Virology 448 (2014) 33–42

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Human herpesvirus-6B protein U19 contains a p53 BOX I homology motif for HDM2 binding and p53 stabilization Emil Kofod-Olsen a,c, Susanne Pettersson b,1, Maura Wallace b, Ahmed Basim Abduljabar a, Bodil Øster a,1, Ted Hupp b, Per Höllsberg a,n a

Department of Biomedicine, Aarhus University, Aarhus, Denmark Institute of Genetics and Molecular Medicine, Cancer Research UK, University of Edinburgh, Edinburgh, Scotland c Department of Infectious Diseases, Aarhus University Hospital, Denmark b

art ic l e i nf o

a b s t r a c t

Article history: Received 30 June 2013 Returned to author for revisions 29 July 2013 Accepted 1 October 2013 Available online 18 October 2013

In order to establish a successful infection, it is of crucial importance for invading viruses to alter the activities of the regulatory protein p53. Beta-herpesviruses stabilize p53 and likely direct its activities towards generation of a replication-friendly environment. We here study the mechanisms behind HHV6B-induced stabilization and inactivation of p53. Stable transgene expression of the HHV-6B protein U19 was sufficient to achieve upregulation of p53. U19 bound directly to the p53-regulating protein HDM2 in vitro, co-precipitated together with HDM2 in lysates, and co-localized with HDM2 in the nucleus when overexpressed. U19 contained a sequence with a putative p53 BOX I-motif for HDM2 binding. Mutation of the two key amino acids within this motif was sufficient to inhibit all the described U19 functions. Our study provides further insight into p53-modulating strategies used by herpesviruses and elucidates a mechanism used by HHV-6B to circumvent the antiviral response. & 2013 Elsevier Inc. All rights reserved.

Keywords: Human herpesvirus-6B p53 U19 Virus–cell interactions HDM2

Introduction During a viral infection, the tumor-suppressor protein p53 plays a major role in the antiviral defense machinery (CollotTeixeira et al., 2004). P53 is a central regulator of cellular fate after genotoxic stress, mediating cellular arrest followed by repair and survival. If the stress is intense or persists, the cell may respond by a programmed suicide usually through apoptosis (Vousden and Lane, 2007; Vousden and Prives, 2009). It is therefore of utmost importance for invading viruses to circumvent the antiviral activities of p53 in order to establish an infection. The beta-herpesvirus human herpesvirus (HHV)-6B and the closely related viruses HHV-6A and -7 are ubiquitously expressed pathogens in the human population (Okuno et al., 1989; Ward et al., 1993). Children are usually infected with HHV-6B within the first 2 years of life leading to the common childhood disease known as 3-day fever (exanthem subitum) (Yamanishi et al., 1988). Recently, HHV-6A and -6B have been associated with Hodgkin's lymphoma (Lacroix et al., 2007, 2010). This has been coupled to the striking feature that p53 is stabilized during HHV-6B infection (Takemoto et al., 2004; Øster et al., 2005). P53 binds to at least one n

Corresponding author. E-mail address: [email protected] (P. Höllsberg). 1 Present address: Högskolan i Skövde, Skövde, Sweden; Bodil Øster, CLC bio, Aarhus, Denmark. 0042-6822/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.virol.2013.10.002

HHV-6B-encoded protein, known as U14, and this protein appears to be transported into the viral particle along with p53 (Takemoto et al., 2005). In addition, it has been suggested that p53 interacts with the protein DR7B (DR6), but a clear association between these proteins remains to be determined (Lacroix et al., 2010; Schleimann et al., 2009). We and others have previously demonstrated that p53 accumulates massively in HHV-6B-infected cells as the infection progresses, and this accumulation is not caused by an increased transcription, but rather from an increased stability of the p53 protein (Takemoto et al., 2004; Øster et al., 2005). This massively accumulated p53 does, however, not appear to retain any transcriptional activity towards cell-cycle arrest or apoptosis during the infection (Øster et al., 2008). We have recently demonstrated that accumulated p53 in HHV-6B-infected cells does not retain any activity after treatment with gamma radiation (Kofod-Olsen et al., 2013). This inhibition is, at least in part, caused by the protein U19, which on its own rescues cells from p53-dependent programmed cell death, but not p53-independent cell death (Kofod-Olsen et al., 2013). However, it still remains unknown exactly how HHV-6B stabilizes p53. Much of our understanding of herpesvirus regulation of the p53 network comes from studying the cytomegaloviruses (CMVs), which also belong to the beta-herpesvirus family. These viruses are important pathogens in many animals including both humans (HCMV) and mice (MCMV). In CMV-infected cells, p53 accumulates in the nucleus

