Recruitment of the tumour suppressor protein p73 by Kaposi’s Sarcoma Herpesvirus latent nuclear antigen contributes to the survival of primary effusion lymphoma cells

Abstract

Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS) and two rare lymphoproliferative disorders, primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman’s disease (MCD). The KSHV latency-associated nuclear antigen-1 (LANA), required for the replication and maintenance of latent viral episomal DNA, is involved in the transcriptional regulation of viral and cellular genes and interacts with different cellular proteins, including the tumour suppressor p53. Here, we report that LANA also recruits the p53-related nuclear transcription factor p73, which influences cellular processes like DNA damage response, cell cycle progression and apoptosis. Both the full-length isoform TAp73α, as well as its dominant negative regulator ΔNp73α, interact with LANA. LANA affects TAp73α stability and sub-nuclear localisation, as well as TAp73α-mediated transcriptional activation of target genes. We observed that the small-molecule inhibitor Nutlin-3, which disrupts the interaction of p53 and p73 with MDM2, induces apoptotic cell death in p53 wild-type, as well as p53-mutant PEL cell lines, suggesting a possible involvement of p73. The small-molecule RETRA, which activates p73 in the context of mutant p53, leads to the induction of apoptosis in p53-mutant PEL cell lines. RNAi-mediated knockdown of p73 confirmed that these effects depend on the presence of the p73 protein. Furthermore, both Nutlin-3 and RETRA disrupt the LANA–p73 interaction in different PEL cell lines. These results suggest that LANA modulates p73 function and that the LANA–p73 interaction may represent a therapeutic target to interfere with the survival of latently KSHV-infected cells.

Introduction

Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS) and two rare lymphoproliferative disorders, primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman’s disease (MCD).1, 2, 3 Even though KSHV is recognised as a tumour virus, the mechanisms underlying its tumourigenic effects are incompletely understood. KSHV infection of primary endothelial cells extends cell survival and induces angiogenesis without resulting in the transformation of endothelial cells,4 and causes chromosomal instability.5 Although not capable of transforming mature B-cells in culture,6 KSHV is required for the survival of PEL cells in culture.7, 8, 9

The majority of KSHV-infected cells is latently infected. Among the few viral genes expressed during latency, latency-associated nuclear antigen-1 (LANA) is required for latent viral replication and segregation of the viral genome during mitosis by tethering the viral genome to cellular chromatin. Furthermore, LANA interacts with many cellular proteins, including the tumour suppressors p53 and pRb, and regulates the expression of several cellular and viral genes.10 Among nearly 200 LANA-regulated cellular genes, seven are related to p53 signalling.11

Two homologues of p53, p63 and p73, also occur in mammalian cells. Although p53 is a well-known tumour suppressor protein, the role of p73 is more diverse and includes regulation of the cell cycle, senescence, apoptosis and an involvement in neuronal development.12 p53 and p73 share a similar protein architecture. In contrast to p53, the p73 gene is expressed in a variety of alternatively spliced variants.13 Internal promoters lead to the expression of transcriptionally active as well as N-terminally truncated (ΔN) isoforms that lack the transactivating (TA) domain and act as dominant negative regulators of p53 and p63 or p73 isoforms possessing the TA domain.14

p53 and p73 can activate an overlapping set of cellular genes affecting cell cycle regulation, DNA damage response and apoptosis, such as p21, gadd45 and bax.12 The key regulator of p53 is mouse double minute-2 (MDM2), promoting ubiquitination and subsequent degradation of p53 (Oliner et al.,15 Haupt et al.,16 Honda et al.17 and Kubbutat et al.18). In contrast, MDM2 associates with p73 and inhibits its transcriptional activity by preventing its acetylation by p300/CPB and subsequent activation.19 Many viruses antagonise p53 and/or p73; examples include adenovirus E1B55K and SV40 large T-antigen,20, 21, 22 HTLV-1 Tax23 and the E6 and E7 proteins of HPV.24, 25

Unlike in the majority of all cancers,26 p53 mutations are infrequent in KSHV-associated malignancies,27, 28 and the role of p53 in KSHV-associated pathogenesis appears to be complex. LANA interacts directly with p53 and represses p53-mediated transcriptional activity.29 Furthermore, p53 was reported to be ubiquitinated and degraded in the presence of LANA.30, 31 However, p53-mediated DNA damage signalling was reported to be intact in PEL cells and activation of the p53 pathway using the MDM2 small-molecule inhibitor Nutlin-3 caused specific activation of apoptosis in PEL cells with wild-type p53 (Petre et al.32 and Sarek et al.33). In PEL cells, LANA exists in a complex with p53 and MDM2, which disintegrates upon activation of p53 by DNA damage or treatment with the MDM2 inhibitor Nutlin-3, thereby restoring the normal function of the p53 tumour suppressor protein.34

Here, we investigate if LANA affects the p53 family member p73. We report that LANA interacts with two p73 isoforms and stabilises both post-transcriptionally. The MDM2 small-molecule inhibitor Nutlin-3, known to disrupt the interaction of MDM2 with p53 and with p73, as well as the small-molecule RETRA, known to release p73 from its interaction with mutant p53, both disrupt the LANA-p73 complex and induce apoptosis in a range of PEL cell lines with mutant or, in the case of Nutlin-3, also wild-type p53. Furthermore, the induction of apoptosis by Nutlin-3 or RETRA in PEL cell lines is substantially diminished if p73 protein levels are transiently reduced by RNAi, especially in p53-mutant cell lines. These results suggest that the ability of LANA to modulate p73 function contributes to the survival of PEL cells, in particular in the absence of functional p53.

Results

LANA interacts with TAp73α and ΔNp73α

Many oncogenic viruses manipulate the tumour suppressor protein p53 and some also affect the p53-family member p73. We investigated if LANA influences p73 function and found that LANA transfected into p53-null H1299 cells co-immunoprecipitates with transfected TAp73α (Figure 1a) and ΔNp73α (Figure 1b). These interactions could be confirmed with co-immunoprecipitation experiments in p53 wild-type HEK 293T cells (Supplementary Figure S1), as well as in naturally KSHV-infected PEL cell lines. In the BC3 PEL cell line, endogenously expressed LANA interacted with microporated TAp73α and ΔNp73α, whereas no interaction could be detected in KSHV-negative BJAB (Figures 1c, d). The interaction of endogenous LANA with microporated TAp73α was reproducibly stronger in comparison with its binding to microporated ΔNp73α (Figure 1d). Furthermore, the interaction of TAp73α and LANA could also be demonstrated with endogenously expressed TAp73α and LANA in all PEL cell lines tested (BC1, BC3, BCP1, BCBL1) (Figure 1e). These results demonstrate that LANA, in addition to the previously reported interaction with p53, interacts with at least two isoforms of p73.

