-HTLV-Luc(Reni) were lysed and luciferase activities were analyzed. (C) Sp1 activates bidirectional HTLV-1 promoter activity. Different amounts of an Sp1-expression vector (1 lg and 5 lg) were cotransfected into CEM cells in the presence of wt pAsLuc(Fire)-HTLV-Luc(Reni). (D) Mutational analysis of the Sp1-binding site D. Cells were transfected with wt or mutated vectors. Results show mean luciferase activity values of three independent samples ± S.D. Results of Student’s t-test are indicated on the graph.

However, our results also show that Tax preferentially promotes sense than antisense transcription. For the HIV-1 LTR, we observed that Tat stimulated sense transcription but had no effect on antisense transcription. This observation confirms our previous result obtained in the context of a full-length Tat-deficient proviral clone (Laverdure et al., 2012). Such a result can be explained by the fact that Tat regulates the process of elongation rather than initiation of transcription by binding to the trans-activation responsive (TAR) element; this LTR R region-derived RNA structure is not formed in HIV-1 antisense transcripts. In this study, bidirectional transcription activity was analyzed on an isolated LTR but the in vivo situation is more

complex since the proviral DNA contains two LTRs able to initiate transcription in both directions. We have previously shown that the presence of the 50 LTR has a negative effect on the antisense transcriptional activity of the 30 LTR (Cavanagh et al., 2006; Landry et al., 2007). Transcriptional interference can explain this negative effect, resulting from the expression of a strong promoter reducing the expression of a weak promoter. It has been clearly demonstrated that collisions between converging elongation complexes lead to the premature termination of the transcriptional progress of one or both complexes (Prescott and Proudfoot, 2002). As Tat and Tax preferentially promote sense transcription, retroviral trans-activator synthesis

50

C. Arpin-André et al. / Plasmid 74 (2014) 45–51

Fig. 4. Complex retroviral trans-activators promote sense transcription. (A) Bidirectional promoter activity of MLV LTR in transfected NIH-3T3 cells. After transfection with pAsLuc(Fire)-MLV-Luc(Reni), pAsLuc(Fire)-HIV-Luc(Reni), or pAsLuc(Fire)-HTLV-Luc(Reni), lysed NIH-3T3 cells were analyzed for luciferase activity. Results show mean luciferase activity values of three independent samples ± S.D. Results of Student’s t-test are indicated on the graph. (B) Tat and Tax preferentially activate sense transcription. CEM cells were cotransfected with the dual luciferase-expressing vector containing either the HIV-1 (left) or the HTLV-1 LTR (right), in the absence or presence of expression vectors for Tat (100 ng of pcDNA-Tat-Flag) or Tax (500 ng of pSG-Tax). Lysed cells were analyzed for luciferase activity. Displayed values correspond to fold stimulations induced by Tat or Tax on Renilla (sense) or Firefly (antisense) activities. Values represent the mean ± S.D. (n = 3). Results of Student’s t-test are also indicated on the graph.

could be an essential step in the regulation of the balance between sense and antisense transcription. We indeed observed that induction of antisense transcript production from the 30 LTR corresponded to an inhibition of Tax expression in HTLV-1-infected cells (Belrose et al., 2011). Furthermore, efficient antisense transcription in HIV-1-infected dendritic cells has been associated with an absence of Gag synthesis (Laverdure et al., 2012), confirming the inverse correlation between production of antisense transcripts from the 30 LTR and Tat-induced stimulation of sense transcription from the 50 LTR. Altogether these observations suggest that retroviral antisense transcription and antisense protein synthesis should be active in cells in which sense transcription from the 50 LTR is down-regulated. This is indeed what is observed in adult T-cell leukemia lymphocytes. Further analyses are warranted to better understand the regulation of LTR bidirectional transcription and how this might act upon establishment of retrovirus infection and consequently pathology development. Acknowledgments This work was supported by institutional grants from the Centre National de la Recherche Scientifique (CNRS) and the Université Montpellier 1 (UM 1), a grant to

J.M.M. from the Agence Nationale de Recherches sur le Sida et les hépatites virales (ANRS). B.B. holds a Canada Research Chair in Human Retrovirology (Tier 2). S.L. was supported by a fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche. We thank Marylène Mougel for providing the MLV LTR.

