Reactivation of Epstein–Barr virus can be triggered by an Rta protein mutated at the nuclear localization signal

Tsuey-Ying Hsu1, Yao Chang1, Pei-Wen Wang1, Mei-Ying Liu2, Mei-Ru Chen1, Jen-Yang Chen1,3 and Ching-Hwa Tsai1

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


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Rta, an immediate-early protein of Epstein–Barr virus (EBV), is a transcriptional activator that induces lytic gene expression and triggers virus reactivation. Being located predominantly in the nucleus, Rta can exert its transactivation function through either direct DNA binding or certain indirect mechanisms mediated by cellular signalling and other transcriptional factors. This study examined whether the subcellular localization of Rta was critical for the induction of target genes. First, 410KRKK413 was identified as a nuclear localization signal (NLS) of Rta. An Rta mutant with the NLS converted to 410AAAA413 showed cytoplasmic localization and failed to activate the promoter of BGLF5. Interestingly, ectopic expression of the Rta mutant still disrupted EBV latency in an epithelial cell line. Reporter gene assays revealed that the NLS-mutated Rta retained the ability to activate two lytic promoters, Zp and Rp, at a considerable level. Thus, the cytoplasmic Rta mutant could induce expression of endogenous Zta and Rta, triggering reactivation of EBV.


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Epstein–Barr virus (EBV) has two phases of infection: a predominant latent state and an inducible lytic state (Kieff & Rickinson, 2001). In contrast to latent infection, where viral expression is restricted to a few latent genes, the lytic cycle shows an expression cascade of numerous lytic genes, leading to amplification of viral genomes and production of progeny virions (Kieff & Rickinson, 2001). EBV reactivation from latency into the lytic cycle can be initiated by two immediate-early viral proteins, BZLF1 (Zta) and BRLF1 (Rta). Both Zta and Rta are potent transcriptional activators that autostimulate their own expression, mutually upregulate each other and cooperatively induce the downstream lytic genes (Adamson et al., 2000; Holley-Guthrie et al., 1990; Liu & Speck, 2003; Ragoczy et al., 1998). Ectopic expression of either Zta or Rta can trigger EBV reactivation and both are essential for completion of the lytic cycle (Feederle et al., 2000; Grogan et al., 1987; Ragoczy et al., 1998; Zalani et al., 1996).

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 GFP–Rta(1–605), the GFP fusion protein containing full-length Rta, was located predominantly in the nucleus (Fig. 1b). GFP–Rta(1–441) and GFP–Rta(100–605), 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 401–441 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). GFP–Rta(NLSm), in which the original 410KRKK413 sequence was mutated to 410AAAA413, was clearly located in the cytoplasm (Fig. 1b). The nuclear localization of GFP–Rta(1–605) and the cytoplasmic localization of GFP–Rta(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.



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Fig. 1. Subcellular localization of Rta and its mutants. (a) Schematic illustration of GFP–Rta fusion constructs. The nuclear (N) or cytoplasmic (C) localization of individual proteins is indicated. (b) Subcellular localization of GFP–Rta fusion proteins in 293 cells. Plasmids expressing GFP or various GFP–Rta fusion proteins were transfected into 293 cells for 24 h and cells were examined under a fluorescence microscope. (c) Subcellular localization of GFP–Rta fusion proteins in EBV-positive NA cells. Plasmids expressing GFP, GFP–Rta(1–605) or GFP–Rta(NLSm) protein were transfected into NA cells for 24 h and cells were examined under a fluorescence microscope. (d) Subcellular localization of Rta and Rta(NLSm) in 293 cells. The vector plasmid pSG5 or plasmids expressing Rta or Rta(NLSm) were transfected into 293 cells for 24 h and the localization of Rta was detected by an immunofluorescence assay using the anti-Rta mAb 8C12.

 
Ectopic expression of Rta can trigger EBV reactivation in NA cells (Chang et al., 2004). To study the induction of EBV reactivation by Rta and its NLS mutant, we transfected NA cells with plasmids expressing various GFP–Rta fusion proteins for 48 h and an immunoblot assay was carried out to detect the expression of BMRF1 as an indicator of the EBV lytic cycle (Chang et al., 2004). Fig. 2(a) shows that GFP–Rta(1–605) was able to induce virus reactivation. The inability of GFP–Rta(1–441) and GFP–Rta(100–605) to induce the lytic cycle (Fig. 2a) was consistent with the notion that Rta-mediated transactivation requires both the amino-terminal region (containing DNA-binding and dimerization domains) and the carboxyl-terminal region (containing a potent transactivation domain) of Rta (Hardwick et al., 1992; Manet et al., 1991). Interestingly and somewhat unexpectedly, GFP–Rta(NLSm) induced expression of BMRF1 at a level similar to that induced by GFP–Rta(1–605) (Fig. 2a), indicating that the cytoplasmic Rta mutant was also able to trigger EBV reactivation. To confirm this phenomenon, plasmids expressing Rta or Rta(NLSm) without the GFP tag were transfected into NA cells for 48 h and the expression of EBV lytic proteins was examined using an immunoblot assay. When Rta and Rta(NLSm) were expressed at a similar level, the induction of Zta, BMRF1 and BGLF5 was also detected at a comparable level (Fig. 2b). Therefore, disruption of the NLS did not abolish the ability of Rta to disrupt EBV latency.