Figure 1
figure1

LANA interacts with TAp73α and ΔNp73α. (a) Co-immunoprecipitation (Co-IP) of LANA with HA-tagged TAp73α. H1299 cells were co-transfected with the indicated expression constructs or pcDNA3.1(−) as empty vector control and lysed after 48 h. LANA was immunoprecipitated from cleared lysates and (co-)immunoprecipitated proteins were analysed by SDS–PAGE and immunoblotting using LANA and HA antibodies. (b) Co-IP of LANA with HA-tagged ΔNp73α. Experiments were performed as described for panel a. (c) Co-IP of endogenous LANA with HA-tagged TAp73α in B-cells. KSHV-negative BJAB and KSHV-positive BC3 were microporated with the expression construct for HA-tagged TAp73α or pcDNA3.1(−) as empty vector control and lysed after 48 h. Co-IP and immunoblotting were performed, as described for a, using antibodies against LANA and TAp73 to detect immunoprecipitated proteins. (d) Co-IP of endogenous LANA with TAp73α and ΔNp73α in BC3 cells. BC3 cells were microporated with the indicated HA-tagged expression constructs or pcDNA3.1(−) as empty vector control and lysed after 48 h. Co-IP was performed, as described in a. (e) Co-IP of endogenous LANA with endogenously expressed TAp73α in different PEL cell lines. Co-IP and IB were performed, as described for a, using antibodies to LANA and TAp73 to detect immunoprecipitated proteins. As a control, duplicate samples were immunoprecipitated with a control IgG.

In agreement with the observed interaction of LANA and TAp73α, gel filtration chromatography of naturally KSHV-infected BC3 revealed that TAp73α eluted in complexes between 400–600 kDa (Supplementary Figure S2A) that also contain LANA, p53 and MDM2. This suggests the existence of higher-order complexes consisting of LANA, MDM2 and p73 or LANA, MDM2 and p53, as previously shown.33, 34 Several viral proteins including KSHV ORF69 (Santarelli et al.)35 and LANA36 are found to be associated with the nuclear matrix, a nuclear substructure that remains after the majority of DNA and soluble as well as chromatin-bound proteins have been removed from the nucleus with high-salt concentrations (Supplementary Figure S2B). Furthermore, p73 is redistributed and translocated to the nuclear matrix in response to ionising radiation.37 To investigate if LANA also affects the nuclear distribution of TAp73α, we performed nuclear matrix fractionations of KSHV-positive B-cell lines and KSHV-negative BJAB cells. In line with Ben-Yehoyada et al.,37 TAp73α is redistributed to the nuclear matrix (fraction IV) of KSHV-negative cells (BJAB) 48 h after ionising irradiation (20Gy) (Supplementary Figure S2C). Although TAp73α was not detectable in the nuclear matrix of untreated BJAB, we could detect TAp73α in the nuclear matrix in all KSHV-positive PEL cell lines tested (Supplementary Figure S2C), as well as in BJAB latently infected with KSHV (BJAB rKSHV.219) (Supplementary Figure S2D, right panel) without the need for irradiation. Employing a BJAB cell line with doxycycline-inducible LANA expression, we observed that, as the expression of TAp73α was only slightly increased in presence of LANA (Supplementary Figure 2D, left panel), the induced expression of LANA alone was sufficient to cause redistribution of TAp73α to the nuclear matrix (Supplementary Figure S2D, right panel). Together, these results underline the interaction of LANA and TAp73α and indicate that LANA and TAp73α form a complex in vivo.

p53, TAp73α and ΔNp73α share a common binding region in the C-terminal domain of LANA

As p53 and p73 share a common domain architecture, we next investigated the binding regions of these proteins in LANA. GST-pulldown experiments with N- and C-terminal truncation mutants of LANA revealed that p53, as well as TAp73α and ΔNp73α, show binding to both regions of LANA (Figures 2a–c). We further analysed the binding of p53 and TAp73α within the C-terminal domain of LANA and observed a similar binding pattern of both proteins to the different truncated LANA GST fusion proteins (Figures 2a–d), in particular to the proteins LANA C, C4, C5, C8 and C8b. Therefore, we concluded that the construct C8b, consisting of amino acids 1026–1055, contains a common binding region for p53 and TAp73α within the C-terminus of LANA (Figure 2a). The transactivation domain of TAp73α appears to be negligible for the interaction, as TAp73α and ΔNp73α both bind to the N- and C-terminus of LANA.

Figure 2
figure2

p53, TAp73α and ΔNp73α share a common binding region in the C-terminal domain of LANA. (a) Schematic diagram of LANA truncation mutants fused to GST. Interaction of LANA constructs with TAp73α and p53 is marked (+, strong binding; −, no/weak binding). (b) Immunoblot of HEK 293T cells transfected with the indicated HA-tagged expression constructs or pcDNA3.1(−) as empty vector control, lysed after 48 h for GST-pulldown experiments (input). (c and d) GST fusion constructs, as depicted in a, were incubated for GST-pulldown assays with HEK 293T lysates, as depicted in b. After extensive washing, protein interaction was analysed by SDS–PAGE and immunoblotting with an HA antibody. Expression of GST-constructs was verified by Ponceau S staining.

LANA stabilises TAp73α and ΔNp73α protein levels

p53 activity is closely related to its stability and LANA was reported to promote p53 degradation.31 Therefore, we investigated the effect of LANA on the stability of p73. After blocking de novo protein synthesis with cycloheximide (CHX) in HeLa cells transfected with HA-TAp73α or HA-ΔNp73α, TAp73α and ΔNp73α levels decreased more slowly in the presence of LANA, suggesting an increased stability of TAp73α and ΔNp73α in the complex with LANA (Figures 3a and b).

Figure 3
figure3

TAp73α and ΔNp73α protein levels are stabilised post-transcriptionally by LANA. (a) HeLa cells were co-transfected with LANA, HA-tagged TAp73α or pcDNA3.1(−) as empty vector control and cycloheximide (CHX) was added 24 h post transfection. At the indicated time points after addition of CHX, cells were lysed and protein levels were analysed by SDS–PAGE and immunoblotted with LANA, HA and actin antibodies. TAp73α and actin levels were digitally quantified by ImageJ, and TAp73α levels were normalised to actin. (b) HeLa cells were co-transfected with LANA, HA-tagged ΔNp73α or pcDNA3.1(−) as empty vector control and the experiment was performed, as described for a.

