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Transcription factor AP1 modulates the internal promoter activity of bovine foamy virus
Virus Research 147 (2010) 139–144
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Transcription factor AP1 modulates the internal promoter activity of bovine foamy virus Wu YaFeng, Tan Juan, Su Yang, Qiao WenTao, Geng YunQi, Chen QiMin ∗ College of Lifesciences, Nankai University, Tianjin 300071, PR China
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Article history: Received 26 June 2009 Received in revised form 6 October 2009 Accepted 9 October 2009 Available online 22 October 2009 Keywords: Bovine foamy virus (BFV) AP1 TPA
a b s t r a c t Foamy virus contains two promoters, which are the canonical long terminal repeat (LTR) promoter and the internal promoter (IP). FV gene expression was considered to initiate at the internal promoter. However, little was known about how basal transcription of IP was triggered by the host cellular factors. Previous studies found some cellular proteins could affect HFV viral replication, but it was no known whether the AP1 signal pathway was involved in the activation of viral replication or not. In this study, we reported that treatment with TPA or AP1 increased basal transcription of IP and did not affect basal transcription of the promoter in the LTR. In addition, the c-Jun mutant blocked the IP activity stimulated by TPA. Two AP1 binding sites located in BFV-IP promoter were found by bioinformatics and mutants of two AP1 binding sites decreased luciferase reporter activity of IP activated by AP1. EMSA assay showed that two AP1 binding sites could bind to c-Jun/c-Fos heterodimeric. We also found TPA and AP1 enhanced BFV3026 replication. Taken together, these data suggested that AP1 was a positive regulator of BFV internal promoter. © 2009 Elsevier B.V. All rights reserved.
Foamy viruses (FV) also called Spumaviruses are complex retroviruses and establish lifelong persistent infections without any accompanying pathologies (Enders and Peebles, 1954; Achong et al., 1971). These viruses infect many mammals including humans (Herchenroder et al., 1994), monkeys (Neumann-Haefelin et al., 1993), horses (Tobaly-Tapiero et al., 2000), cats (Lutz, 1990; Mochizuki et al., 1990), and cattle (Johnson et al., 1983). Regulation of FV replication is controlled by two promoters: long terminal repeats (LTR) (Maurer et al., 1988; Schmidt et al., 1997) and internal promoter (IP) (Lochelt et al., 1993; Campbell et al., 1994; Mergia, 1994; Winkler et al., 1997) within the env gene. The genomes of foamy viruses direct viral transcription through both promoters in the LTR and IP. The foamy viruses display different regulatory elements as compared with other retroviruses, thereby there is an intriguing possibility that the mechanisms involved in foamy virus gene expression may be unique and complex. The FV genome encodes Tas, a transactivator protein, required for transcription from both the LTR promoter and the internal promoter IP (Lochelt et al., 1991; Baunach et al., 1993; Yu and Linial, 1993). Tas is not found in viral particles and foamy virus has the transcriptional capacity from an internal promoter, indicating the activation of IP depends
∗ Corresponding author at: College of Lifesciences, Room 414, 94 WeiJin Road, Nankai District, Tianjin 300071, PR China. Tel.: +86 22 23501783; fax: +86 22 23501783. E-mail address: [email protected] (Q. Chen). 0168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2009.10.005
on the host cellular factors when FV Tas has not been expressed at the earliest stage. The precise mechanism of IP transcription at the earliest stage where the potential cellular factors involved remains to be elucidated. Heretofore, little is known about how basal transcription of bovine foamy virus (BFV) IP was triggered by the host cellular factors (Renshaw et al., 1991; Renshaw and Casey, 1994; Wang et al., 2000). In this study, we demonstrated that AP1 was one of the potential cellular factors to regulate BFV-IP activity. The AP-1 transcriptional factor is mainly composed of Jun/Fos protein dimmers (Angel et al., 1987; Lee et al., 1987), and highly conserved between the human and bovine. AP-1 regulates the activity of a variety of cellular and viral genes (Chiu et al., 1988; Lucibello et al., 1988; Sassone-Corsi et al., 1988; Schonthal et al., 1988; Maurer et al., 1991). AP-1 directs an activity that controls both basal and inducible transcription of several genes containing AP-1 sites (consensus sequence 5 -TGAG/CTCA-3 ), which are also known as TPA-responsive elements (TREs) (Angel and Karin, 1991). Previously, it was reported that the induction of persistently infected cells with TPA greatly enhanced HFV replication, but it was not determined whether the AP1 signal pathway was involved in the activation of viral replication (Meiering and Claudia Rubio, 2001; Meiering et al., 2002). Based on these observations, we wanted to know whether the TPA-induced AP1 pathway exerted an effect on BFV reactivation by activating viral gene expression. To directly examine whether TPA stimulation affected the activity of IP and LTR promoter, luciferase reporter assay was used. The pGL3-Luc, pGL3-6 × AP1-Luc (positive control including
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Fig. 1. AP1 enhanced BFV-IP transcriptional activation. (A) Schematic representation of plasmids used in the Luciferase reporter assay. Shaded box: 6 × AP1; black box: BFV-IP; Gray box: BFV-LTR. (B) TPA was able to activate BFV-IP. (C) AP1 was able to activate BFV-IP. Gray box: no c-Jun and c-Fos; open box: 100 ng c-Jun and c-Fos; black box: 250 ng c-Jun and c-Fos. (D) c-Jun mutant inhibited the activity of BFV-IP activated by TPA. Gray box: pGL3-Luc; open box: pGL3-6 × AP1-Luc; black box: pGL3-BFV-IP–Luc. All transfections were performed in triplicate and the error bars represented standard deviations.