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early during infection (Kovacs et al., 1996; Wang et al., 2000, 2001). This accumulation and stabilization of the p53 protein is concomitant with a decrease in p53 ubiquitination by the E3-ligase murine double-minute 2 (MDM2, known as HDM2 in humans) (Chen et al., 2007). During HCMV infection, HDM2 interacts with the protein IE2-86 facilitating shuttling to the cytoplasm and proteasome-independent degradation (Zhang et al., 2006). Stabilization of p53 is an early event, as the virus needs p53 activity at an early time-point during the infection, where p53 may function as a transcription factor for viral genes and possibly cellular genes that may provide a replication-friendly environment (Casavant et al., 2006; Hannemann et al., 2009). HDM2-targeted degradation of p53 is one of the main regulatory mechanisms controlling the level and the activity of p53. HDM2 binds p53 at two sites, a small alpha-helix in the p53-BOX1 domain as well as a site in the p53-DB domain (Wallace et al., 2006). This binding facilitates HDM2-mediated K48-linked polyubiquitin chains to be added to p53, targeting it for proteasomal degradation (Kubbutat et al., 1998). Upon p53-activation signals, such as DNA-damage, p53 is modified by multiple posttranslational modifications. Among these are essential phosphorylations in the BOX1-domain, leading to the disruption of the p53– HDM2 interaction, and thereby stabilizing the p53 protein (Canman et al., 1998; Shieh et al., 2000). In this paper, we describe how p53 stabilization and accumulation may occur during HHV-6B infection. We identify the HHV-6Bencoded protein U19 as sufficient for stabilizing p53. We moreover uncover a motif within U19 that mimics the p53 Box I motif for HDM2 binding and demonstrates that this motif is essential for the p53 stabilizing activity of U19.

Results U19 stabilizes p53 in the cytoplasm We have previously found that the U19 protein encoded by the HHV-6B U19 ORF leads to inhibition of p53-induced programmed cell death and that U19 expression appeared to accumulate p53 in the nucleus (Kofod-Olsen et al., 2013). In an earlier study, we demonstrated U19 mRNA expression from HHV-6B-infected MOLT3 and HCT116 cells (Kofod-Olsen et al., 2008). To confirm that a U19 protein was actually expressed during infection, HHV6B-infected cells (24, 48, and 72 hpi) were analyzed by Western blotting (WB) using a polyclonal rabbit antibody raised against a U19 peptide. This antibody identified a protein product expressed during infection, with an expression profile corresponding to the early gene kinetics previously reported for U19 (Fig. 1A) (KofodOlsen et al., 2008; Tsao et al., 2009). A band was only observed in lysates from infected cells that also stained positive using a separate viral marker, DR6 (Fig. 1A). The specificity of the U19 antibody was further confirmed by blocking with a U19-derived peptide, which significantly reduced recognition of U19 by the antibody (Fig. 1B). To determine if the U19 expression influenced the level of p53, we performed WB analysis on an HCT116 clone stably expressing the U19 gene (HCT116-U19-S) (Kofod-Olsen et al., 2008). This clone had increased level of p53 similar to that observed during HHV-6B infection (Fig. 1C). WB with antibodies against p53 demonstrated that HCT116-U19-S cells contained a band with lower MW than p53, which was also observed during HHV-6B infection. The identity of this band is unknown, but may correspond to one of the p53 isoforms or alternatively to a p53degradation product. Separation of cytoplasmic/nuclear fractions from HCT116 wt and U19-S cells demonstrated that p53 almost exclusively accumulated in the cytoplasm in the HCT116-U19-S

cells (Fig. 1D). This was also observed in HHV-6B-infected cells (48 hpi), although with a relatively larger proportion of nuclear accumulation of p53 (Fig. 1E). To determine whether the increase in p53 protein was due to increased stabilization of the protein or increased mRNA levels, we performed real-time PCR with primers specific for p53 mRNA (Fig. 1F). There was no increase in p53 mRNA levels, and the increased p53 levels are thus likely due to increased protein stabilization. In order to test whether the p53 levels during HHV-6B infection were dependent on U19, we introduced a pool of four U19-specific siRNAs into HCT116 cells by nucleofection. Cells were grown for 24 h after transfection and infected with HHV-6B for additional 48 h. The transfection procedure itself induces an upregulation of p53, which is also observed in the mock-treated group (Fig. 2A, lane 1). The presence of siRNA against U19 resulted in a knockdown of U19 (75% drop), concomitantly with a reduction in the level of p53 (30% drop), with only a minor decrease (10%) in the expression of a control protein, 7C7, not targeted by the siRNA (Fig. 2A and B). Whether this minor decrease is caused by the treatment of the cells or is caused by alterations in U19 or p53 expression remains to be determined. The observed reduction in p53 levels is less than the observed reduction in U19, thus this experiment does not rule out the contribution of other viral proteins in the stabilization of p53. In any case, expression of U19 siRNA is sufficient for a reduction in the levels of p53 during HHV-6B infection. HHV-6B and U19 does not block p53 ubiquitination To determine if p53 was still able to be poly-ubiquitinated during infection, cells were infected with HHV-6B for 24 h and subsequently treated with the proteasome inhibitor MG132. Immunoblotting demonstrated ubiquitinated p53 as multiple bands above p53 (Fig. 3A). The relative level of Ub-p53 did not increase when the HHV-6B-infected cells were treated with MG132 (Fig. 3B). To address whether this observation was due to a defect in the ubiquitination system, HHV-6B-infected cells were treated with MG132 and analyzed for total ubiquitination levels (Fig. 3C). Both mock-treated and HHV-6B-infected cells accumulated ubiquitinated proteins after blocking of the proteasome with MG132, indicating that infection did not block ubiquitination in general. These data are supported by previous findings by Takemoto et al. (2004). To determine if p53 behaved in a similar manner in U19-expressing cells, we treated wt and U19-S cells with MG132 and performed immunoblotting for p53. Similar to our observations with HHV-6B, U19 did not prevent polyubiquitination of p53, but did prevent an accumulation during MG132 treatment (Fig. 3D and E). U19 binds HDM2 To test the possibility that U19 could interact directly with the E3-ubiquitin ligase HDM2, which is responsible for p53-degradation, we examined whether U19 contained the p53-binding motif for HDM2. Importantly, U19 contained a sequence with strong similarity to the p53 HDM2-interacting short alpha-helix within box I (Fig. 4A). We have previously reported that U19 is present at PML-nuclear-body-like structures (PML-NB, also known as ND10), where a part of the p53-activation occurs through binding of the HDM2-p53 complex to PML (Kofod-Olsen et al., 2008). To determine if the predicted short alpha-helix of U19 could bind to HDM2, we generated an ELISA with recombinant HDM2 protein and U19 peptide. This showed that the short U19 alpha-helix could indeed bind to recombinant HDM2, whereas a scrambled peptide did not (Fig. 4B). To extend this analysis, we generated recombinant 6xHIS labeled U19 (U19-HIS) and conducted ELISA binding