Small molecules Nutlin-3 and RETRA induce apoptotic cell death in KSHV-positive PEL cell lines

To investigate the functional importance of the LANA–p73 complex, we employed two small-molecule compounds shown to affect p73 activity. Nutlin-3 disrupts the interaction between MDM2 and p53, as well as between MDM2 and p73, leading to the activation of p53 and p73 and their downstream targets.38, 39 Nutlin-3 had already been tested on PEL cell lines and found to cause cell death in PEL cell lines with wild-type p53 status, but not in KSHV-uninfected B-cell lines.32, 33, 34 In cells with mutated p53, the small-molecule compound RETRA releases p73 from an inhibitory complex with mutant p53 and thereby also restores p73 function.40

We first investigated the effect of Nutlin-3 on the metabolic activity of PEL cells with either wild-type (BC3, BC1) or mutant p53 (BCP1, BCBL1, CroAP/6), as well as KSHV-negative B-cell lines with mutant p53 (BJAB, DG75), using a MTT assay. Although the CroAP/6 cell line had been described as p53 wild-type in the gene segment where most mutations occur in other cancers,41 we found, by sequencing the entire p53 gene, a deletion of 16 bp within intron 3 that might affect splicing (Supplementary Table 1). All PEL cell lines tested, independently of their p53 status, were sensitive to Nutlin-3 treatment at the dosage of 7 μM: the metabolic activity was decreased by 70–90% after 96 h of Nutlin-3 treatment in all PEL cell lines except BCBL1, which showed a moderate decrease of 40% in metabolic activity (Figure 4a). We confirmed functional signalling up- and downstream of p53 and p73 in Nutlin-3-treated cell lines. Treatment of the p53 wild-type PEL cell lines (BC3, BC1) with Nutlin-3 for 12 h led to an increase in p53, MDM2 and p21 protein levels, as well as to phosphorylation of p53 on Ser15, and increased the levels of phosphorylated H2AX (γH2AX), a marker of DNA damage, although the mutant p53, KSHV-negative BJAB and DG75 showed no such response (Figure 4b). Interestingly, the KSHV-infected, p53-mutant PEL cell lines (BCBL1, BCP1, CroAP/6) also responded to Nutlin-3 treatment by increased levels of p53, phospho-p53(Ser15), MDM2 and p21.

Figure 4
figure4

Small-molecule inhibitor Nutlin-3 leads to the activation of p53/p73 downstream targets and causes apoptosis in KSHV-positive PEL cell lines independent of their p53 status. (a) Nutlin-3 causes reduced metabolic activity in PEL cell lines. PEL cell lines with wild-type p53 (BC3, BC1) or mutant p53 (BCBL1, BCP1, CroAP/6) and KSHV-negative, mutant p53 cells (BJAB and DG75) were cultured with Nutlin-3 (7 μM) or vehicle control (DMSO). Metabolic activity was evaluated at the indicated time points by MTT assay and results are shown as curves displaying the percentage relative to the vehicle-treated sample from the same time point. Results are presented as the mean of triplicate samples±s.d., and the experiment shown is representative of at least three independent experiments. (b) Nutlin-3 induces p53, MDM2 and p53/p73 downstream target- and DNA damage response factors in PEL cell lines independently of their p53 status. Cells were incubated for 12 h with Nutlin-3 (7 μM) (+) or vehicle control (DMSO) (−) and whole-cell lysates were analysed by SDS–PAGE and immunoblotting using the indicated antibodies. (c) Nutlin-3 induces apoptosis in PEL cell lines. PEL cell lines with wild-type p53 (BC3, BC1), mutant p53 (BCBL1, BCP1) and KSHV-negative, mutant p53 cells (BJAB and DG75) cultured for 24 h with Nutlin-3 (7 μM) or vehicle control (DMSO) were analysed by a luminescent Caspase 3/7 activity assay. Samples were normalised to cell viability as assessed by trypan blue exclusion assay and the activity of the DMSO sample was subtracted as background. Results are presented as the mean of two independent experiments±s.d. (d) Nutlin-3 induces PARP-1 cleavage. Cells were incubated for 24 h with Nutlin-3 (7 μM) (+) or vehicle control (DMSO) (−) and whole-cell lysates were analysed by SDS–PAGE and immunoblotting using the indicated antibodies. The arrow indicates a PARP-1 cleavage product.

Trypan blue exclusion assay indicated that Nutlin-3 treatment leads to induced cell death in all PEL cell lines tested (Supplementary Figure S3A). Furthermore, we performed an effector Caspase 3/7 activity assay to analyse Nutlin-3-mediated apoptosis induction directly. We observed an increased Caspase 3/7 activity following Nutlin-3 treatment in all PEL cell lines tested irrespective of their p53 status, but not in KSHV-negative B-cell lines; however, the increase was two to four times higher in p53 wild-type cell lines (BC3, BC1) compared with p53 mutant PEL cell lines (BCBL1, BCP1) (Figure 4c). The induction of apoptosis could be confirmed by immunoblotting for the Caspase 3 target PARP-1 (Poly-(ADP-ribose)-polymerase 1), which showed PARP-1 cleavage, a marker for apoptosis,42 in all PEL cell lines tested, but not in the KSHV-uninfected B-cell lines (Figure 4d). Furthermore, we observed increased levels of the p53/p73 downstream target and pro-apoptotic BAX (Bcl-2-associated X) protein in all PEL cell lines analysed. The susceptibility of both p53 wild-type and p53 mutant PEL cell lines to Nutlin-3 suggests that an additional pathway beyond p53 can be activated by Nutlin-3 in all PEL cell lines.

To further investigate the ability of active TAp73α to induce cell death in PEL cell lines, we tested the effect of the small-molecule RETRA, which is known to suppress mutant p53 cancer cells through a p73-dependent pathway,40 on the proliferation of PEL cell lines with wild-type and mutated p53. RETRA decreased metabolic activity in p53-mutant PEL cell lines (BCBL1, BCP1, CroAP/6) as assessed by MTT assay, whereas no effect was observed in either PEL cell lines with wild-type p53 (BC3, BC1) or KSHV-negative B-cell lines with mutant p53 (BJAB, DG75) (Figure 5a). Furthermore, RETRA induced γH2AX phosphorylation and decreased p53 levels in all PEL cell lines with mutant p53, but not in PEL cell lines with wild-type p53; it also increased the levels of the p53/p73 target p21 in two of the PEL cell lines with mutant p53 (Figure 5b).