six repeats AP1-responsive elements), pGL3-BFV-IP-Luc (including BFV-IP sequence from 8572 to 9509) and pGL3-BFV-LTR-Luc (including BFV-LTR sequence from −55 to 1257) were cloned into the pGL3-basic luciferase reporter vector (Fig. 1A). HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37 ◦ C, 5% CO2 . For luciferase reporter assays, HeLa cells were seeded at 2 × 105 cells/well in 12-well cell culture plates. After 24 h, HeLa cells were transfected alone with 25 ng reporter plasmids, and then treated with 0–50 nM TPA (Sigma) at 24 h posttransfection. Transient transfections were performed by a polyethyleneimine (Sigma) method. Luciferase reporter activities were measured after 24 h. Cell lysates were prepared with lysis buffer (Promega). Lysate was added with the luciferase substrate and analyzed by the 96 Microplate Luminometer (Promega). A remarkable increase of luciferase reporter activity was observed in HeLa cells transfected with pGL3-6 × AP1-luc or pGL3-BFV-IP-luc expression constructs prior to be treated with TPA, and a further dose-dependent manner was also observed, whereas no significant change in Hela cells transfected with pGL3-BFV-LTR-Luc expression construct (Fig. 1B). TPA could activate AP1 by regulating the phosphorylation and activation of c-Jun. Therefore, it was possible that TPA activated BFV-IP through the transcriptional factor AP1 (c-Jun-plus-c-Fos heterodimers) pathway. Here, luciferase reporter assay was also used to investigate the effect of AP1 on the activity of BFV-IP and LTR. The c-Jun and c-Fos expressing plasmids were cotransfected with the 100 ng plasmids as shown in Fig. 1A. Transfected HeLa
cells expressing c-Jun and c-Fos showed a remarkable increase of luciferase reporter activity either in pGL3-6 × AP1-luc or pGL3BFV-IP-luc expression constructs, as compared with control cells transfected alone with the vector pGL3-luc (Fig. 1C). However, cells cotransfected with equal amounts of c-Jun and c-Fos showed no effect in Hela cells transfected with pGL3-BFV-LTR-Luc expression construct. There is one perfect AP1 binding site (TGAGTCA) and one similar AP1 binding site (TGGCTCA) at position 789–795 and 497–503 of BFV-LTR, respectively. However, the LTR could not be activated by PMA or AP1 according to the experiments. In this study, our results indicated AP1 could activate BFV-IP, but had no effect on the activity of BFV-LTR. HeLa cells were cotransfected with increasing c-Jun (S63/73A) (an inactive mutant of c-Jun constructed into eukaryotic expression vector pSG5) and 100 ng pGL3-Luc, pGL3-6 × AP1-luc or pGL3-BFVIP-luc reporter plasmids respectively. Cells treated with 25 nM TPA at 24 h posttransfection. The c-Jun (S63/73A) was able to inhibit the activity of the pGL3-BFV-IP-Luc transactivated by TPA and a further dose-dependent manner was observed (Fig. 1D). The results indicated that TPA activated the BFV-IP promoter by AP1 signal pathway. To determine which region of BFV-IP promoter plays an important role in AP1 activation, a series of deletion mutants of BFV-IP was used in the luciferase reporter assay (Fig. 2A). Position 1 was defined as the first nucleotide of the BFV proviral genome (GenBank accession no. AY134750) in this paper. BFV-IP deletion mutants at
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the amount of 25 ng were transfected with or without c-Jun/c-Fos in HeLa cells. Similar to BFV-IP (8572–9509), BFV-IP1 (9053–9509) retained the same ability in response to c-Jun and c-Fos activation (Fig. 2B), suggesting that IP promoter from 9053 to 9509 was essential for the AP1 activation. Meanwhile, BFV-IP2 (9117–9509) and BFV-IP3 (9205–9509) lost the ability in response to c-Jun and c-Fos activation. All together, there is the AP1-responsive element within the region from 9053 to 9117. Compared with AP1 consensus oligonucleotide, bioinformatics displayed that two AP1-like elements located at positions of nucleotides 9099–9105 and 9109–9115 (Fig. 3A). Mutation reporter plasmids, BFV-IP-AP1m-a (9099-atACTGTGAATGACTCT9115), BFV-IP-AP1m-b (9099-TGACTGTGAAatACTCT-9115), BFVIP-AP1m-ab (9099-atACTGTGAAatACTCT-9115) were mutated by Quikchange Site-Directed Mutagenesis Kit (Stratagene) (Fig. 3B). Cotransfection of 100 ng reporter plasmids with c-Jun/c-Fos expression plasmid was performed in HeLa cells. BFV-IP-AP1m-a and BFV-IP-AP1m-b possessed only about 40% luciferase activity compared with BFV-IP. However, c-Jun and c-Fos almost did not activate BFV-IP-AP1m-ab (Fig. 3C). The results indicated that the sequences at positions of nucleotides 9099–9105 and 9109–9115 were AP1 binding sites within the BFV-IP promoter. To test the ability of two AP1 binding sites where Jun-Fos complexes bind to AP1, the DIG gel shift kit (Roche) was used in this experiment. AP1 consensus, AP1 mutant, BFV-IP-AP1-a and BFVIP-AP1-b oligonucleotides were synthesized (Sangon, Shanghai, China) (Fig. 4A). Mix single strand oligonucleotide at the molar ratio of 1:1 in TEN-buffer. The oligonucleotide was incubated at 95 ◦ C for 3 min and then cooled slowly to 15–25 ◦ C. The double strand oligonucleotides at the amount of 100 ng was added into DIG-ddUTP mix solution incubating at 37 ◦ C for 15 min and put on ice for 2 min; 2 l 0.2 M EDTA (pH 8.0) was added into the mixer to stop the reaction. The c-Jun and c-Fos proteins were expressed from the bacteria and then purified. In the gel shift reaction, labeled oligonucleotide without purified proteins, labeled oligonucleotide with purified proteins and unlabeled oligonucleotide for specific competition were prepared for polyacrylamide gel electrophoresis. Following the electrophoretic separation the oligonucleotide–protein complexes were blotted and detected by
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Fig. 2. Identification of the AP1-responsive region in BFV-IP. (A) Schematic representation of BFV-IP deletion mutants used in the Luciferase reporter assay. Black boxes: BFV-IP sequences. (B) Identification of the region responsible for the AP1 activation. Open box: no c-Jun and c-Fos; black box: 250 ng c-Jun and c-Fos. The numbers next to the bars refer to the activity fold. All transfections were performed in triplicate and the error bars represented standard deviations.