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Fig. 1. U19 expression stabilizes p53. (A) Western blot analysis of HCT116 cells mock-treated or HHV-6B-infected for 24, 48, and 72 h. The membrane was probed with antibodies against U19 and DR6. (B) Western blot analysis of HCT116 cells infected with HHV-6B for 24 or 48 h. The membrane was probed with antibodies against U19 and GAPDH (loading control) in the presence or absence of a U19 peptide. (C) Western blot analysis of HCT116 wt cells, cells stably expressing U19 (U19-S), mock-treated cells or cells infected with HHV-6B for 48 h. The membrane was probed with antibodies against p53 and GAPDH (loading control). p53Δ indicates expected positions for alternatively spliced p53 proteins (D) Western blot analysis of nuclear and cytoplasmic fractions from wt HCT116 cells or HCT116 U19-S. The membrane was probed with antibodies against U19, p53, and the nuclear/cytoplasmic markers RCC1 and GAPDH. (E) Western blot analysis of nuclear and cytoplasmic fractions from HCT116 cells mocktreated or HHV-6B-infected for 48 h. The membrane was probed with antibodies against 7C7 (infection control), p53, and the nuclear/cytoplasmic markers RCC1 and GAPDH. (F) Real-time PCR on HCT116 wt and U19S cDNA with p53 specific primers. Error bars represent SD of three replicates. Experiments are representatives of at least two independent experiments.

Fig. 2. U19 stabilizes p53 during infection. (A) Western blot analysis of HCT116 cells infected with HHV-6B and treated with different concentrations of a pool of U19 siRNA. The membrane was probed with antibodies against p53, U19, 7C7 (infection control) and GAPDH (loading control). (B) Quantification of band-intensity of Western blot shown in panel (A). The reduction in p53, U19 and 7C7 is shown as percentage drop relative to GAPDH. The quantification is an average of four independent experiments.

assay with recombinant U19 and recombinant HDM2. U19-HIS bound to HDM2, but failed to bind to BSA (Fig. 4C). Furthermore, recombinant U20-HIS (another HHV-6B-encoded protein generated by similar extractions) did not bind to the HDM2coated wells. This indicated that U19 bound directly to HDM2 in vitro. To test whether or not U19 could also interact with HDM2 in vivo, HCT116 cells were transfected with U19-FLAG and immunoprecipitated with anti-HDM2 antibodies and immunoblotted with an anti-FLAG antibody (Fig. 4D). A band of expected size was clearly identified in U19-transfected cells. A band of lesser intensity was visible in the lane with mock-transfected cells. This band is likely the heavy

chain of the precipitated antibody exposed as a result of antibody cross-reactivity. Taken together these data suggested that U19 coimmunoprecipitated with HDM2. The amino acids F19 and W23 within p53 box I are critical for the binding of p53 to HDM2 (Kussie et al., 1996). To examine whether the corresponding amino acids F124 and W128 in U19 were equally important, we generated a U19 peptide with F124S and W128S mutations (U19mut). AlphaScreen proximity ligation assays were performed to determine the interactions between recombinant HDM2 protein and p53, p53mut, U19, or U19mutderived peptides (Fig. 4E). This demonstrated that U19 peptide bound to HDM2 specifically and dependent on F124 and W128.

