1 Department of Microbiology, College of Medicine, National Taiwan University, Room 722, Number 1, Section 1, Jen-Ai Road, Taipei, Taiwan
2 Center of General Education, National Taipei College of Nursing, Taipei, Taiwan
3 Extramural Research Affairs Department, National Health Research Institute, Taipei, Taiwan
Correspondence
Tsuey-Ying Hsu
tyhsu{at}ha.mc.ntu.edu.tw
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
It is well known that Zta exerts its transactivation function through direct binding to consensus DNA sequences present in target promoters, including the Zta promoter (Zp), the Rta promoter (Rp) and the promoter of BMRF1 (EBV DNA polymerase processivity factor) (Flemington & Speck, 1990; Holley-Guthrie et al., 1990
; Sinclair et al., 1991
). In contrast, Rta may utilize multiple mechanisms to induce gene expression. Previous studies have shown that Rta can bind directly to the promoters of some EBV lytic genes, such as BMRF1, BMLF1 (Mta) and BALF2 (EBV major DNA-binding protein) (Hung & Liu, 1999
; Kenney et al., 1989
; Quinlivan et al., 1993
). Recently we also found that the promoter of BGLF5 (EBV DNase) contains an Rta-responsive region that can be bound by Rta directly (unpublished data). The identified Rta-binding elements are generally GC-rich but the DNA sequences are quite diverse (Gruffat et al., 1992
; Gruffat & Sergeant, 1994
; Hung & Liu, 1999
). Notably, some cases of Rta-mediated transactivation do not involve direct binding of Rta to the target promoters. For example, Rta activates Zp, Rp and the promoter of BALF5 (EBV DNA polymerase) in certain indirect, non-DNA-binding mechanisms (Adamson et al., 2000
; Darr et al., 2001
; Liu et al., 1996
; Ragoczy & Miller, 2001
).
Without direct DNA binding, Rta is likely to use more than one way of inducing gene transcription. For activation of Zp, Rta triggers cellular signalling pathways involving mitogen-activated kinases, resulting in activation of the ATF2 transcription factor targeted to the ZII element of Zp (Adamson et al., 2000). In addition, activation of phosphatidylinositol 3-kinase is required for Rta-induced Zta expression (Darr et al., 2001
). For autostimulation of Rp, the Sp1/Sp3-binding sites in the promoter are involved in Rta-mediated activation, and it has been suggested that Rta may affect the post-translational regulation or the binding partners of Sp1/Sp3 (Ragoczy & Miller, 2001
). Another study has shown that Rta activates the promoter of BALF5 through USF and E2F transcription factors (Liu et al., 1996
). Therefore, it is generally considered that the so-called indirect mechanisms of Rta-induced transactivation are mediated by cellular signalling or other transcription factors. The detailed process by which Rta affects cellular factors, however, is largely unclear.
Here we have provided another approach to delineate and dissect the mechanisms of Rta-mediated transactivation. Rta is located predominantly in the nucleus but cytoplasmic localization of Rta has also been observed (Cox et al., 1990; Darr et al., 2001
). It was of interest to determine whether the subcellular localization of Rta was critical for the induction of target genes. If the transactivation involved binding of Rta to DNA or to nuclear factors, nuclear localization of Rta might be required. On the other hand, if the transactivation was mediated through interaction of Rta with cytoplasmic proteins to trigger signalling transduction, a cytoplasmic Rta should be sufficient for gene induction. In this study, we first identified a nuclear localization signal (NLS) of Rta. An Rta mutant showing cytoplasmic localization was generated by disruption of the NLS. Notably, the cytoplasmic Rta mutant was able to induce expression of endogenous Zta and Rta in EBV-infected cells, triggering virus reactivation.