Immunological and Chemiluminescent signal (according the protocol of Dig gel shift kit, Roche). We performed EMSA with DIG-labeled oligonucleotides probes and purified c-Jun and c-Fos from bacterial lysates. Full-length c-
Fig. 3. Identification of the AP1 binding sites in BFV-IP. (A) Schematic representation of AP1 binding sites. (B) Schematic representation of BFV-IP site-directed mutagenesis used in the luciferase reporter assay. (C) Identification of AP1-responsive elements within the BFV-IP promoter. Open box: no c-Jun and c-Fos; black box: 500 ng c-Jun and c-Fos. All transfections were performed in triplicate and the error bars represented standard deviations.
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Fig. 4. The relative protein-binding affinity of AP1 binding sites within BFV-IP. (A) Sequences of the oligonucleotides used for EMSA. Underlined sequences represented typical or mutant AP1 sequences. (B) c-Jun/c-Fos heterodimers bond to AP1 consensus motif: lane 1, control; lane 2, purified c-Jun alone; lane 3, purified c-Fos alone; lane 4, mixed c-Jun and c-Fos; lane 5, 100 × cold competitor. (C) c-Jun/c-Fos heterodimers could not bind to AP1 mutant motif: lane 6, control; lane 7, purified c-Jun alone; lane 8, purified c-Fos alone; lane 9, mixed c-Jun and c-Fos; lanes 10 and 11, AP1 consensus probes used as a positive control (D) c-Jun/c-Fos heterodimers bond to BFV-IP-AP1-a motif: lane 12, control; lane 13, purified c-Jun alone; lane 14, purified c-Fos alone; lane 15, mixed c-Jun and c-Fos; lane 16, 100 × cold competitor. (E) c-Jun/c-Fos heterodimers bond to BFV-IP-AP1-b motif: lane 17, control; lane 18, purified c-Jun alone; lane 19; purified c-Fos alone; lane 20, mixed c-Jun and c-Fos; lane 21, 100 × cold competitor.
fos and c-jun genes were amplified by PCR. The PCR product was treated with the enzyme EcoR I-Xho I. The 1143 bp fragment of cfos or 996 bp fragment of c-jun was inserted into the EcoR I-Xho I site of the bacterial expression vector pGEX-6P-1. The c-Jun and c-Fos proteins were expressed with pGEX-6P-1 expression system. GSTfusion proteins were cleaved with PreScission Protease. Purified proteins (c-Jun and c-Fos) were analyzed by SDS-PAGE and used in the EMSA (Maurer et al., 1991). Neither c-Jun nor c-Fos showed any binding affinity for the AP1 consensus probes (Fig. 4B, lanes 2 and 3); while, when c-Jun and c-Fos were mixed, the heterodimers of c-Jun and c-Fos formed a typical AP1 shifted band (Fig. 4B, lane 4). The AP1 shifted band was not observed by competition with a 100-fold molar excess of unlabeled AP1 consensus oligonucleotides (Fig. 4B, lane 5). The results indicated that the binding of c-Fos-plus-c-Jun heterodimers to DNA was more stable than binding of homodimers. Consistently, we got the same results with BFV-IP-AP1-a, BFV-IP-AP1-b probes (Fig. 4D
and E). The AP1 shifted band was observed in BFV-IP-AP1-a (Fig. 4D, lane 15) and BFV-IP-AP1-b (Fig. 4E, lane 20). In the negative control, we could not find AP1 shifted band in AP1 mutant probes (Fig. 4C, lane 9) compared with AP1 consensus probes (Fig. 4C, lane 11). These data confirmed the capability of AP1 proteins to bind to the two AP1 binding sites within BFV-IP promoter. The potential of AP1 and TPA to enhance BFV replication were examined by western blot and coculture assay. BFV3026 was isolated from the BIV seropositive cattle by our lab previously (Liu et al., 1997). HeLa cells were infected with BFV3026 and then treated with TPA after 24 h. At 2 days postinfection, viral antigen Btas expression was monitored by Western Blot. Cell extracts were electrophoresed in a 12% SDS-PAGE at 80 V for 2 h. Separated proteins were electrophoretically transferred to PVDF membranes at 100 V for 1 h (Millipore). Membranes were blocked in PBS with 5% skim milk for 1 h. After blocking, the membrane was incubated with the anti-Btas antibody (separated from immunized mouse by our lab)
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Fig. 5. AP1 enhanced the viral replication. (A) TPA increased the BFV Btas protein expression. Western blot analysis of protein expression used anti-Btas and anti-Actin antisera. The control represents the same dose of solvent. (B) The c-Jun and c-Fos increased the BFV Btas protein expression. Western blot analysis of protein expression used anti-Btas, and anti-Actin antisera. The control represents the equal amounts of empty vector. (C) TPA increased the luciferase expression of BFVL. The control represents the same dose of solvent. (D) The c-Jun and c-Fos increased the luciferase expression of BFVL. The control represents the equal amounts of empty vector.
in PBST for 2 h. The membrane was washed for three times with PBST and incubated with the goat-anti-mouse IgG second antibody (Santa Cruz) at room temperature for 1 h. The membrane was washed for five times with PBST and the bound antibodies were detected with the Western Blotting Luminol Reagent (Santa Cruz). We found that TPA greatly increased the level of Btas expression compared with control (Fig. 5A). HeLa cells were transfected with cJun and c-Fos expression plasmid, and then infected with BFV3026 after 24 h. At 2 days postinfection, viral antigen Btas expression was monitored by Western Blot. We found that the transfection of HeLa cells with c-Jun and c-Fos greatly increased the level of Btas expression compared with the control (Fig. 5B). To test the virus replication, an indicator cell line was used. BHK21 cells were transfected with the luciferase expression gene that was driven by the BFV-LTR for establishing an indicator cell line named BFVL. The virus titer of BFV was determined by detecting expression of the luciferase reporter gene driven by LTR. HeLa cells were infected with BFV3026 and then treated with TPA after 24 h. At 3 days postinfection, 1/20 of the cells were cocultured with BFVL cells, which were tested by luciferase reporter assay after 96 h. As shown in Fig. 5C, treatment with TPA was able to increase luciferase expression. To investigate the influence of AP1 on viral replication, HeLa cells were transfected with c-Jun and cFos expression plasmids and then infected with BFV3026 after 24 h. At 3 days postinfection, 1/20 of the cells were cocultured with BFVL cells, which were tested by luciferase reporter assay after 96 h. As shown in Fig. 5D, overexpression of c-Jun and c-Fos was found to be associated with higher Luciferase expression in BFVL indicator cells.