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Fig. 3. (A) Western blot analysis on HCT116 cells infected with HHV-6B for 24 h and mock-treated or treated with MG132 (10 mM) for additional 24 h. The membrane was probed with antibodies against p53 and GAPDH (loading control). The top panel shows an overexposed p53-probed membrane. (B) Quantification of the fraction of ubiquitinated p53 relative to the total amount of p53 as shown in panel (A). (C) Lysates from (A) probed with an antibody against ubiquitin and GAPDH (loading control). (D) Western blot analysis on HCT116 wt and U19-S cells mock-treated or treated with MG132 (10 mM) for 24 h. The membrane was probed with antibodies against p53 and GAPDH (loading control). (E) Quantification of the fraction of ubiquitinated p53 relative to the total amount of p53 as shown in panel (D). Experiments are representatives of at least two separate experiments.

This demonstrated that U19 contained a p53 box I motif for binding to HDM2. The binding of U19 peptide was of lower affinity than that of p53 peptide and required approximately twice as high concentrations and reached only half the maximum binding to HDM2. It may, however, be in the interest of the virus to only partially inhibit HDM2, as low levels of HDM2 is known to facilitate p53 shuttling to the cytoplasm (Li et al., 2003). We next wanted to examine whether U19 located to the same complex as HDM2 in vivo. We have previously shown that U19 colocalize with PML in small nuclear foci resembling ND10 (KofodOlsen et al., 2008). An HDM2-expressing plasmid was introduced into HCT116 cells together with U19-FLAG. When co-expressed with an empty control plasmid, HDM2 was distributed all over the nucleus (Fig. 5A, row ii). When HDM2 was co-expressed with U19 it was likewise distributed diffusely in the nucleus, but did now also stain ND10 dot-like structures that co-localized with U19 (Fig. 5A, row iii). The co-staining between U19 and HDM2 was not caused by staining in two different z-planes, as z-stack images also revealed the same punctuate staining pattern (Fig. 5A, row iii last panel). To determine whether or not the F124S-W128S double-mutant U19 co-localized with HDM2, a FLAG-tagged version (U19mut-F) was generated. This construct expressed a protein of similar size to wt U19-F (Fig. 5B). HCT116 cells co-transfected with both HDM2 and U19mut-F were then analyzed by confocal microscopy. U19mut-F accumulated exclusively in the cytoplasm (Fig. 5C, row i). HDM2, however, still located to the nucleus (Fig. 5C, row ii), but did not localize to ND10-like foci (Fig. 5C, row iii), as was seen when expressed with U19 (Fig. 5A, row iii). This demonstrated that the predicted HDM2-binding motif in U19 was necessary for the translocation of U19 to the nucleus, whereas

HDM2 translocated normally, and thus did not interact with the mutant. U19-dependent inactivation of p53 is dependent on the HDM2-binding domain To explore the importance of the possible p53-binding alphahelix in U19 for its inhibitory function on p53, we generated lentiviral U19 and U19-F124S-W128S double-mutant packaging vectors. We used a lentiviral transduction system to generate a mixed U19-expressing HCT116 population (U19-LV) and a mixed U19mut expressing population (U19mut-LV). Transduction efficiencies were determined using a GFP-LV vector with the same sequence background as the U19-LV and U19mut-LV vectors. Vectors were used in titers with efficiencies higher than 95%, determined by flow cytometry (data not shown). Similar to what we have previously shown, the mixed U19 population showed increased levels of p53 when compared to a mock population (Fig. 6A). The U19mut population showed similar levels as the mock population, emphasizing a link between the predicted short helix motif of U19 and the stabilization of p53. To analyze whether the mutant could inhibit p53 activity, U19-LV and U19mut-LV cells were γ-irradiated and analyzed for PARP cleavage. U19mut cells showed clear cleavage of PARP after γ-radiation, whereas U19 cells displayed no PARP cleavage upon this treatment (Fig. 6B). To further address the U19-dependent inhibition of γ-radiation, we performed quantitative cell viability analysis, by measuring intracellular ATP levels, on mock-LV, U19-LV, and U19mut-LV cells. Whereas U19 expression rescued the cells from dying, U19mut expression restored a normal sensitive phenotype (Fig. 6C). The kinetics of this death induction was examined by flow cytometry