To map the NLS of Rta, a series of green fluorescence protein (GFP)Rta fusion constructs was generated by cloning the Rta-coding sequences into the pEGFP-C1 plasmid (Clontech) (Fig. 1a). The plasmids were then transfected into 293 cells using Lipofectamine 2000 reagent (Invitrogen) and the cells were examined under a fluorescence microscope at 24 h post-transfection. The localization of GFP was shown as a diffuse pattern in both the nucleus and the cytoplasm, while GFPRta(1605), the GFP fusion protein containing full-length Rta, was located predominantly in the nucleus (Fig. 1b
). GFPRta(1441) and GFPRta(100605), the carboxyl-terminally and amino-terminally truncated Rta fusion proteins, respectively, also showed nuclear localization (Fig. 1b
). Further deletion analysis narrowed the location of the NLS to aa 401441 of the Rta protein (Fig. 1b
). By careful examination of the sequences within the region, we found four consecutive basic residues, 410KRKK413, which perfectly matched the criterion for a conventional NLS (Kalderon et al., 1984
). GFPRta(NLSm), in which the original 410KRKK413 sequence was mutated to 410AAAA413, was clearly located in the cytoplasm (Fig. 1b
). The nuclear localization of GFPRta(1605) and the cytoplasmic localization of GFPRta(NLSm) were also confirmed in NA cells, an EBV-positive epithelial cell line generated in a previous study (Chang et al., 1999
) (Fig. 1c
). To rule out any possible effect of GFP on the subcellular localization, we constructed plasmids expressing full-length Rta or Rta(NLSm) without fusion with GFP. Both constructs were derived from the pSG5 plasmid (Stratagene). After transfection into 293 cells for 24 h, protein localization was examined by an immunofluorescence assay using the anti-Rta mAb 8C12 (Argene). Wild-type Rta was detected predominantly in the nucleus, while Rta(NLSm) showed cytoplasmic localization (Fig. 1d
). Therefore, we concluded that 410KRKK413 is a critical NLS of Rta.
|
|
To compare further the abilities of Rta and Rta(NLSm) to activate viral promoters, a reporter gene assay was carried out in 293 cells. Zp-Luc, Rp-Luc and BGLF5p-Luc are reporter plasmids that contain Zp, Rp and the promoter of BGLF5, respectively, to drive the firefly luciferase gene (Chang & Liu, 2000; and unpublished data). Zp-Luc and Rp-Luc were derived from the pGL2-basic plasmid (Promega), while BGLF5p-Luc was derived from the pGL3-basic plasmid (Promega). Each reporter plasmid was co-transfected with effector plasmids using a calcium phosphate method, and cells were subjected to a luciferase activity assay at 48 h post-transfection. For activation of Zp and Rp, Rta(NLSm) maintained about 5060 % of the transactivation activity of wild-type Rta (Fig. 3
a, b). Using a series of smaller amounts of the effector plasmids, we found that the activity of Rta(NLSm) was maintained relative to that of Rta, even at the lower expression levels (Fig. 3d
f). Therefore, the activity of Rta(NLSm) was unlikely to be due to nuclear leakage of the overexpressed proteins. On the other hand, Rta(NLSm) almost completely lost its ability to activate the promoter of BGLF5 compared with wild-type Rta (Fig. 3c
). These results clearly indicated that, although the cytoplasmic Rta mutant failed to activate the promoter of BGLF5 and possibly some other viral genes, it retained the ability to activate Zp and Rp at a considerable level, thus explaining how this mutant was still able to trigger EBV reactivation.
|
Although it is generally considered that Rta may utilize multiple ways of inducing gene expression, the detailed mechanisms remain largely unknown. For transactivation through direct DNA binding, it has not yet been resolved how Rta is able to bind to diverse DNA sequences present in the various promoters (Gruffat et al., 1992; Gruffat & Sergeant, 1994
; Hung & Liu, 1999
). For the indirect mechanisms without DNA binding, the bigger mystery of how Rta may affect cellular signalling and other transcription factors also remains (Adamson et al., 2000
; Darr et al., 2001
). Here, we examined whether the nuclear localization of Rta was required for transactivation of the target genes, and the results provided some interesting information.