The Spumaviruses are a subfamily of the retroviruses which include complex retroviruses of human, simian, feline, cat and bovine origin. The regulation of FV gene expression appears to be different from that of other complex retroviruses. Foamy viruses establish latency infections in the absence of accompanying pathology in a long-term (Cullen, 1992). Here we found TPA greatly enhanced the basal activity of BFV-IP and could not enhance the basal activity of BFV-LTR. The positive effect of TPA on the BFV-IP indicated that it might be a consequence of the fact that TPA activated AP1 signal pathway. Therefore, AP1 was used to activate BFV promoters, and we found that AP1 signal pathway contributed to the transcriptional activation of IP. In addition, c-Jun (S63/73A) was able to repress the transcriptional activity of IP activated by TPA, which suggested that TPA activated the BFV-IP promoter by AP1 signal pathway. We found two AP1-like elements within IP by bioinformatics. To validate the two AP1-like elements, the deletion mutations and site-directed mutagenesis of BFV-IP were used in the luciferase reporter assay. In addition, the capability of AP1 proteins to bind to AP1-like elements was confirmed by EMSA assay. The results indicated that the sequences at positions of nucleotides 9099–9105 and 9109–9115 were AP1responsive elements within BFV-IP promoter. It has been reported that AP1 resulted in an augmentation of the early gene expression in many viruses. Hess reported that the AP-1 binding sites in the visna virus LTR contribute significantly to its basal and transactivator-mediated activity (Hess et al., 1989). Shizhen elucidated early activation of the kaposi’s sarcoma-associated herpesvirus RTA, RAP, and MTA promoters by the TPA-induced AP1 pathway (Wang and Wu, 2004). AP1 also
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played a crucial role during human papillomavirus (HPV) early gene expression (Mack and Laimins, 1991). Moreover, AP-1 binding sites within the HIV-1 LTR contributed to transactivation of the HIV-1 provirus (Roebuck et al., 1996). FV gene expression was considered to initiate at the internal promoter and included the switch from an early IP-directed phase of Tas gene expression to a late phase of LTR-driven structural gene expression (Bodem et al., 1997; Yang et al., 1997). Therefore, FV replication might be regulated by mechanisms that involved a temporal pattern of gene expression. Although it was known that the virus used the host cellular factors to complete the replication at earlier stage, the factors that regulated this process were poorly understood. Our studies showed that AP1 was a positive regulator of BFV basal IP activity, which suggested that AP1 was one of the activation factors in the BFV earlier gene expression. Because the IP promoter had a higher basal transcription level than the LTR promoter, the IP might be the early promoter for FV gene expression that provided the essential Tas transactivator for viral replication. Here we hypothesized that the transcription factor AP1 (c-Jun-plusc-Fos heterodimers) signal pathway contributed to the activation of the BFV-IP in earlier stage. Taken together, our results suggested that AP1 was a positive regulator of basal IP activity. More experiments were needed to verify the role of AP1 in the BFV temporal pattern of gene expression. We hoped that our research could direct further studies in the elucidation of the complex regulation mechanism of foamy virus and contribute to the research of viral vectors. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30770097, 30770080) and the Natural Science Foundation of TianJin (08JCZDJC21000). We gratefully thank Dr. Emily Wang and Kong XiaoHong for the help of the experiment design and writing assistance. References Achong, B.G., Mansell, W.A., Epstein, M.A., Clifford, P., 1971. An unusual virus in cultures from a human nasopharyngeal carcinoma. J. Natl. Cancer Inst. 46, 299–307. Angel, P., Karin, M., 1991. The role of Jun, Fos and the AP-1 complex in cellproliferation and transformation. Biochim. Biophys. Acta 1072, 129–157. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R.J., Rahmsdorf, H.J., Jonat, C., Herrlich, P., Karin, M., 1987. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49, 729– 739. Baunach, G., Maurer, B., Hahn, H., Kranz, M., Rethwilm, A., 1993. Functional analysis of human foamy virus accessory reading frames. J. Virol. 67, 5411–5418. Bodem, J., Löchelt, M., Yang, P., Flügel, R.M., 1997. Regulation of gene expression by human foamy virus and potentials of foamy viral vectors. Stem Cells 15 (Suppl 1), 141–147. Campbell, M., Renshaw-Gegg, L., Renne, R., Luciw, P.A., 1994. Characterization of the internal promoter of simian foamy viruses. J. Virol. 68, 4811–4820. Chiu, R., Boyle, W.J., Meek, J., Smeal, T., Hunter, T., Karin, M., 1988. The c-Fos protein interacts with c-Jun/AP-1 to stimulate transcription of AP-1 responsive genes. Cell 54, 541–552. Cullen, B.R., 1992. Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiol. Rev. 56 (3), 375–394. Enders, J., Peebles, T., 1954. Propagation in tissue culture of cytopathogenic agents from patients with measles. Proc. Soc. Biol. Med. 86, 277–287. Herchenroder, O., Renne, R., Loncar, D., Cobb, E.K., Murthy, K.K., Schneider, J., Mergia, A., Luciw, P.A., 1994. Isolation, cloning, and sequencing of simian foamy viruses from chimpanzees (SFVcpz): high homology to human foamy virus (HFV). Virology 201, 187–199.