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Fig. 4. U19 binds to HDM2. (A) Sequence of HDM2 binding BOX I motif in p53 and the homologous motif in U19. Residues within the binding motif are in red, with critical residues in blue. Below, a model of the short p53–HDM2 binding alpha-helix and the presumed structure of the homologous domain in U19 modeled on the p53 structure. Arrows indicate essential amino acid residues F and W. The model was made in Swiss-viewer and PyMol. (B) ELISA with recombinant HDM2. The wells were coated with the indicated peptides, incubated with recombinant HDM2 and followed by incubation with an HDM2-specific antibody. (C) ELISA with recombinant proteins U19-HIS and U20HIS. The wells were coated with recombinant HDM2 or BSA and incubated with U19-HIS or U20-HIS, followed by incubation with a 6  HIS-specific antibody. Measurements were performed in triplicate is shown. Error bars indicate SD. (D) Co-immunoprecipitation of U19 with HDM2. Mock or U19-FLAG-transfected HCT116 cells were immunoprecipitated with anti-HDM2 antibodies and immunoblotted with an anti-FLAG antibody. Left part of the figure shows input lysate and right part of the figure shows immunoprecipitates of beads, Ab control, mock and U19 plasmid-transfected lysates. (E) AlphaScreen proximity ligation assay was performed to determine interactions between HDM2 and p53-, p53mut-, U19- and U19mut-derived peptides. Principle of the assay is indicated on top of the graph, showing donor bead A that is excited and releases singlets of oxygen. If protein–peptide interaction occurs, these singlets of oxygen may reach and excite the acceptor bead B, which emits light. Graph demonstrates the interaction between HDM2 and the indicated peptides as a function of peptide concentration. Experiments are representatives of at least two independent experiments.

analyses with 7AAD staining on mock-LV, U19-LV, and U19mut-LV cells treated with UV- and γ-radiation. None of these cell lines survived UV treatment, whereas U19-LV cells were rescued from γ-radiation-induced cell death (Fig. 6D–I). Nutlin-3 treatment is known to induce cell death in HCT116 cells by binding into the binding-grove in HDM2 (Vassilev et al., 2004). To address whether or not Nutlin-3 treatment could drive U19-expressing cells into cell death, we performed ATP viability assay analysis on mock-LV, U19-LV, and p53
/
cells treated with Nutlin-3 or γ-radiation. Whereas mock-LV cells showed reduced viability after treatment with either Nutlin-3, γ-radiation, or a combination of both, U19-LV and p53
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cells tolerated these treatments (Fig. 7A–D). This indicates that the binding-grove in HDM2 is already blocked in cells expressing U19.

Discussion Invading dsDNA viruses presumably induce a DNA-damage-like status in the nucleus, thereby leading to the activation of p53 (Lilley et al., 2010). Moreover, p53 may be activated through the actions of DNA-sensors in the cytoplasm. Either way it is of utmost importance that the virus gains control of the activities elicited by p53 in order to prevent a programmed suicide by the infected cell. It may, however, not be sufficient to simply block all the activities of p53, as it is likely involved in maintaining a state-of-repair in the infected cell, and thus helps to shape a replication-friendly environment. This certainly appears to be the case during CMV infection, where p53 is used as a transcription factor for viral genes (Casavant et al., 2006; Hannemann et al., 2009). Furthermore, p53 has recently been linked to a growing

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Fig. 5. U19mut fails to translocate to the nucleus. (A) Confocal microscopy images of HCT116 cells co-transfected with U19-FLAG-expressing and HDM2-expressing plasmids. The cells were stained with antibodies against FLAG-tag (green), HDM2 (red) or DAPI stain (blue). The images to the right and under the last panel represent the z-plane along the red and green lines respectively. (B) Western blot analysis of HCT116 cells transfected with a U19-FLAG (U19-F), a U19mut-FLAG (U19mut-F) or a mock plasmid and analyzed with antibodies against the FLAG-tag and GAPDH (loading control). (C) Confocal microscopy images of HCT116 cells co-transfected with U19mut-FLAG and HDM2 plasmids. The cells were stained with antibodies against U19-FLAG-tag (green) and HDM2 (red), or stained with DAPI (blue). Experiments are representatives of at least two independent experiments.

number of cellular homeostatic activities, making complete p53inhibition a poor infection strategy (Vousden and Prives, 2009). HHV-6B appears to alter p53 functions through numerous proteins including U14, U19 and possibly DR6 (Kashanchi et al., 1997; Kofod-Olsen et al., 2013; Takemoto et al., 2005). The observation that U14 brings p53 into the particle supports the notion that the virus needs a certain form of p53 immediately after the viral entry. Our findings show that U19 is likely one of the key proteins involved in shaping the p53-network towards a virusfriendly state. If p53 is needed to help to shape the replication environment, it is important that it is stabilized and redirected in infected cells. During HCMV infection this is achieved by sequestering HDM2 in the cytoplasm and directing it towards degradation. We show that a different strategy is used by HHV-6B, where U19 binds directly to HDM2 and sequesters it to small nuclear foci. This is sufficient to cause the accumulation of p53, primarily in the cytoplasm.