Figure 5
figure5

Small-molecule RETRA leads to the activation of p53/p73 downstream targets and causes apoptosis in KSHV-positive PEL cell lines with p53 mutant status. (a) RETRA causes reduced metabolic activity in PEL cell lines. PEL cell lines with wild-type p53 (BC3, BC1) or mutant p53 (BCBL1, BCP1, CroAP/6) and KSHV-negative, mutant p53 cells (BJAB and DG75) were cultured with RETRA (4 μM) or vehicle control (DMSO). Metabolic activity was evaluated at the indicated time points by MTT assay and results are shown as curves displaying the percentage, relative to the vehicle-treated sample from the same time point. Results are presented as the mean of triplicate samples±s.d and the experiment shown is representative of at least three independent experiments. (b) RETRA induces the p53/p73 downstream target p21 and DNA damage marker γH2AX in PEL cell lines with mutant p53 status. Cells were incubated for 12 h with RETRA (4 μM) (+) or vehicle control (DMSO) (−) and whole-cell lysates were analysed by SDS–PAGE and immunoblotting using the indicated antibodies. (c) RETRA induces apoptosis in p53-mutant PEL cell lines. PEL cell lines with wild-type p53 (BC3, BC1), mutant p53 (BCBL1, BCP1) and KSHV-negative, mutant p53 cells (BJAB and DG75) cultured for 24 h with RETRA (5 μM) or vehicle control (DMSO) were analysed by a luminescent Caspase 3/7 activity assay. Samples were normalised to cell viability as assessed by trypan blue exclusion assay and the activity of the DMSO control sample was subtracted as background. Results are presented as the mean of two independent experiments±s.d. (d) RETRA induces PARP-1 cleavage in p53-mutant PEL cell lines. Cells were incubated for 24 h with RETRA (5 μM) (+) or vehicle control (DMSO) (−) and whole-cell lysates were analysed by SDS–PAGE and immunoblotting using the indicated antibodies. The arrow indicates a PARP-1 cleavage product.

The inhibition of metabolic activity and induction of p53/p73-dependent genes observed with RETRA was accompanied by a marked increase in cell death in the p53-mutant PEL cell lines, as assessed by trypan blue exclusion assay (Supplementary Figure S3B). Furthermore, we observed a strong increase of Caspase 3/7 activity (Figure 5c), specifically in the p53-mutant BCBL1 and BCP1 PEL cell lines. Induction of apoptosis by RETRA could be further confirmed by immunoblot revealing increased PARP-1 cleavage in these cell lines, as well as an increase of the extra-large isoform of BIM (Bcl-1-interacting mediator of apoptosis), a BH3-only pro-apoptotic member of the Bcl-2 family (Figure 5d).

Nutlin-3 and RETRA induced apoptosis in PEL cell lines is reduced after p73 knockdown

To elucidate the impact of p73 on the observed effects mediated by Nutlin-3 and RETRA in PEL cell lines, we transiently transfected siRNA targeting p73 24 h before the treatment with Nutlin-3 or RETRA and performed a Caspase 3/7 activity assay. The microporation of PEL cell lines with p73 siRNA led to a decrease of TAp73α protein levels by 60–85%, whereas the expression of p53 or LANA was not affected (Figure 6a). Caspase 3/7 activity was substantially reduced in the p53-mutant cell lines BCBL1 and BCP1 24 h after the treatment with Nutlin-3 (Figure 6b) or RETRA (Figure 6c), confirming the impact of p73 on the observed induction of apoptosis. Interestingly, we observed a moderate reduction of Nutlin-3-induced Caspase 3/7 activity also for the p53 wild-type cell line BC3, suggesting that p73 might also be involved in Nutlin-3-mediated cell death when wild-type p53 is present.

Figure 6
figure6

Nutlin-3 and RETRA-induced apoptosis in PEL cell lines is dependent on TAp73α. (a) PEL cell lines with wild-type p53 (BC3), or mutant p53 (BCBL1, BCP1) were microporated with siRNA targeting all isoforms of p73 or non-targeting siRNA (siCntr) and whole-cell lysates were analysed after 48 h by SDS–PAGE and immunoblotting using the indicated antibodies. (b) PEL cell lines with wild-type p53 (BC3) or mutant p53 (BCP1) were microporated with siRNA targeting all isoforms of p73 or non-targeting siRNA (siCntr) and after 24 h, cells were treated with Nutlin-3 (7 μM) or vehicle control (DMSO). 24 hours after treatment, apoptosis induction was analysed by a luminescent Caspase 3/7 activity assay. Samples were normalised to cell viability as assessed by trypan blue exclusion assay and the activity of the DMSO control was subtracted as background. Results are presented as the mean of three independent experiments±s.d.; statistical analysis was performed using a one-sample t-test (two-tailed) and differences were considered to be significant with P<0.05. (c) PEL cell lines with mutant p53 (BCBL1, BCP1) were microporated with siRNA targeting all isoforms of p73 or non-targeting siRNA (siCntr) and after 24 h, were treated with RETRA (5 μM) or vehicle control (DMSO). 24 h after treatment, apoptosis induction was analysed, as described in b.

Nutlin-3 and RETRA disrupt the LANA–TAp73α complex

The apoptotic effects mediated by Nutlin-3 and RETRA on PEL cell lines were accompanied by a disruption of the LANA–p73 complex. As shown in Figure 7a, treatment of the p53 wild-type PEL cell line BC3 with Nutlin-3 reduced the amount of endogenous TAp73α co-immunoprecipitating with LANA. Likewise, treatment of the p53 mutant PEL cell lines BCP1 and BCBL1 with RETRA abolished the interaction of endogenous TAp73α and LANA (Figure 7b and data not shown). These results suggest that, in particular in p53 mutant PEL cells, disruption of LANA–TAp73α complex restores p73-dependent expression of cellular genes such as p21 or BAX, involved in cell cycle arrest or apoptosis induction, and thereby leads to induction of cell death in PEL cells.

Figure 7
figure7

Small-molecules Nutlin-3 and RETRA disrupt the LANA–TAp73α complex. Input and Co-IP of endogenous LANA with endogenously expressed TAp73α in (a) p53 wild-type BC3 cells, treated with Nutlin-3 (7 μM) (+) or vehicle (DMSO) (−) or (b) p53 mutant BCP1 cells, treated with RETRA (4 μM) (+) or vehicle (DMSO) (−), 12 h before harvest. CoIP was performed, as described in Figure 1e.