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Short communication
Transcription factor AP1 modulates the internal promoter activity of bovine foamy virus Wu YaFeng, Tan Juan, Su Yang, Qiao WenTao, Geng YunQi, Chen QiMin ∗ College of Lifesciences, Nankai University, Tianjin 300071, PR China
a r t i c l e
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Article history: Received 26 June 2009 Received in revised form 6 October 2009 Accepted 9 October 2009 Available online 22 October 2009 Keywords: Bovine foamy virus (BFV) AP1 TPA
a b s t r a c t Foamy virus contains two promoters, which are the canonical long terminal repeat (LTR) promoter and the internal promoter (IP). FV gene expression was considered to initiate at the internal promoter. However, little was known about how basal transcription of IP was triggered by the host cellular factors. Previous studies found some cellular proteins could affect HFV viral replication, but it was no known whether the AP1 signal pathway was involved in the activation of viral replication or not. In this study, we reported that treatment with TPA or AP1 increased basal transcription of IP and did not affect basal transcription of the promoter in the LTR. In addition, the c-Jun mutant blocked the IP activity stimulated by TPA. Two AP1 binding sites located in BFV-IP promoter were found by bioinformatics and mutants of two AP1 binding sites decreased luciferase reporter activity of IP activated by AP1. EMSA assay showed that two AP1 binding sites could bind to c-Jun/c-Fos heterodimeric. We also found TPA and AP1 enhanced BFV3026 replication. Taken together, these data suggested that AP1 was a positive regulator of BFV internal promoter. © 2009 Elsevier B.V. All rights reserved.
Foamy viruses (FV) also called Spumaviruses are complex retroviruses and establish lifelong persistent infections without any accompanying pathologies (Enders and Peebles, 1954; Achong et al., 1971). These viruses infect many mammals including humans (Herchenroder et al., 1994), monkeys (Neumann-Haefelin et al., 1993), horses (Tobaly-Tapiero et al., 2000), cats (Lutz, 1990; Mochizuki et al., 1990), and cattle (Johnson et al., 1983). Regulation of FV replication is controlled by two promoters: long terminal repeats (LTR) (Maurer et al., 1988; Schmidt et al., 1997) and internal promoter (IP) (Lochelt et al., 1993; Campbell et al., 1994; Mergia, 1994; Winkler et al., 1997) within the env gene. The genomes of foamy viruses direct viral transcription through both promoters in the LTR and IP. The foamy viruses display different regulatory elements as compared with other retroviruses, thereby there is an intriguing possibility that the mechanisms involved in foamy virus gene expression may be unique and complex. The FV genome encodes Tas, a transactivator protein, required for transcription from both the LTR promoter and the internal promoter IP (Lochelt et al., 1991; Baunach et al., 1993; Yu and Linial, 1993). Tas is not found in viral particles and foamy virus has the transcriptional capacity from an internal promoter, indicating the activation of IP depends
∗ Corresponding author at: College of Lifesciences, Room 414, 94 WeiJin Road, Nankai District, Tianjin 300071, PR China. Tel.: +86 22 23501783; fax: +86 22 23501783. E-mail address: [email protected] (Q. Chen). 0168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2009.10.005
on the host cellular factors when FV Tas has not been expressed at the earliest stage. The precise mechanism of IP transcription at the earliest stage where the potential cellular factors involved remains to be elucidated. Heretofore, little is known about how basal transcription of bovine foamy virus (BFV) IP was triggered by the host cellular factors (Renshaw et al., 1991; Renshaw and Casey, 1994; Wang et al., 2000). In this study, we demonstrated that AP1 was one of the potential cellular factors to regulate BFV-IP activity. The AP-1 transcriptional factor is mainly composed of Jun/Fos protein dimmers (Angel et al., 1987; Lee et al., 1987), and highly conserved between the human and bovine. AP-1 regulates the activity of a variety of cellular and viral genes (Chiu et al., 1988; Lucibello et al., 1988; Sassone-Corsi et al., 1988; Schonthal et al., 1988; Maurer et al., 1991). AP-1 directs an activity that controls both basal and inducible transcription of several genes containing AP-1 sites (consensus sequence 5 -TGAG/CTCA-3 ), which are also known as TPA-responsive elements (TREs) (Angel and Karin, 1991). Previously, it was reported that the induction of persistently infected cells with TPA greatly enhanced HFV replication, but it was not determined whether the AP1 signal pathway was involved in the activation of viral replication (Meiering and Claudia Rubio, 2001; Meiering et al., 2002). Based on these observations, we wanted to know whether the TPA-induced AP1 pathway exerted an effect on BFV reactivation by activating viral gene expression. To directly examine whether TPA stimulation affected the activity of IP and LTR promoter, luciferase reporter assay was used. The pGL3-Luc, pGL3-6 × AP1-Luc (positive control including
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Fig. 1. AP1 enhanced BFV-IP transcriptional activation. (A) Schematic representation of plasmids used in the Luciferase reporter assay. Shaded box: 6 × AP1; black box: BFV-IP; Gray box: BFV-LTR. (B) TPA was able to activate BFV-IP. (C) AP1 was able to activate BFV-IP. Gray box: no c-Jun and c-Fos; open box: 100 ng c-Jun and c-Fos; black box: 250 ng c-Jun and c-Fos. (D) c-Jun mutant inhibited the activity of BFV-IP activated by TPA. Gray box: pGL3-Luc; open box: pGL3-6 × AP1-Luc; black box: pGL3-BFV-IP–Luc. All transfections were performed in triplicate and the error bars represented standard deviations.