Nutlin-3 is known to displace p53 from HDM2 with IC50 of 90 nM (Shangary and Wang, 2009). Since U19 appears to bind to HDM2 with a lower affinity than p53, Nutlin-3 is also expected to displace U19 from HDM2. Nevertheless, Nutlin-3 does not rescue the cells from the U19 phenotype. This suggests that besides the U19–HDM2 interaction, U19 may have additional effects on p53. These potential effects also appear to require the HDM2-binding site, since the mutant U19 lacks the function of U19. In this respect, it is also known, that Nutlin-3 has additional affects at high concentrations that are independent on p53 (Shangary and Wang, 2009). The accumulated p53 does not appear to maintain activity towards cell-cycle arrest or apoptosis, even after γ-radiation. A model for p53 translocation to the cytoplasm argues that high levels of HDM2 leads to poly-ubiquitin chain attachment to p53, leading to its degradation, whereas small amounts of HDM2 lead to multiple mono-ubiquitin modifications of p53 causing cytoplasmic translocation and stabilization (Li et al., 2003). Thus it is

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Fig. 6. U19 rescues cells from γ-radiation-induced cell death in a manner dependent on the p53 BOX I homology motif for HDM2 binding. (A) Western blot analysis of HCT116 cells transduced with a lentiviral U19 vector, a lentiviral U19 mutant (F124S-W128S) or a lentiviral control vector (mock). The membrane was probed with antibodies against p53, U19 and GAPDH (loading control). (B) Western blot analysis of HCT116 cells transduced with lentiviral U19 (LV-U19), lentiviral U19mut (LV-U19mut), treated with γ-irradiation (30 Gy followed by 24 h of incubation). The membrane was probed with antibodies against PARP and GAPDH (loading control). (C) ATP Cell Viability assay. HCT116 mock, LV-U19 or LV-U19mut cells were treated with γ-radiation followed by 24 h of incubation and luciferase measurements. Measurements were performed in triplicates. Error bars represents SD. P values (t-test) by comparison with mock: U19 24 h: p o 0.008; U19mut 24 h: p 40.1; U19 48 h: po 0.006; U19mut 48 h: p 40.7. (D–I) Flow cytometry analyses of cell death in wt (LV-mock), LV-U19 and LV-U19mut cells treated with mock, γ-radiation (30 Gy followed by 24 h of incubation) or UV (240 J/cm2) and analyzed by 7AAD staining 0, 3, 6, 24, 48, and 72 h post treatment. (E), (G), and (I) show the histograms from 0 and 72 h measurements in (D), (F), and (H), respectively. Experiments are representatives of at least two independent experiments.

also possible that U19-binding of HDM2 leaves a small amount of free HDM2, and that this is sufficient to maintain high cytoplasmic levels of p53, which is not directed towards cell death pathways. The U19-induced inhibition of p53 function is dependent on the amino acids F124 and W128, suggesting that U19–HDM2 interactions

are needed. As the U19 double-mutant fails to translocate to the nucleus, we suggests that the lack of p53 inhibition by the mutant is likely due to failed nuclear localization. One possibility is that U19 needs to bind HDM2 in the cytoplasm and that this binding facilitates its nuclear import. In this respect it is noteworthy that U19 does not

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Fig. 7. U19 inhibits Nutlin-3-induced cell death. (A–C) ATP Cell Viability assay on HCT116 mock, LV-U19 or p53
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cells, treated with Nutlin-3 (Nut3) alone for 1 h, γ-irradiated (γ–IR) 30 Gy or both Nutlin-3 treated and γ-irradiated (N/γ), followed by luciferase measurements after 24 h of incubation. Measurements were performed in triplicates. (D) P values from A to C. P values were generated using two-tailed, unpaired students t-tests. Error bars represent SD. Experiments are representatives of two independent experiments.

contain any known nuclear translocation signals. We cannot rule out that the U19mut protein is misfolded; however, p53 has been shown to fold correctly despite mutations in the BOX I motif. Based on our findings we suggest a model where U19 binds HDM2 and is shuttled to the nucleus. In the nucleus, U19 associates with ND10-like foci together with PML and recruits HDM2. Perhaps this may leave a minor fraction of p53 free to perform non-defensive activities in the infected cell, such as shaping the cellular environment in a more replication-friendly direction. Regulation of cell-cycle progression is in part controlled by p53. Being an important tumor suppressor, p53 is mutated in more than 50% of human cancers. Viral interference with this protein may therefore promote the development of various cancers. Over the last decade, HHV-6B has been associated with different kinds of cancer, predominantly with Hodgkin's lymphoma. There are little data to support the notion that this is due to direct transforming activities. Rather, it is likely that HHV-6B infection augments the transforming activities of other infectious agents, such as EBV. Lacroix et al. (2007) showed that a large number of patients with Hodgkin's lymphoma had a combined HHV-6B/EBV infection. If HHV-6B is important for the development of certain cancers, our findings suggest that U19 may be one of the mechanisms employed by the virus.