Furthermore, the repressive effect of LANA on p53-mediated activation of the p53/p73 downstream target Bax (Wong et al.43 and Supplementary Figure S4A) could also be observed after activation of the bax promoter by TAp73α in co-transfection experiments performed in p53-null H1299 cells (Supplementary Figure S4B), indicating that LANA generally affects not only p53- but also p73-mediated transcriptional regulation.

Taken together, our findings illustrate that the interaction of LANA with TAp73α results in the inhibition of TAp73α without reducing TAp73α protein levels. Disruption of the LANA–TAp73α complex could therefore represent a possible approach to restore TAp73α function.

Discussion

The mechanisms involved in KSHV-associated tumourigenesis are poorly understood. The tumour suppressor p53 has a key role in the DNA damage response, cell cycle regulation and apoptosis when DNA is damaged. p53 is affected by latent (LANA) and lytic (RTA, vIRF-1, -3, K-bZIP) KSHV proteins, indicating its importance for the KSHV life cycle.44, 45, 46, 47 Although p53 interacts with LANA, leading to repressed transcriptional activation of p53-dependent target genes and perhaps its degradation,29, 31 p53-dependent signalling due to DNA damage seems to be intact in KSHV-infected PEL cell lines, as the MDM2 inhibitor Nutlin-3 was shown to induce cell death in KSHV-infected PEL cells.32, 33 This observation has been interpreted to indicate that p53-dependent pathways can still be activated by pharmacological means.

The p53-family member p73 also has a role in response to stress and DNA damage, as TAp73 knockout mice show increased genomic instability, infertility and are tumour prone.48, 49 The structural and functional similarities of p53 and p73 led us to investigate the effects of KSHV on different p73 isoforms.

We found that LANA interacts with at least two different p73 isoforms, the full-length TAp73α and its dominant negative form ΔNp73α. This interaction of LANA and TAp73α is independent of the presence of p53, as we could detect the interaction in p53-null (H1299) and p53 wild-type (HEK 293T) cell lines. TAp73α and p53 share a common binding region in the C-terminal domain of LANA, reflecting their similar domain structure. Furthermore, LANA causes the relocalisation of TAp73α to the nuclear matrix and seems to be present in higher-order complexes with p53 and with TAp73α in PEL cells.

Unlike p53, which has been reported to be ubiquitinated and degraded by the ability of LANA to recruit an EC5S ubiquitin complex,31 TAp73α and ΔNp73α levels appear to be stabilised by LANA. This could be the result of the recruitment of p73 into a LANA-containing complex that would protect p73 against proteolytic degradation. However, despite stabilising TAp73α, LANA inhibits at least some of its functional activity, in particular the ability of TAp73α to activate the p53- and p73-responsive promoter of the pro-apoptotic Bax protein. This effect was specific for TAp73α, as no effect of LANA on ΔNp73α-mediated transcriptional regulation could be observed (data not shown).

The current standard treatment for PEL includes doxorubicin, which activates a p53-dependent DNA damage response in PEL cells.32 Because of its ability to reactivate p53-dependent signalling, Nutlin-3 was suggested as an additional treatment option for patients with wild-type p53 status.32, 33 As Nutlin-3 also affects TAp73α–MDM2 complexes39 and as we observed induction of apoptotic cell death mediated by Nutlin-3 in p53 wild-type and p53 mutant cell lines, we investigated the effect of RETRA on PEL cell growth. RETRA acts by disrupting a complex of mutated p53 and p73, and thereby restores p73 function.40 We observed that RETRA reactivates p73-dependent signalling in p53-mutant PEL cell lines, revealed by the increased expression of the p53/p73-dependent cell cycle regulator p21, and leads to the induction of apoptosis in these cell lines. Furthermore, RETRA induces the expression of BIMEL, a pro-apoptotic member of the Bcl-2 family, shown to be specifically activated by p73 in different experimental settings.50, 51, 52

By silencing p73 expression with the help of siRNA, we could confirm the involvement of p73 in the Nutlin-3 and RETRA-mediated induction of apoptosis in p53 mutant PEL cell lines. The moderate decrease of Nutlin-3-induced Caspase 3/7 activity also in the p53 wild-type cell line BC3 after knockdown of p73 could suggest that p73 might, at least partly, be involved in Nutlin-3-mediated apoptosis in p53 wild-type cells. This potential interplay of p53 and p73 might explain the two to fourfold higher induction of Caspase 3/7 activity after Nutlin-3 treatment in the BC3 and BC1 p53 wild-type cell lines in comparison to p53 mutant BCBL1 and BCP1. As the siRNA used in our experiments targets all p73 isoforms, we cannot exclude that other isoforms than TAp73α might be involved in the observed effect.

The moderate effects of Nutlin-3 on the p53-mutant BCBL1 cell line might be explained by the high basal levels of early and late KSHV lytic proteins detected in this cell line (data not shown), as the induction of the lytic cycle was shown recently to diminish effects of Nutlin-3 on PEL cell lines.53

We also found that both Nutlin-3 and RETRA disrupt the LANA–p73 complex in different cell lines, suggesting that releasing p73 from its interaction with LANA could restore its function. Disruption of the interaction between KSHV LANA and TAp73α with Nutlin-3 and RETRA might antagonise the LANA-mediated post-transcriptional stabilisation of TAp73α, and thereby explain the observation that TAp73α protein levels were not increased after Nutlin-3 or RETRA treatment in PEL cell lines, as described in other experimental systems in the absence of LANA.39, 40

The results shown here could provide an explanation for the seemingly controversial observation that LANA appears to induce p53 degradation,31 yet p53-dependent signalling and DNA damage responses can be restored by compounds such as Nutlin-3. As Nutlin-3 also restores p73-dependent signalling,39 releases p73 from its complex with LANA and induces reduced apoptosis induction after transient p73 knockdown, it seems to act, at least in part, through an activation of the p73 pathway. Our observations suggest that the ability of LANA to neutralise the function of TAp73α, in addition to that of p53, may have an important role in the survival of virus-infected cells, and long-term latent persistence. As p53 and p73 both appear to bind to the same 30aa-region of LANA (amino acids 1026–1055), it is conceivable that they share the same binding site in LANA and that this site could represent a therapeutic target to interfere with viral persistence.

Materials and Methods

DNA expression constructs

The expression vectors for full-length LANA, as well as LANA GST fusion proteins, were described previously.54, 55 In addition, the constructs C8a, C8b and LANA N were generated by PCR (see Supplementary Table 2 for primer sequences). The PCR products were inserted into pGEX4T1 and all constructs were sequenced.