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Fig. 2. Induction of EBV lytic proteins by ectopic expression of Rta or an Rta NLS mutant in NA cells. (a) Induction of BMRF1 by GFP–Rta fusion proteins. NA cells were transfected with plasmids expressing GFP or various GFP–Rta fusion proteins for 48 h and then subjected to an immunoblot assay for detection of the indicated proteins. Antibodies used in this assay included JL-8 (anti-GFP; BD Biosciences Clontech), 88A9 (anti-BMRF1; Tsai et al., 1991) and AC-15 (anti-{beta}-actin; Sigma). NA cells treated with 12-O-tetradecanoylphorbol-13-acetate and sodium n-butyrate (TPA+SB) were used as a positive control for BMRF1 expression. (b) Induction of EBV lytic proteins by Rta and Rta(NLSm). NA cells were transfected with the vector plasmid pSG5 or with plasmids expressing Rta or Rta(NLSm) for 48 h and then subjected to an immunoblot assay for detection of the indicated proteins. Antibodies used in this assay included 467 (anti-Rta), 4F10 (anti-Zta; Tsai et al., 1997), 88A9, 311H (anti-BGLF5; Tsai et al., 1997) and AC-15. (c) Expression kinetics of EBV lytic proteins induced by GFP–Rta and GFP–Rta(NLSm). NA cells were transfected with plasmids expressing GFP–Rta or GFP–Rta(NLSm) for the indicated times (h) and then subjected to an immunoblot assay for detection of the indicated proteins. Antibodies used in this assay included 467, 4F10, 88A9, 311H, AC-15 and a polyclonal antibody against BALF2.

 
Detailed analysis of the lytic expression kinetics induced by GFP–Rta(1–605) and GFP–Rta(NLSm) in NA cells, however, revealed some notable differences. While GFP–Rta(1–605) and GFP–Rta(NLSm) induced endogenous Zta, Rta and BMRF1 with similar kinetics, GFP–Rta(NLSm)-mediated induction of BGLF5 and BALF2 showed a significant delay in comparison with that induced by GFP–Rta(1–605) (Fig. 2c). Such a delay is likely to reflect the fact that the cytoplasmic Rta mutant cannot induce these genes by itself and, instead, it may upregulate these genes by induced endogenous Rta. In support of this explanation, activation of both BGLF5 and BALF2 promoters is through a mechanism involving direct DNA binding of Rta, and could not be achieved by the cytoplasmic Rta mutant (Hung & Liu, 1999; and unpublished data). Accordingly, while GFP–Rta(1–605) induced BGLF5 and BALF2 from 16 h post-transfection, GFP–Rta(NLSm) could not upregulate these genes until 24 h post-transfection, when endogenous Rta might have been induced and accumulated (Fig. 2c).

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 50–60 % of the transactivation activity of wild-type Rta (Fig. 3a, 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.



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Fig. 3. Activation of viral promoters by Rta and Rta(NLSm). 293 cells were transfected with reporter plasmids containing Zp (a, d), Rp (b, e) or the promoter of BGLF5 (c) in conjunction with the vector plasmid pSG5 or plasmids expressing Rta or Rta(NLSm). At 48 h post-transfection, a luciferase assay was performed. The promoterless reporter plasmids pGL2-basic and pGL3-basic were used as controls. The experiment was carried out in duplicate and error bars are given. To titrate the transactivation activities of Rta and Rta(NLSm), a series of smaller amounts of the effector plasmids was used as indicated (d, e) and the expression levels of Rta and Rta(NLSm) were examined in an immunoblot assay (f).

 
In summary, we identified 410KRKK413 as a critical NLS of Rta and generated an Rta NLS mutant showing cytoplasmic localization. The Rta mutant lost the ability to induce BGLF5 but still activated Zp and Rp and initiated the EBV lytic cycle. This is the first study to demonstrate that there are differential requirements for the subcellular localization of Rta to induce target genes.

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 GFP–Rta(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 GFP–Rta(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
 
We thank Dr S. Diane Hayward for providing the original Rta-expressing plasmid and Dr Shih-Tung Liu for providing the reporter plasmids Zp-Luc and Rp-Luc. The excellent technical assistance of Chia-Sui Chen, Sz-Chi Chen, Sheng-Yen Hwang, Yi-Chieh Lin and Chung-Chun Wu is deeply appreciated. This work was supported by the National Health Research Institutes (NHRI-EX89-B708P and NHRI-EX90-9012BP) and National Science Council (NSC-89-2320-B002-201 and NSC-91-2320-B002-179). Y. C. was a recipient of an NHRI Postdoctoral Fellowship Award (RE90N003) from June 2001 to May 2003.


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Received 27 August 2004; accepted 1 November 2004.