six repeats AP1-responsive elements), pGL3-BFV-IP-Luc (including BFV-IP sequence from 8572 to 9509) and pGL3-BFV-LTR-Luc (including BFV-LTR sequence from −55 to 1257) were cloned into the pGL3-basic luciferase reporter vector (Fig. 1A). HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37 ◦ C, 5% CO2 . For luciferase reporter assays, HeLa cells were seeded at 2 × 105 cells/well in 12-well cell culture plates. After 24 h, HeLa cells were transfected alone with 25 ng reporter plasmids, and then treated with 0–50 nM TPA (Sigma) at 24 h posttransfection. Transient transfections were performed by a polyethyleneimine (Sigma) method. Luciferase reporter activities were measured after 24 h. Cell lysates were prepared with lysis buffer (Promega). Lysate was added with the luciferase substrate and analyzed by the 96 Microplate Luminometer (Promega). A remarkable increase of luciferase reporter activity was observed in HeLa cells transfected with pGL3-6 × AP1-luc or pGL3-BFV-IP-luc expression constructs prior to be treated with TPA, and a further dose-dependent manner was also observed, whereas no significant change in Hela cells transfected with pGL3-BFV-LTR-Luc expression construct (Fig. 1B). TPA could activate AP1 by regulating the phosphorylation and activation of c-Jun. Therefore, it was possible that TPA activated BFV-IP through the transcriptional factor AP1 (c-Jun-plus-c-Fos heterodimers) pathway. Here, luciferase reporter assay was also used to investigate the effect of AP1 on the activity of BFV-IP and LTR. The c-Jun and c-Fos expressing plasmids were cotransfected with the 100 ng plasmids as shown in Fig. 1A. Transfected HeLa
cells expressing c-Jun and c-Fos showed a remarkable increase of luciferase reporter activity either in pGL3-6 × AP1-luc or pGL3BFV-IP-luc expression constructs, as compared with control cells transfected alone with the vector pGL3-luc (Fig. 1C). However, cells cotransfected with equal amounts of c-Jun and c-Fos showed no effect in Hela cells transfected with pGL3-BFV-LTR-Luc expression construct. There is one perfect AP1 binding site (TGAGTCA) and one similar AP1 binding site (TGGCTCA) at position 789–795 and 497–503 of BFV-LTR, respectively. However, the LTR could not be activated by PMA or AP1 according to the experiments. In this study, our results indicated AP1 could activate BFV-IP, but had no effect on the activity of BFV-LTR. HeLa cells were cotransfected with increasing c-Jun (S63/73A) (an inactive mutant of c-Jun constructed into eukaryotic expression vector pSG5) and 100 ng pGL3-Luc, pGL3-6 × AP1-luc or pGL3-BFVIP-luc reporter plasmids respectively. Cells treated with 25 nM TPA at 24 h posttransfection. The c-Jun (S63/73A) was able to inhibit the activity of the pGL3-BFV-IP-Luc transactivated by TPA and a further dose-dependent manner was observed (Fig. 1D). The results indicated that TPA activated the BFV-IP promoter by AP1 signal pathway. To determine which region of BFV-IP promoter plays an important role in AP1 activation, a series of deletion mutants of BFV-IP was used in the luciferase reporter assay (Fig. 2A). Position 1 was defined as the first nucleotide of the BFV proviral genome (GenBank accession no. AY134750) in this paper. BFV-IP deletion mutants at
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the amount of 25 ng were transfected with or without c-Jun/c-Fos in HeLa cells. Similar to BFV-IP (8572–9509), BFV-IP1 (9053–9509) retained the same ability in response to c-Jun and c-Fos activation (Fig. 2B), suggesting that IP promoter from 9053 to 9509 was essential for the AP1 activation. Meanwhile, BFV-IP2 (9117–9509) and BFV-IP3 (9205–9509) lost the ability in response to c-Jun and c-Fos activation. All together, there is the AP1-responsive element within the region from 9053 to 9117. Compared with AP1 consensus oligonucleotide, bioinformatics displayed that two AP1-like elements located at positions of nucleotides 9099–9105 and 9109–9115 (Fig. 3A). Mutation reporter plasmids, BFV-IP-AP1m-a (9099-atACTGTGAATGACTCT9115), BFV-IP-AP1m-b (9099-TGACTGTGAAatACTCT-9115), BFVIP-AP1m-ab (9099-atACTGTGAAatACTCT-9115) were mutated by Quikchange Site-Directed Mutagenesis Kit (Stratagene) (Fig. 3B). Cotransfection of 100 ng reporter plasmids with c-Jun/c-Fos expression plasmid was performed in HeLa cells. BFV-IP-AP1m-a and BFV-IP-AP1m-b possessed only about 40% luciferase activity compared with BFV-IP. However, c-Jun and c-Fos almost did not activate BFV-IP-AP1m-ab (Fig. 3C). The results indicated that the sequences at positions of nucleotides 9099–9105 and 9109–9115 were AP1 binding sites within the BFV-IP promoter. To test the ability of two AP1 binding sites where Jun-Fos complexes bind to AP1, the DIG gel shift kit (Roche) was used in this experiment. AP1 consensus, AP1 mutant, BFV-IP-AP1-a and BFVIP-AP1-b oligonucleotides were synthesized (Sangon, Shanghai, China) (Fig. 4A). Mix single strand oligonucleotide at the molar ratio of 1:1 in TEN-buffer. The oligonucleotide was incubated at 95 ◦ C for 3 min and then cooled slowly to 15–25 ◦ C. The double strand oligonucleotides at the amount of 100 ng was added into DIG-ddUTP mix solution incubating at 37 ◦ C for 15 min and put on ice for 2 min; 2 l 0.2 M EDTA (pH 8.0) was added into the mixer to stop the reaction. The c-Jun and c-Fos proteins were expressed from the bacteria and then purified. In the gel shift reaction, labeled oligonucleotide without purified proteins, labeled oligonucleotide with purified proteins and unlabeled oligonucleotide for specific competition were prepared for polyacrylamide gel electrophoresis. Following the electrophoretic separation the oligonucleotide–protein complexes were blotted and detected by
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Fig. 2. Identification of the AP1-responsive region in BFV-IP. (A) Schematic representation of BFV-IP deletion mutants used in the Luciferase reporter assay. Black boxes: BFV-IP sequences. (B) Identification of the region responsible for the AP1 activation. Open box: no c-Jun and c-Fos; black box: 250 ng c-Jun and c-Fos. The numbers next to the bars refer to the activity fold. All transfections were performed in triplicate and the error bars represented standard deviations.