HA-tagged human p53 in pcDNA was kindly provided by M Dobbelstein (University of Göttingen, Germany), HA-tagged TAp73α by Curt M Horvarth (Northwestern University, Evanston, IL, USA) and used after sub-cloning into a pcDNA3.1 vector, and HA-tagged ΔNp73α in pcDNA by M Tomassino (IARC, Lyon, France).

The luciferase reporter construct containing the p53-responsive promoter region of the human gene bax (in pGL3Basic) was kindly provided by K Vousden (Beatson Institute for Cancer Research, Glasgow, UK).

Cell culture and reagents

HEK 293T, HeLa and p53-null H1299 cells were cultured in DMEM, 10% FCS, 50 IU/ml penicillin and 50 μg/ml streptomycin. The KSHV- and EBV-negative B-cell lines DG75 and BJAB, BJAB stably infected with KSHV (S Kati et al., in preparation) and the KSHV-infected PEL cell lines (BC1, BC3, BCBL1, BCP1, CroAP/6) were maintained in RPMI 1640, 20% FCS, 50 IU/ml penicillin and 50 μg/ml streptomycin, and in case of BJAB rKSHV.219, 4.2 μg/ml puromycin. For the doxycycline-inducible eGFP or eGFP-LANA BJAB cell lines, retroviral vectors encoding eGFP or eGFP-LANA as a fusion protein were used to transduce BJAB cells (details available on request). To determine the p53 status of the CroAP/6 cell line, genomic DNA was isolated using the DNA Blood Kit (Qiagen, Hilden, Germany) and the entire p53 gene was sequenced. Nutlin-3 (Enzo Life Sciences, Plymouth, PA, USA) and RETRA (Tocris Bioscience, Bristol, UK) were diluted in DMSO.

Transient transfection and protein stability assay

Adherent cells seeded in six-well plates were transiently transfected with FuGENE transfection reagent (Promega, Madison, WI, USA), whereas B-cells were microporated using the Neon transfection system (Invitrogen, Carlsbad, CA, USA). For CHX kinetics, CHX (50 μl/ml) was added 24 h after transfection and cells were lysed at the indicated time points in TBST lysis buffer (20 mM Tris–HCl, pH 7.5; 1 mM EDTA; 100 mM NaCl; 1% Triton X-100) supplemented with protease inhibitors. Protein amounts were determined using Bradford assay; samples were adjusted accordingly and analysed by SDS–PAGE and immunoblot. For transient siRNA transfection, B-cells were microporated with non-targeting or p73 siRNA (Dharmacon, Lafayette, CO, USA) using the Neon transfection system (Invitrogen).

Binding assays

Production of GST fusion proteins and GST-pulldown assays were performed as previously described.56 For immunoprecipitation assays, the monoclonal antibody α-LANA (ABI, Columbia, MD, USA) was immobilised to Protein G-sepharose beads (GE Healthcare, Uppsala, Sweden). Cells were lysed in TBST lysis buffer with protease inhibitors, and the cleared lysate was incubated with prepared beads overnight at 4 °C. Samples were washed extensively with TBST supplemented with 1% sodium deoxycholate. Beads were resuspended in loading buffer and the samples were analysed by SDS–PAGE and immunoblot.

Immunoblotting

SDS–PAGE and subsequent immunoblotting were performed using the following antibodies: rat α-LANA (ABI), mouse α-p73 (IMG-246, Imgenex, San Diego, CA, USA) (the antibody detects all TAp73 isoforms), rat α-HA (clone 3F10, Roche, Indianapolis, IN, USA), mouse α-p53 (Ab-6, Calbiochem, Nottingham, UK), rabbit α-p53 (FL-393, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse α-phospho p53 (Ser 15) (Cell Signaling Technology, Beverly, MA, USA), mouse α-actin (Sigma, St Louis, MO, USA), mouse α-γH2AX (Upstate, Lake Placid, NY, USA), mouse α-p21 (BD Biosciences Pharmingen, San Jose, CA, USA), mouse α-MDM2 (BD Biosciences Pharmingen), mouse α-PARP-1 (F2, Santa Cruz), rabbit α-BIM (C34C5, Cell Signaling Technology), rabbit α-BAX (D2E11, Cell Signaling Technology). The ImageJ 1.42q software (National Institute of Health, Bethesda, MD, USA) was used to digitally quantify signal intensities of Western blot bands.

MTT cell proliferation assay

Cells (4 × 104 cells per 96-well) were incubated with the indicated inhibitors or vehicle control (DMSO). At the designated time point thereafter, cells were incubated with 50 μg/ml MTT (Sigma) in OPTI-MEM for 2 h at 37 °C. The reaction was stopped with isopropanol and MTT metabolism was measured at an OD550 and normalised to OD620. Results are based on triplicate samples and assays were independently repeated at least three times. Graphs display the average relative to the vehicle-treated sample from the same time point. Error bars represent the s.d.

Caspase 3/7 activity assay

Cells (1.8 × 104 cells per 96-well) were incubated with the indicated inhibitors or vehicle control (DMSO). Caspase 3/7 activity was analysed after 24 h with the Caspase-Glo 3/7 assay (Promega). Results were normalised to cell viability as assessed by trypan blue exclusion assay using a 0.4% trypan blue solution (Sigma). Experiments were performed in duplicate samples. For statistical analysis, one-sample t-test (two-tailed) was performed using GraphPad Prism 5.00 (GraphPad Software, San Diego, CA, USA).

References

  1. 1

    Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 1994; 266: 1865–1869.

  2. 2

    Cesarman E, Chang Y, Moore PS, Said JW, Knowles DM . Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N Engl J Med 1995; 332: 1186–1191.

  3. 3

    Soulier J, Grollet L, Oksenhendler E, Cacoub P, Cazals-Hatem D, Babinet P et al. Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman’s disease. Blood 1995; 86: 1276–1280.

  4. 4

    Wang L, Damania B . Kaposi’s sarcoma-associated herpesvirus confers a survival advantage to endothelial cells. Cancer Res 2008; 68: 4640–4648.

  5. 5

    Pan H, Zhou F, Gao SJ . Kaposi’s sarcoma-associated herpesvirus induction of chromosome instability in primary human endothelial cells. Cancer Res 2004; 64: 4064–4068.

  6. 6

    Kliche S, Kremmer E, Hammerschmidt W, Koszinowski U, Haas J . Persistent infection of Epstein-Barr virus-positive B lymphocytes by human herpesvirus 8. J Virol 1998; 72: 8143–8149.