Materials and methods Cells and viruses HCT116 wt and HCT116-p53
/
cells were a gift from Bert Vogelstein and Kenneth Kinzler (Brattain et al., 1981; Bunz et al.,

1998). HCT116-U19S cells were generated as previously described (Kofod-Olsen et al., 2008). HCT116 cells were grown in McCoy's 5A medium supplemented with 10% fetal calf serum, glutamine (0.2 g/L), streptavidine (0.2 g/L), penicillin (0.2 g/L) and HEPES (10 mM). HCT116-U19S cells were grown under the presence of 0.6 mg/ml G418 geneticin (Life Technologies, Europe, BV). Lentiviral transduced U19-LV, U19mut-LV and Luc-LV cells were generated as previously described (Kofod-Olsen et al., 2013). HHV-6B (PL-1 strain) were propagated in MOLT3 cells and purified as previously described (Øster and Höllsberg, 2002). Viral titers were determined using the 3H-thymidine incorporation method as previously described (Turcanova et al., 2009). Purified virus was used in dilutions of 1:10, corresponding to a titer of 220 IU/ml. Antibodies and reagents Antibodies used for fluorescence imaging were goat antiDDDDK FITC-conjugated pAb (Abcam Europe, UK) and mouse anti-HDM2 mAb (2A10). Secondary antibodies for fluorescence microscopy were F(ab0 )2 antibody fragment conjugated with Alexa 488 or 546 (Life Technologies). Antibodies used for Western blotting were mouse anti-p53 mAb (DO-7) (Life Technologies), rabbit anti-GAPDH mAb (FL335) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), goat anti-RCC1 mAb (N-19) (Santa Cruz Biotechnology), rabbit anti-PUMA pAb (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-p21 mAb (12D1) (Cell Signaling Technology) and rabbit anti-DDDDK-tag pAb (Abcam). DR6 was visualized using a rabbit anti-DR6 peptide pAb (1:200), a custom made antibody from GenScript, Piscataway, NJ, USA (Schleimann et al., 2009). A polyclonal rabbit anti-U19 was generated for the U19 peptide sequence: LSRHTQTDRSEAMC using peptide-KLH conjugation (GenScript). The antibody was affinity purified to a

E. Kofod-Olsen et al. / Virology 448 (2014) 33–42

purity of 92% and had a titer of 1:512,000 in ELISA using the peptide as an antigen. Secondary antibodies were horseradish peroxidase-conjugated swine anti-rabbit pAb (P0217) (DAKO, Glostrup, Denmark), horseradish peroxidase-conjugated rabbit anti-mouse pAb (P0260) (DAKO) and horseradish peroxidaseconjugated rabbit anti-goat pAb (P0449) (DAKO). Membranes were developed with Chemiluminescence femto (Pierce, Thermo Scientific, Slangerup, Denmark). All antibodies were used in 5% skimmed milk. Nutlin-3 was purchased from Sigma Aldrich and used at concentrations of 10 mM (Sigma Aldrich, Milwaukee, WI). Cells treated with MG132 (Sigma Aldrich) were incubated for 24 h at a concentration of 10 mM. Cells exposed to γ radiation were treated with 30 Gy using a cesium γ-source, followed by the additional culture for 24 h before use. Cells exposed to UV radiation were treated with UV-C light (60 J/cm2) in minimal amounts of media. Immunoprecipitation and Western blotting HCT116 cells were transfected with U19-FLAG (Kofod-Olsen et al., 2008). Cell pellets were lysed in 0.1% Triton lysis buffer and precleared with protein G beads. Protein concentration was determined by Bradford assays following preclearing. Total cell lysate (7 mg) was incubated with 4B2 HDM2 antibody overnight for immunoprecipitation and U19-FLAG protein was detected with an anti-FLAG antibody. Protein G Fast Flow beads were added to lysate antibody mix and incubated for 1 h. Beads were washed and proteins eluted by boiling in a sample buffer. Eluates were separated on a 4–12% NuPAGE gel in MOPS buffer. All other lysates were run on 10% or 12% BIS-TRIS Criterion XT precast gels (Biorad) at 175 V, blotted to nitrocellulose membranes for 105 min at 300 mA, blocked in 5% skimmed milk in 0.01% Tween/TBS or 5% BSA/TBS. Primary antibodies were suspended in 5% skimmed milk/ 0.01% Tween/TBS or 5% BSA/TBS and membranes were incubated overnight at 4 1C, and washed four times in 0.01% Tween/TBS. Secondary antibodies were suspended in 5% skimmed milk/0.01% Tween/TBS for 1 h at room temperature. Protein bands were visualized on a LAS4000 imager using FEMTO chemiluminisens (Pierce).