Immunological and Chemiluminescent signal (according the protocol of Dig gel shift kit, Roche). We performed EMSA with DIG-labeled oligonucleotides probes and purified c-Jun and c-Fos from bacterial lysates. Full-length c-
Fig. 3. Identification of the AP1 binding sites in BFV-IP. (A) Schematic representation of AP1 binding sites. (B) Schematic representation of BFV-IP site-directed mutagenesis used in the luciferase reporter assay. (C) Identification of AP1-responsive elements within the BFV-IP promoter. Open box: no c-Jun and c-Fos; black box: 500 ng c-Jun and c-Fos. All transfections were performed in triplicate and the error bars represented standard deviations.
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Fig. 4. The relative protein-binding affinity of AP1 binding sites within BFV-IP. (A) Sequences of the oligonucleotides used for EMSA. Underlined sequences represented typical or mutant AP1 sequences. (B) c-Jun/c-Fos heterodimers bond to AP1 consensus motif: lane 1, control; lane 2, purified c-Jun alone; lane 3, purified c-Fos alone; lane 4, mixed c-Jun and c-Fos; lane 5, 100 × cold competitor. (C) c-Jun/c-Fos heterodimers could not bind to AP1 mutant motif: lane 6, control; lane 7, purified c-Jun alone; lane 8, purified c-Fos alone; lane 9, mixed c-Jun and c-Fos; lanes 10 and 11, AP1 consensus probes used as a positive control (D) c-Jun/c-Fos heterodimers bond to BFV-IP-AP1-a motif: lane 12, control; lane 13, purified c-Jun alone; lane 14, purified c-Fos alone; lane 15, mixed c-Jun and c-Fos; lane 16, 100 × cold competitor. (E) c-Jun/c-Fos heterodimers bond to BFV-IP-AP1-b motif: lane 17, control; lane 18, purified c-Jun alone; lane 19; purified c-Fos alone; lane 20, mixed c-Jun and c-Fos; lane 21, 100 × cold competitor.
fos and c-jun genes were amplified by PCR. The PCR product was treated with the enzyme EcoR I-Xho I. The 1143 bp fragment of cfos or 996 bp fragment of c-jun was inserted into the EcoR I-Xho I site of the bacterial expression vector pGEX-6P-1. The c-Jun and c-Fos proteins were expressed with pGEX-6P-1 expression system. GSTfusion proteins were cleaved with PreScission Protease. Purified proteins (c-Jun and c-Fos) were analyzed by SDS-PAGE and used in the EMSA (Maurer et al., 1991). Neither c-Jun nor c-Fos showed any binding affinity for the AP1 consensus probes (Fig. 4B, lanes 2 and 3); while, when c-Jun and c-Fos were mixed, the heterodimers of c-Jun and c-Fos formed a typical AP1 shifted band (Fig. 4B, lane 4). The AP1 shifted band was not observed by competition with a 100-fold molar excess of unlabeled AP1 consensus oligonucleotides (Fig. 4B, lane 5). The results indicated that the binding of c-Fos-plus-c-Jun heterodimers to DNA was more stable than binding of homodimers. Consistently, we got the same results with BFV-IP-AP1-a, BFV-IP-AP1-b probes (Fig. 4D
and E). The AP1 shifted band was observed in BFV-IP-AP1-a (Fig. 4D, lane 15) and BFV-IP-AP1-b (Fig. 4E, lane 20). In the negative control, we could not find AP1 shifted band in AP1 mutant probes (Fig. 4C, lane 9) compared with AP1 consensus probes (Fig. 4C, lane 11). These data confirmed the capability of AP1 proteins to bind to the two AP1 binding sites within BFV-IP promoter. The potential of AP1 and TPA to enhance BFV replication were examined by western blot and coculture assay. BFV3026 was isolated from the BIV seropositive cattle by our lab previously (Liu et al., 1997). HeLa cells were infected with BFV3026 and then treated with TPA after 24 h. At 2 days postinfection, viral antigen Btas expression was monitored by Western Blot. Cell extracts were electrophoresed in a 12% SDS-PAGE at 80 V for 2 h. Separated proteins were electrophoretically transferred to PVDF membranes at 100 V for 1 h (Millipore). Membranes were blocked in PBS with 5% skim milk for 1 h. After blocking, the membrane was incubated with the anti-Btas antibody (separated from immunized mouse by our lab)
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Fig. 5. AP1 enhanced the viral replication. (A) TPA increased the BFV Btas protein expression. Western blot analysis of protein expression used anti-Btas and anti-Actin antisera. The control represents the same dose of solvent. (B) The c-Jun and c-Fos increased the BFV Btas protein expression. Western blot analysis of protein expression used anti-Btas, and anti-Actin antisera. The control represents the equal amounts of empty vector. (C) TPA increased the luciferase expression of BFVL. The control represents the same dose of solvent. (D) The c-Jun and c-Fos increased the luciferase expression of BFVL. The control represents the equal amounts of empty vector.
in PBST for 2 h. The membrane was washed for three times with PBST and incubated with the goat-anti-mouse IgG second antibody (Santa Cruz) at room temperature for 1 h. The membrane was washed for five times with PBST and the bound antibodies were detected with the Western Blotting Luminol Reagent (Santa Cruz). We found that TPA greatly increased the level of Btas expression compared with control (Fig. 5A). HeLa cells were transfected with cJun and c-Fos expression plasmid, and then infected with BFV3026 after 24 h. At 2 days postinfection, viral antigen Btas expression was monitored by Western Blot. We found that the transfection of HeLa cells with c-Jun and c-Fos greatly increased the level of Btas expression compared with the control (Fig. 5B). To test the virus replication, an indicator cell line was used. BHK21 cells were transfected with the luciferase expression gene that was driven by the BFV-LTR for establishing an indicator cell line named BFVL. The virus titer of BFV was determined by detecting expression of the luciferase reporter gene driven by LTR. HeLa cells were infected with BFV3026 and then treated with TPA after 24 h. At 3 days postinfection, 1/20 of the cells were cocultured with BFVL cells, which were tested by luciferase reporter assay after 96 h. As shown in Fig. 5C, treatment with TPA was able to increase luciferase expression. To investigate the influence of AP1 on viral replication, HeLa cells were transfected with c-Jun and cFos expression plasmids and then infected with BFV3026 after 24 h. At 3 days postinfection, 1/20 of the cells were cocultured with BFVL cells, which were tested by luciferase reporter assay after 96 h. As shown in Fig. 5D, overexpression of c-Jun and c-Fos was found to be associated with higher Luciferase expression in BFVL indicator cells.