References Adachi, N., Lieber, M.R., 2002. Bidirectional gene organization: a common architectural feature of the human genome. Cell 109 (7), 807–809. Barbeau, B., Mesnard, J.M., 2011. Making sense out of antisense transcription in human T-cell lymphotropic viruses (HTLVs). Viruses 3, 456–468. Basbous, J., Bazarbachi, A., Granier, C., Devaux, C., Mesnard, J.M., 2003. The central region of human T-cell leukemia virus type 1 Tax protein contains distinct domains involved in subunit dimerization. J. Virol. 77, 13028–13035. Belrose, G., Gross, A., Olindo, S., Lezin, A., Dueymes, M., Komla-Soukha, I., Smadja, D., Tanaka, Y., Willems, L., Mesnard, J.-M., Peloponese, J.-M., Cesaire, R., 2011. Opposite effects of valproate on Tax and HBZ expressions in T-lymphocytes from HTLV-1 asymptomatic carriers and HAM/TSP patients. Blood 118, 2483–2491. Bentley, K., Deacon, N., Sonza, S., Zeichner, S., Churchill, M., 2004. Mutational analysis of the HIV-1 LTR as a promoter of negative sense transcription. Arch. Virol. 149, 2277–2294. Briquet, S., Richardson, J., Vanhee-Brossollet, C., Vaquero, C., 2001. Natural antisense transcripts are detected in different cell lines and tissues of cats infected with feline immunodeficiency virus. Gene 267, 157–164.