First, in the cases of BGLF5 and BALF2, where Rta-mediated transactivation is through direct DNA binding, the nuclear localization of Rta should be required (Hung & Liu, 1999; and unpublished data). Consistent with this notion, induction of BGLF5 and BALF2 by GFPRta(NLSm) showed a significant delay in NA cells (Fig. 2c
), and Rta(NLSm) was almost unable to activate the promoter of BGLF5 (Fig. 3c
). It should be noted that the promoter of BMRF1 also contains an Rta-binding element, but induction of this gene is solely dependent on Zta in epithelial cells (Chang et al., 2004
; Holley-Guthrie et al., 1990
; Quinlivan et al., 1993
). As expression of Zta was induced by wild-type and NLS-mutated Rta with similar kinetics, the induction of BMRF1 by GFPRta(NLSm) was not delayed in NA cells (Fig. 2c
).
The most notable finding was that the Rta NLS mutant could still activate Zp and Rp (Fig. 3a, b). It has been reported that both promoters are activated by Rta without direct binding of Rta to the DNA, but it remains unknown whether Rta exerts this indirect transactivation in the nucleus or in the cytoplasm (Adamson et al., 2000
; Darr et al., 2001
; Liu & Speck, 2003
; Ragoczy & Miller, 2001
). As we found that the Rta NLS mutant retained the transactivation function at a considerable level, Rta may be functional in the cytoplasm to trigger cellular signalling, leading to activation of Zp and Rp. On the other hand, the roles of nuclear Rta in activating Zp and Rp cannot be ruled out, since the transactivation mediated by wild-type Rta was about twice the level of Rta(NLSm) (Fig. 3a, b, d and e
). In the nucleus, Rta may interact with transcription factors or other nuclear proteins, elevating the activities of Zp and Rp in an indirect manner. Therefore, Rta-mediated activation of Zp and Rp should be achieved by more than one mechanism: the cytoplasmic Rta and the nuclear Rta are likely to exert some potent effects separately or cooperatively.
Rta(NLSm) is a useful tool to dissect the complicated mechanisms of Rta-mediated transactivation. Our current work aims to identify cellular proteins that interact with Rta and Rta(NLSm), which hopefully may help to elucidate the mechanisms by which Rta induces gene expression in such a variety of ways.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Chang, L.-K. & Liu, S.-T. (2000). Activation of the BRLF1 promoter and lytic cycle of EpsteinBarr virus by histone acetylation. Nucleic Acids Res 28, 39183925.
Chang, Y., Tung, C.-H., Huang, Y.-T., Lu, J., Chen, J.-Y. & Tsai, C.-H. (1999). Requirement for cell-to-cell contact in EpsteinBarr virus infection of nasopharyngeal carcinoma cells and keratinocytes. J Virol 73, 88578866.
Chang, Y., Chang, S.-S., Lee, H.-H., Doong, S.-L., Takada, K. & Tsai, C.-H. (2004). Inhibition of the EpsteinBarr virus lytic cycle by Zta-targeted RNA interference. J Gen Virol 85, 13711379.
Cox, M. A., Leahy, J. & Hardwick, J. M. (1990). An enhancer within the divergent promoter of EpsteinBarr virus responds synergistically to the R and Z transactivators. J Virol 64, 313321.[Medline]
Darr, C. D., Mauser, A. & Kenney, S. (2001). EpsteinBarr virus immediate-early protein BRLF1 induces the lytic form of viral replication through a mechanism involving phosphatidylinositol-3 kinase activation. J Virol 75, 61356142.
Feederle, R., Kost, M., Baumann, M., Janz, A., Drouet, E., Hammerschmidt, W. & Delecluse, H. J. (2000). The EpsteinBarr virus lytic program is controlled by the co-operative functions of two transactivators. EMBO J 19, 30803089.