The Spumaviruses are a subfamily of the retroviruses which include complex retroviruses of human, simian, feline, cat and bovine origin. The regulation of FV gene expression appears to be different from that of other complex retroviruses. Foamy viruses establish latency infections in the absence of accompanying pathology in a long-term (Cullen, 1992). Here we found TPA greatly enhanced the basal activity of BFV-IP and could not enhance the basal activity of BFV-LTR. The positive effect of TPA on the BFV-IP indicated that it might be a consequence of the fact that TPA activated AP1 signal pathway. Therefore, AP1 was used to activate BFV promoters, and we found that AP1 signal pathway contributed to the transcriptional activation of IP. In addition, c-Jun (S63/73A) was able to repress the transcriptional activity of IP activated by TPA, which suggested that TPA activated the BFV-IP promoter by AP1 signal pathway. We found two AP1-like elements within IP by bioinformatics. To validate the two AP1-like elements, the deletion mutations and site-directed mutagenesis of BFV-IP were used in the luciferase reporter assay. In addition, the capability of AP1 proteins to bind to AP1-like elements was confirmed by EMSA assay. The results indicated that the sequences at positions of nucleotides 9099–9105 and 9109–9115 were AP1responsive elements within BFV-IP promoter. It has been reported that AP1 resulted in an augmentation of the early gene expression in many viruses. Hess reported that the AP-1 binding sites in the visna virus LTR contribute significantly to its basal and transactivator-mediated activity (Hess et al., 1989). Shizhen elucidated early activation of the kaposi’s sarcoma-associated herpesvirus RTA, RAP, and MTA promoters by the TPA-induced AP1 pathway (Wang and Wu, 2004). AP1 also
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played a crucial role during human papillomavirus (HPV) early gene expression (Mack and Laimins, 1991). Moreover, AP-1 binding sites within the HIV-1 LTR contributed to transactivation of the HIV-1 provirus (Roebuck et al., 1996). FV gene expression was considered to initiate at the internal promoter and included the switch from an early IP-directed phase of Tas gene expression to a late phase of LTR-driven structural gene expression (Bodem et al., 1997; Yang et al., 1997). Therefore, FV replication might be regulated by mechanisms that involved a temporal pattern of gene expression. Although it was known that the virus used the host cellular factors to complete the replication at earlier stage, the factors that regulated this process were poorly understood. Our studies showed that AP1 was a positive regulator of BFV basal IP activity, which suggested that AP1 was one of the activation factors in the BFV earlier gene expression. Because the IP promoter had a higher basal transcription level than the LTR promoter, the IP might be the early promoter for FV gene expression that provided the essential Tas transactivator for viral replication. Here we hypothesized that the transcription factor AP1 (c-Jun-plusc-Fos heterodimers) signal pathway contributed to the activation of the BFV-IP in earlier stage. Taken together, our results suggested that AP1 was a positive regulator of basal IP activity. More experiments were needed to verify the role of AP1 in the BFV temporal pattern of gene expression. We hoped that our research could direct further studies in the elucidation of the complex regulation mechanism of foamy virus and contribute to the research of viral vectors. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30770097, 30770080) and the Natural Science Foundation of TianJin (08JCZDJC21000). We gratefully thank Dr. Emily Wang and Kong XiaoHong for the help of the experiment design and writing assistance. References Achong, B.G., Mansell, W.A., Epstein, M.A., Clifford, P., 1971. An unusual virus in cultures from a human nasopharyngeal carcinoma. J. Natl. Cancer Inst. 46, 299–307. Angel, P., Karin, M., 1991. The role of Jun, Fos and the AP-1 complex in cellproliferation and transformation. Biochim. Biophys. Acta 1072, 129–157. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R.J., Rahmsdorf, H.J., Jonat, C., Herrlich, P., Karin, M., 1987. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49, 729– 739. Baunach, G., Maurer, B., Hahn, H., Kranz, M., Rethwilm, A., 1993. Functional analysis of human foamy virus accessory reading frames. J. Virol. 67, 5411–5418. Bodem, J., Löchelt, M., Yang, P., Flügel, R.M., 1997. Regulation of gene expression by human foamy virus and potentials of foamy viral vectors. Stem Cells 15 (Suppl 1), 141–147. Campbell, M., Renshaw-Gegg, L., Renne, R., Luciw, P.A., 1994. Characterization of the internal promoter of simian foamy viruses. J. Virol. 68, 4811–4820. Chiu, R., Boyle, W.J., Meek, J., Smeal, T., Hunter, T., Karin, M., 1988. The c-Fos protein interacts with c-Jun/AP-1 to stimulate transcription of AP-1 responsive genes. Cell 54, 541–552. Cullen, B.R., 1992. Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiol. Rev. 56 (3), 375–394. Enders, J., Peebles, T., 1954. Propagation in tissue culture of cytopathogenic agents from patients with measles. Proc. Soc. Biol. Med. 86, 277–287. Herchenroder, O., Renne, R., Loncar, D., Cobb, E.K., Murthy, K.K., Schneider, J., Mergia, A., Luciw, P.A., 1994. Isolation, cloning, and sequencing of simian foamy viruses from chimpanzees (SFVcpz): high homology to human foamy virus (HFV). Virology 201, 187–199.
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