  7. 7

    Guasparri I, Keller SA, Cesarman EKSHV . vFLIP is essential for the survival of infected lymphoma cells. J Exp Med 2004; 199: 993–1003.

  8. 8

    Godfrey A, Anderson J, Papanastasiou A, Takeuchi Y, Boshoff C . Inhibiting primary effusion lymphoma by lentiviral vectors encoding short hairpin RNA. Blood 2005; 105: 2510–2518.

  9. 9

    Wies E, Mori Y, Hahn A, Kremmer E, Sturzl M, Fleckenstein B et al. The viral interferon-regulatory factor-3 is required for the survival of KSHV-infected primary effusion lymphoma cells. Blood 2008; 111: 320–327.

  10. 10

    Renne R, Barry C, Dittmer D, Compitello N, Brown PO, Ganem D . Modulation of cellular and viral gene expression by the latency-associated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus. J Virol 2001; 75: 458–468.

  11. 11

    An FQ, Compitello N, Horwitz E, Sramkoski M, Knudsen ES, Renne R . The latency-associated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus modulates cellular gene expression and protects lymphoid cells from p16 INK4A-induced cell cycle arrest. J Biol Chem 2005; 280: 3862–3874.

  12. 12

    Melino G, Lu X, Gasco M, Crook T, Knight RA . Functional regulation of p73 and p63: development and cancer. Trends Biochem Sci 2003; 28: 663–670.

  13. 13

    Moll UM, Slade N . p63 and p73: roles in development and tumor formation. Mol Cancer Res 2004; 2: 371–386.

  14. 14

    Grob TJ, Novak U, Maisse C, Barcaroli D, Luthi AU, Pirnia F et al. Human delta Np73 regulates a dominant negative feedback loop for TAp73 and p53. Cell Death Differ 2001; 8: 1213–1223.

  15. 15

    Oliner JD, Pietenpol JA, Thiagalingam S, Gyuris J, Kinzler KW, Vogelstein B . Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53. Nature 1993; 362: 857–860.

  16. 16

    Haupt Y, Maya R, Kazaz A, Oren M . Mdm2 promotes the rapid degradation of p53. Nature 1997; 387: 296–299.

  17. 17

    Honda R, Tanaka H, Yasuda H . Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett 1997; 420: 25–27.

  18. 18

    Kubbutat MH, Jones SN, Vousden KH . Regulation of p53 stability by Mdm2. Nature 1997; 387: 299–303.

  19. 19

    Zeng X, Chen L, Jost CA, Maya R, Keller D, Wang X et al. MDM2 suppresses p73 function without promoting p73 degradation. Mol Cell Biol 1999; 19: 3257–3266.

  20. 20

    Marin MC, Jost CA, Irwin MS, DeCaprio JA, Caput D, Kaelin WG . Viral oncoproteins discriminate between p53 and the p53 homolog p73. Mol Cell Biol 1998; 18: 6316–6324.

  21. 21

    Roth J, Konig C, Wienzek S, Weigel S, Ristea S, Dobbelstein M . Inactivation of p53 but not p73 by adenovirus type 5 E1B 55-kilodalton and E4 34-kilodalton oncoproteins. J Virol 1998; 72: 8510–8516.

  22. 22

    Steegenga WT, Shvarts A, Riteco N, Bos JL, Jochemsen AG . Distinct regulation of p53 and p73 activity by adenovirus E1A, E1B, and E4orf6 proteins. Mol Cell Biol 1999; 19: 3885–3894.

  23. 23

    Kaida A, Ariumi Y, Ueda Y, Lin JY, Hijikata M, Ikawa S et al. Functional impairment of p73 and p51, the p53-related proteins, by the human T-cell leukemia virus type 1 Tax oncoprotein. Oncogene 2000; 19: 827–830.

  24. 24

    Park JS, Kim EJ, Lee JY, Sin HS, Namkoong SE, Um SJ . Functional inactivation of p73, a homolog of p53 tumor suppressor protein, by human papillomavirus E6 proteins. Int J Cancer 2001; 91: 822–827.

  25. 25

    Accardi R, Dong W, Smet A, Cui R, Hautefeuille A, Gabet AS et al. Skin human papillomavirus type 38 alters p53 functions by accumulation of deltaNp73. EMBO Rep 2006; 7: 334–340.

  26. 26

    Hollstein M, Sidransky D, Vogelstein B, Harris CC . p53 mutations in human cancers. Science 1991; 253: 49–53.

  27. 27

    Nador RG, Cesarman E, Chadburn A, Dawson DB, Ansari MQ, Sald J et al. Primary effusion lymphoma: a distinct clinicopathologic entity associated with the Kaposi’s sarcoma-associated herpes virus. Blood 1996; 88: 645–656.

  28. 28

    Katano H, Sato Y, Sata T . Expression of p53 and human herpesvirus-8 (HHV-8)-encoded latency-associated nuclear antigen with inhibition of apoptosis in HHV-8-associated malignancies. Cancer 2001; 92: 3076–3084.

  29. 29

    Friborg J, Kong W, Hottiger MO, Nabel GJ . p53 inhibition by the LANA protein of KSHV protects against cell death. Nature 1999; 402: 889–894.

  30. 30

    Si H, Robertson ES . Kaposi’s sarcoma-associated herpesvirus-encoded latency-associated nuclear antigen induces chromosomal instability through inhibition of p53 function. J Virol 2006; 80: 697–709.

  31. 31

    Cai QL, Knight JS, Verma SC, Zald P, Robertson ES . EC5S ubiquitin complex is recruited by KSHV latent antigen LANA for degradation of the VHL and p53 tumor suppressors. PLoS Pathog 2006; 2: e116.

  32. 32

    Petre CE, Sin SH, Dittmer DP . Functional p53 signaling in Kaposi’s sarcoma-associated herpesvirus lymphomas: implications for therapy. J Virol 2007; 81: 1912–1922.

  33. 33

    Sarek G, Kurki S, Enback J, Iotzova G, Haas J, Laakkonen P et al. Reactivation of the p53 pathway as a treatment modality for KSHV-induced lymphomas. J Clin Invest 2007; 117: 1019–1028.

  34. 34

    Chen W, Hilton IB, Staudt MR, Burd CE, Dittmer DP . Distinct p53, p53:LANA, and LANA complexes in Kaposi’s Sarcoma-associated Herpesvirus Lymphomas. J Virol 2010; 84: 3898–3908.