Flemington, E. & Speck, S. H. (1990). Autoregulation of EpsteinBarr virus putative lytic switch gene BZLF1. J Virol 64, 12271232.[Medline]
Grogan, E., Jenson, H., Countryman, J., Heston, L., Gradoville, L. & Miller, G. (1987). Transfection of a rearranged viral DNA fragment, WZhet, stably converts latent EpsteinBarr viral infection to productive infection in lymphoid cells. Proc Natl Acad Sci U S A 84, 13321336.[Abstract]
Gruffat, H. & Sergeant, A. (1994). Characterization of the DNA-binding site repertoire for the EpsteinBarr virus transcription factor R. Nucleic Acids Res 22, 11721178.[Abstract]
Gruffat, H., Duran, N., Buisson, M., Wild, F., Buckland, R. & Sergeant, A. (1992). Characterization of an R-binding site mediating the R-induced activation of the EpsteinBarr virus BMLF1 promoter. J Virol 66, 4652.[Abstract]
Hardwick, J. M., Tse, L., Applegren, N., Nicholas, J. & Veliuona, M. A. (1992). The EpsteinBarr virus R transactivator (Rta) contains a complex, potent activation domain with properties different from those of VP16. J Virol 66, 55005508.[Abstract]
Holley-Guthrie, E. A., Quinlivan, E. B., Mar, E. C. & Kenney, S. (1990). The EpsteinBarr virus (EBV) BMRF1 promoter for early antigen (EA-D) is regulated by the EBV transactivators, BRLF1 and BZLF1, in a cell-specific manner. J Virol 64, 37533759.[Medline]
Hung, C.-H. & Liu, S.-T. (1999). Characterization of the EpsteinBarr virus BALF2 promoter. J Gen Virol 80, 27472750.
Kalderon, D., Roberts, B. L., Richardson, W. D. & Smith, A. E. (1984). A short amino acid sequence able to specify nuclear location. Cell 39, 499509.[Medline]
Kenney, S., Holley-Guthrie, E., Mar, E. C. & Smith, M. (1989). The EpsteinBarr virus BMLF1 promoter contains an enhancer element that is responsive to the BZLF1 and BRLF1 transactivators. J Virol 63, 38783883.[Medline]
Kieff, E. & Rickinson, A. B. (2001). EpsteinBarr virus and its replication. In Fields Virology, 4th edn, pp. 25112573. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
Liu, P. & Speck, S. H. (2003). Synergistic autoactivation of the EpsteinBarr virus immediate-early BRLF1 promoter by Rta and Zta. Virology 310, 199206.[Medline]
Liu, C., Sista, N. D. & Pagano, J. S. (1996). Activation of the EpsteinBarr virus DNA polymerase promoter by the BRLF1 immediate-early protein is mediated through USF and E2F. J Virol 70, 25452455.[Abstract]
Manet, E., Rigolet, A., Gruffat, H., Giot, J. F. & Sergeant, A. (1991). Domains of the EpsteinBarr virus (EBV) transcription factor R required for dimerization, DNA binding and activation. Nucleic Acids Res 19, 26612667.[Abstract]
Quinlivan, E. B., Holley-Guthrie, E. A., Norris, M., Gutsch, D., Bachenheimer, S. L. & Kenney, S. C. (1993). Direct BRLF1 binding is required for cooperative BZLF1/BRLF1 activation of the EpsteinBarr virus early promoter, BMRF1. Nucleic Acids Res 21, 19992007.[Abstract]
Ragoczy, T. & Miller, G. (2001). Autostimulation of the EpsteinBarr virus BRLF1 promoter is mediated through consensus Sp1 and Sp3 binding sites. J Virol 75, 52405251.
Ragoczy, T., Heston, L. & Miller, G. (1998). The EpsteinBarr virus Rta protein activates lytic cycle genes and can disrupt latency in B lymphocytes. J Virol 72, 79787984.
Sinclair, A. J., Brimmell, M., Shanahan, F. & Farrell, P. J. (1991). Pathways of activation of the EpsteinBarr virus productive cycle. J Virol 65, 22372244.[Medline]
Tsai, C.-H., Williams, M. V. & Glaser, R. (1991). Characterization of two monoclonal antibodies to EpsteinBarr virus diffuse early antigen which react to two different epitopes and have different biological function. J Virol Methods 33, 4752.[CrossRef][Medline]
Tsai, C.-H., Liu, M.-T., Chen, M.-R., Lu, J., Yang, H.-L., Chen, J.-Y. & Yang, C.-S. (1997). Characterization of monoclonal antibodies to the Zta and DNase proteins of EpsteinBarr virus. J Biomed Sci 4, 6977.[Medline]
Zalani, S., Holley-Guthrie, E. & Kenney, S. (1996). EpsteinBarr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism. Proc Natl Acad Sci U S A 93, 91949199.
Received 27 August 2004;
accepted 1 November 2004.