C. Arpin-André et al. / Plasmid 74 (2014) 45–51 Briquet, S., Vaquero, C., 2002. Immunolocalization studies of an antisense protein in HIV-1-infected cells and viral particles. Virology 292, 177– 184. Bukrinsky, M.I., Etkin, A.I., Ivanovsky, D.I., 1990. Plus strand of the HIV provirus DNA is expressed at early stages of infection. AIDS Res. Hum. Retroviruses 6, 425–426. Burnett, J.C., Miller-Jensen, K., Shah, P.S., Arkin, A.P., Schaffer, D.V., 2009. Control of stochastic gene expression by host factors at the HIV promoter. PLoS Pathog. 5 (1), e1000260. Carninci, P., Sandelin, A., Lenhard, B., Katayama, S., Shimokawa, K., Ponjavic, J., Semple, C.A., Taylor, M.S., Engstrom, P.G., Frith, M.C., Forrest, A.R., Alkema, W.B., Tan, S.L., Plessy, C., Kodzius, R., Ravasi, T., Kasukawa, T., Fukuda, S., Kanamori-Katayama, M., Kitazume, Y., Kawaji, H., Kai, C., Nakamura, M., Konno, H., Nakano, K., MottaguiTabar, S., Arner, P., Chesi, A., Gustincich, S., Persichetti, F., Suzuki, H., Grimmond, S.M., Wells, C.A., Orlando, V., Wahlestedt, C., Liu, E.T., Harbers, M., Kawai, J., Bajic, V.B., Hume, D.A., Hayashizaki, Y., 2006. Genome-wide analysis of mammalian promoter architecture and evolution. Nat. Genet. 38, 626–635. Cavanagh, M.-H., Landry, S., Audet, B., Arpin-Andre, C., Hivin, P., Paré, M.E., Thete, J., Wattel, E., Marriott, S.J., Mesnard, J.-M., Barbeau, B., 2006. HTLV-I antisense transcripts initiating in the 30 LTR are alternatively spliced and polyadenylated. Retrovirology 3, 15. Clerc, I., Hivin, P., Rubbo, P.A., Lemasson, I., Barbeau, B., Mesnard, J.-M., 2009. 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 391, 195– 202. Clerc, I., Laverdure, S., Torresilla, C., Landry, S., Borel, S., Vargas, A., ArpinAndre, C., Gay, B., Briant, L., Gross, A., Barbeau, B., Mesnard, J.M., 2011. Polarized expression of the membrane ASP protein derived from HIV1 antisense transcription in T cells. Retrovirology 8, 74. Clerc, I., Polakowski, N., Andre-Arpin, C., Cook, P., Barbeau, B., Mesnard, J.M., Lemasson, I., 2008. An interaction between the human T cell leukemia virus type 1 basic leucine zipper factor (HBZ) and the KIX domain of p300/CBP contributes to the down-regulation of taxdependent viral transcription by HBZ. J. Biol. Chem. 283, 23903– 23913. Gaudray, G., Gachon, F., Basbous, J., Biard-Piechaczyk, M., Devaux, C., Mesnard, J.M., 2002. The complementary strand of HTLV-1 RNA genome encodes a bZIP transcription factor that down-regulates the viral transcription. J. Virol. 76, 12813–12822. Gazon, H., Lemasson, I., Polakowski, N., Cesaire, R., Matsuoka, M., Barbeau, B., Mesnard, J.M., Peloponese Jr., J.M., 2012. Human T-cell leukemia virus type 1 (HTLV-1) bZIP factor requires cellular transcription factor JunD to upregulate HTLV-1 antisense transcription from the 3’ long terminal repeat. J. Virol. 86, 9070–9078. Halin, M., Douceron, E., Clerc, I., Journo, C., Ko, N.L., Landry, S., Murphy, E.L., Gessain, A., Lemasson, I., Mesnard, J.M., Barbeau, B., Mahieux, R., 2009. Human T-cell leukemia virus type 2 produces a spliced antisense transcript encoding a protein that lacks a classical bZIP domain but still inhibits Tax2-mediated transcription. Blood 114, 2427–2437. Han, Y., Lin, Y.B., An, W., Xu, J., Yang, H.C., O’Connell, K., Dordai, D., Boeke, J.D., Siliciano, J.D., Siliciano, R.F., 2008. Orientation-dependent regulation of integrated HIV-1 expression by host gene transcriptional readthrough. Cell Host Microbe 4 (2), 134–146. Hivin, P., Arpin-André, C., Clerc, I., Barbeau, B., Mesnard, J.M., 2006. A modified version of a Fos-associated cluster in HBZ affects Jun transcriptional potency. Nucleic Acids Res. 34, 2761–2772. Kobayashi-Ishihara, M., Yamagishi, M., Hara, T., Matsuda, Y., Takahashi, R., Miyake, A., Nakano, K., Yamochi, T., Ishida, T., Watanabe, T., 2012. HIV-1-encoded antisense RNA suppresses viral replication for a prolonged period. Retrovirology 9 (1), 38. Landry, S., Halin, M., Lefort, S., Audet, B., Vaquero, C., Mesnard, J.M., Barbeau, B., 2007. Detection, characterization and regulation of antisense transcripts in HIV-1. Retrovirology 4, 71. Landry, S., Halin, M., Vargas, A., Lemasson, I., Mesnard, J.M., Barbeau, B., 2009. Upregulation of human T-cell leukemia virus type 1 antisense transcription by the viral tax protein. J. Virol. 83, 2048–2054. Larocca, D., Chao, L.A., Seto, M.H., Brunck, T.K., 1989. Human T-cell leukemia virus minus strand transcription in infected T-cells. Biochem. Biophys. Res. Commun. 163, 1006–1013.