  35. 35

    Santarelli R, Farina A, Granato M, Gonnella R, Raffa S, Leone L et al. Identification and characterization of the product encoded by ORF69 of Kaposi’s sarcoma-associated herpesvirus. J Virol 2008; 82: 4562–4572.

  36. 36

    Ohsaki E, Suzuki T, Karayama M, Ueda K . Accumulation of LANA at nuclear matrix fraction is important for Kaposi’s sarcoma-associated herpesvirus replication in latency. Virus Res 2009; 139: 74–84.

  37. 37

    Ben-Yehoyada M, Ben-Dor I, Shaul Y . c-Abl tyrosine kinase selectively regulates p73 nuclear matrix association. J Biol Chem 2003; 278: 34475–34482.

  38. 38

    Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004; 303: 844–848.

  39. 39

    Lau LM, Nugent JK, Zhao X, Irwin MS . HDM2 antagonist Nutlin-3 disrupts p73-HDM2 binding and enhances p73 function. Oncogene 2008; 27: 997–1003.

  40. 40

    Kravchenko JE, Ilyinskaya GV, Komarov PG, Agapova LS, Kochetkov DV, Strom E et al. Small-molecule RETRA suppresses mutant p53-bearing cancer cells through a p73-dependent salvage pathway. Proc Natl Acad Sci USA 2008; 105: 6302–6307.

  41. 41

    Carbone A, Cilia AM, Gloghini A, Capello D, Fassone L, Perin T et al. Characterization of a novel HHV-8-positive cell line reveals implications for the pathogenesis and cell cycle control of primary effusion lymphoma. Leukemia 2000; 14: 1301–1309.

  42. 42

    D’Amours D, Sallmann FR, Dixit VM, Poirier GG . Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis. J Cell Sci 2001; 114: 3771–3778.

  43. 43

    Wong LY, Matchett GA, Wilson AC . Transcriptional activation by the Kaposi’s sarcoma-associated herpesvirus latency-associated nuclear antigen is facilitated by an N-terminal chromatin-binding motif. J Virol 2004; 78: 10074–10085.

  44. 44

    Gwack Y, Hwang S, Byun H, Lim C, Kim JW, Choi EJ et al. Kaposi’s sarcoma-associated herpesvirus open reading frame 50 represses p53-induced transcriptional activity and apoptosis. J Virol 2001; 75: 6245–6248.

  45. 45

    Seo T, Park J, Lee D, Hwang SG, Choe J . Viral interferon regulatory factor 1 of Kaposi’s sarcoma-associated herpesvirus binds to p53 and represses p53-dependent transcription and apoptosis. J Virol 2001; 75: 6193–6198.

  46. 46

    Rivas C, Thlick AE, Parravicini C, Moore PS, Chang Y . Kaposi’s sarcoma-associated herpesvirus LANA2 is a B-cell-specific latent viral protein that inhibits p53. J Virol 2001; 75: 429–438.

  47. 47

    Park J, Seo T, Hwang S, Lee D, Gwack Y, Choe J . The K-bZIP protein from Kaposi’s sarcoma-associated herpesvirus interacts with p53 and represses its transcriptional activity. J Virol 2000; 74: 11977–11982.

  48. 48

    Tomasini R, Tsuchihara K, Wilhelm M, Fujitani M, Rufini A, Cheung CC et al. TAp73 knockout shows genomic instability with infertility and tumor suppressor functions. Genes Dev 2008; 22: 2677–2691.

  49. 49

    Rosenbluth JM, Pietenpol JA . The jury is in: p73 is a tumor suppressor after all. Genes Dev 2008; 22: 2591–2595.

  50. 50

    Toh WH, Nam SY, Sabapathy K . An essential role for p73 in regulating mitotic cell death. Cell Death Differ 2010; 17: 787–800.

  51. 51

    Busuttil V, Droin N, McCormick L, Bernassola F, Candi E, Melino G et al. NF-kappaB inhibits T-cell activation-induced, p73-dependent cell death by induction of MDM2. Proc Natl Acad Sci USA 2010; 107: 18061–18066.

  52. 52

    Amin AR, Paul RK, Thakur VS, Agarwal ML . A novel role for p73 in the regulation of Akt-Foxo1a-Bim signaling and apoptosis induced by the plant lectin, Concanavalin A. Cancer Res 2007; 67: 5617–5621.

  53. 53

    Sarek G, Ma L, Enbäck J, Järviluoma A, Moreau P, Haas J et al. Kaposi‘s sarcoma herpesvirus lytic replication compromises apoptotic response to p53 reactivation in virus-induced lymphomas. Oncogene 2013; 32: 1091–1098.

  54. 54

    Viejo-Borbolla A, Kati E, Sheldon JA, Nathan K, Mattsson K, Szekely L et al. A domain in the C-terminal region of latency-associated nuclear antigen 1 of Kaposi’s sarcoma-associated Herpesvirus affects transcriptional activation and binding to nuclear heterochromatin. J Virol 2003; 77: 7093–7100.

  55. 55

    Platt GM, Simpson GR, Mittnacht S, Schulz TF . Latent nuclear antigen of Kaposi’s sarcoma-associated herpesvirus interacts with RING3, a homolog of the Drosophila female sterile homeotic (fsh) gene. J Virol 1999; 73: 9789–9795.

  56. 56

    Viejo-Borbolla A, Ottinger M, Bruning E, Burger A, Konig R, Kati E et al. Brd2/RING3 interacts with a chromatin-binding domain in the Kaposi’s Sarcoma-associated herpesvirus latency-associated nuclear antigen 1 (LANA-1) that is required for multiple functions of LANA-1. J Virol 2005; 79: 13618–13629.

Download references

Acknowledgements

We thank Magdalena Weidner-Glunde for cloning of the GST fusion construct LANA N, as well as Eva Gellermann for cloning the GST fusion constructs C8a and C8b. This work was supported by the DFG IRTG 1273, the EU Integrated Project INCA (LSHC-CT-18730) and the collaborative research centre (CRC) 900 of the Deutsche Forschungsgemeinschaft.

Author information

Correspondence to T F Schulz.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Santag, S., Jäger, W., Karsten, C. et al. Recruitment of the tumour suppressor protein p73 by Kaposi’s Sarcoma Herpesvirus latent nuclear antigen contributes to the survival of primary effusion lymphoma cells. Oncogene 32, 3676–3685 (2013). https://doi.org/10.1038/onc.2012.385

Download citation

Keywords

  • KSHV
  • primary effusion lymphoma
  • LANA
  • p73
  • Nutlin-3
  • RETRA

Further reading