51

Larocque, E., Halin, M., Landry, S., Marriott, S., Switzer, W.M., Barbeau, B., 2011. HTLV-3- and HTLV-4-derived antisense transcripts encode proteins with similar Tax-inhibiting function but distinct subcellular localization. J. Virol. 85, 12673–12684. Laverdure, S., Gross, A., Arpin-Andre, C., Clerc, I., Beaumelle, B., Barbeau, B., Mesnard, J.M., 2012. HIV-1 antisense transcription is preferentially activated in primary monocyte-derived cells. J. Virol. 86, 13785– 13789. Lemasson, I., Lewis, M.R., Polakowski, N., Hivin, P., Cavanagh, M.H., Thebault, S., Barbeau, B., Nyborg, J.K., Mesnard, J.M., 2007. Human Tcell leukemia virus type 1 (HTLV-1) bZIP protein interacts with the cellular transcription factor CREB to inhibit HTLV-1 transcription. J. Virol. 81, 1543–1553. Li, M., Green, P.L., 2007. Detection and quantitation of HTLV-1 and HTLV-2 mRNA species by real-time RT-PCR. J. Virol. Methods 142, 159–168. Livengood, J.A., Nyborg, J.K., 2004. The high-affinity Sp1 binding site in the HTLV-1 promoter contributes to Tax-independent basal expression. Nucleic Acids Res. 32 (9), 2829–2837. Ludwig, L.B., Ambrus, J.L., Krawczyk, K.A., Sharma, S., Brooks, S., Hsiao, C.B., Schwartz, S.A., 2006. Human immunodeficiency virus-type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products. Retrovirology 3, 80. Melamed, A., Laydon, D.J., Gillet, N.A., Tanaka, Y., Taylor, G.P., Bangham, C.R., 2013. Genome-wide determinants of proviral targeting, clonal abundance and expression in natural HTLV-1 infection. PLoS Pathog. 9 (3), e1003271. Michael, N.L., Vahey, M.T., d’Arcy, L., Ehrenberg, P.K., Mosca, J.D., Rappaport, J., Redfield, R.R., 1994. Negative-strand RNA transcripts are produced in human immunodeficiency virus type 1-infected cells and patients by a novel promoter downregulated by Tat. J. Virol. 68, 979–987. Murata, K., Hayashibara, T., Sugahara, K., Uemura, A., Yamaguchi, T., Harasawa, H., Hasegawa, H., Tsuruda, K., Okazaki, T., Koji, T., Miyanishi, T., Yamada, Y., Kamihira, S., 2006. A novel alternative splicing isoform of human T-cell leukemia virus type 1 bZIP factor (HBZ-SI) targets distinct subnuclear localization. J. Virol. 80, 2495– 2505. Peeters, A., Lambert, P.F., Deacon, N.J., 1996. A fourth Sp1 site in the human immunodeficiency virus type 1 long terminal repeat is essential for negative-sense transcription. J. Virol. 70, 6665–6672. Prescott, E.M., Proudfoot, N.J., 2002. Transcriptional collision between convergent genes in budding yeast. Proc. Natl. Acad. Sci. U.S.A. 99 (13), 8796–8801. Rasmussen, M.H., Ballarin-Gonzalez, B., Liu, J., Lassen, L.B., Fuchtbauer, A., Fuchtbauer, E.M., Nielsen, A.L., Pedersen, F.S., 2010. Antisense transcription in gammaretroviruses as a mechanism of insertional activation of host genes. J. Virol. 84, 3780–3788. Suemori, K., Fujiwara, H., Ochi, T., Ogawa, T., Matsuoka, M., Matsumoto, T., Mesnard, J.M., Yasukawa, M., 2009. HBZ is an immunogenic protein, but not a target antigen for human T-cell leukemia virus type 1specific cytotoxic T lymphocytes. J. Gen. Virol. 90, 1806–1811. Torresilla, C., Larocque, E., Landry, S., Halin, M., Coulombe, Y., Masson, J.Y., Mesnard, J.M., Barbeau, B., 2013. Detection of the HIV-1 minusstrand-encoded antisense protein and its association with autophagy. J. Virol. 87 (9), 5089–5105. Trinklein, N.D., Aldred, S.F., Hartman, S.J., Schroeder, D.I., Otillar, R.P., Myers, R.M., 2004. An abundance of bidirectional promoters in the human genome. Genome Res. 14 (1), 62–66. Usui, T., Yanagihara, K., Tsukasaki, K., Murata, K., Hasegawa, H., Yamada, Y., Kamihira, S., 2008. Characteristic expression of HTLV-1 basic zipper factor (HBZ) transcripts in HTLV-1 provirus-positive cells. Retrovirology 5, 34. Vanhee-Brossollet, C., Thoreau, H., Serpente, N., D’Auriol, L., Levy, J.P., Vaquero, C., 1995. A natural antisense RNA derived from the HIV-1 env gene encodes a protein which is recognized by circulating antibodies of HIV+ individuals. Virology 206, 196–202. Wierstra, I., 2008. Sp1: emerging roles–beyond constitutive activation of TATA-less housekeeping genes. Biochem. Biophys. Res. Commun. 372 (1), 1–13. Yoshida, M., Satou, Y., Yasunaga, J., Fujisawa, J.-I., Matsuoka, M., 2008. Transcriptional control of spliced and unspliced HTLV-1 bZIP factor gene. J. Virol. 82, 9359–9368.