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with shaking for 1 h at room temperature and read on a Fluoroskan plate reader (Perkin Elmer, Waltham, MA). The biotinylated peptides derived from U19 and p53 had an N-terminal SGSG spacer and a C-terminal amide (U19 wt-peptide: SGSGSPTFSDFWTTAMEFS; U19 mutant peptide: SGSGSPTASDFATTAMEFS (alanine mutated at the critical hydrophobic residues required for HDM2 binding); p53-wt peptide (SGSGPPLSQ ETFSDLWKLLP) and the p53mut peptide: SGSGPPLSQETASD LWKLLP) were obtained from Mimitopes (Clayton, Victoria, Australia) and resuspended in DMSO to a stock concentration of 5 mg/ ml. HDM2 protein was purified as described previously (Shimizu et al., 2002). Flow cytometry Mock (wt) or U19-transduced HCT116 cells were detached by trypsin treatment, harvested, washed twice with PBS and stained for 20 min with 7-amino-actinomycin D (7AAD). Cells were then washed twice with PBS, and analyzed on a Cytomics FC 500 flow cytometer (Beckman Coulter) using the green 488 nm laser. Data were analyzed using the FlowJo software. Confocal microscopy HCT116 cells were grown on poly-L-lysin-coated (Sigma) coverslips (0.17 mm) for 24 h. The cells were washed twice in PBS, fixed in 4% formalin for 30 min, permeabilized in 0.02% Triton-X 100 for 10 min and blocked for 30 min in 5% BSA/PBS. After blocking, cells were incubated for 1 h with primary and secondary antibodies in 1% BSA/PBS, washed three times in PBS and incubated for 1 h with second primary and secondary antibodies, washed twice in PBS and once for 15 min in DAPI/PBS (300 nM). After staining, the glasses were mounted on objectiveslides in a mounting medium (30% glycerol, 8% polyvinylalcohol (Mowiol 4-88), 0.2 M Tris–HCl and 2.5% 1.4-diazobicyclo-[2.2.2]-octane (DABCO)). The slides were imaged with a Carl Zeiss 710 CLSM confocal microscope using the 488 nm line of a Helium–Neon laser, the 546 nm line of an Argon laser and the 405 nm line of a Violet 405 nm diode laser.

ELISA with recombinant proteins

Cell viability assay

U19-HIS and U20-HIS were expressed in Escherichia coli BL21STAR cells by IPTG treatment and extracted on ProBond Ni þ columns following the manufacturer's instructions (Quiagen). The extracted protein was subsequently dried and resuspended in PBS. The 96-well microtiter plates were coated with a 40 ng recombinant HDM2 or BSA overnight at 4 1C. The plates were washed three times in PBS þ0.05% Tween and blocked with PBS þ 0.05% Tweenþ1% BSA for 2 h at 4 1C followed by incubation with recombinant U19-HIS or U20-HIS for 2 h at 4 1C. The plate was incubated with HRP-conjugated anti-HIS antibody for 20 min followed by the addition of a developing reagent (Bio-Rad, Hercules, CA). Reactions were stopped by the addition of 1 M H2SO4 and analyzed on an ELISA-reader at 450 nm.

Cellular viability after treatment was determined using the CellTiter-Glos Luminicent Cell Viability Assay according to the manufacturer's instructions (Promega, Madison, WI, USA). Cells were grown in 50 ml flat-sided growth-tubes, treated and incubated as described in the text, following by resuspension in 1 ml media. For sample measurements, a 100 ml cell sample solution was mixed with a 100-ml CellTiter-Glos Luminicent Cell Viability Assay solution. Luminescence was measured as relative light units using an Ascent Luminoscan with 1 s sampling.

AlphaScreen proximity ligation assay The ability of HDM2 to interact directly with U19 was evaluated using purified components. AlphaScreen proximity ligation assay was performed as described previously (Wallace et al., 2006). Increasing amounts of the indicated biotinylated peptides (in nanograms) were incubated with Streptavidin Donor beads and then incubated with Protein A Acceptor beads pre-coated with the Anti-HDM2 mAb (2A10) and full-length untagged HDM2 protein (200 ng per reaction). The samples were mixed and incubated

Acknowledgments This work was supported by grants from Doctor Sofus Carl Emil Friis and wife Olga Doris Friis’ award and the AU Ideas program, Aarhus University. We thank Bert Vogelstein and Kenneth Kinzler for the kind gift of HCT116 wt and HCT116-p53
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cells, Paulo Lusso for the PL-1 strain of HHV-6B, and Bettina Bundgaard for technical assistance. References Brattain, M.G., Fine, W.D., Khaled, F.M., Thompson, J., Brattain, D.E., 1981. Heterogeneity of malignant cells from a human colonic carcinoma. Cancer Research 41, 1751–1756.

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Bunz, F., Dutriaux, A., Lengauer, C., Waldman, T., Zhou, S., Brown, J.P., Sedivy, J.M., Kinzler, K.W., Vogelstein, B., 1998. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282, 1497–1501. Canman, C.E., Lim, D.S., Cimprich, K.A., Taya, Y., Tamai, K., Sakaguchi, K., Appella, E., Kastan, M.B., Siliciano, J.D., 1998. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281, 1677–1679. Casavant, N.C., Luo, M.H., Rosenke, K., Winegardner, T., Zurawska, A., Fortunato, E.A., 2006. Potential role for p53 in the permissive life cycle of human cytomegalovirus. Journal of Virology 80, 8390–8401. Chen, Z., Knutson, E., Wang, S., Martinez, L.A., Albrecht, T., 2007. Stabilization of p53 in human cytomegalovirus-initiated cells is associated with sequestration of HDM2 and decreased p53 ubiquitination. Journal of Biological Chemistry 282, 29284–29295. Collot-Teixeira, S., Bass, J., Denis, F., Ranger-Rogez, S., 2004. Human tumor suppressor p53 and DNA viruses. Reviews in Medical Virology 14, 301–319. Hannemann, H., Rosenke, K., O’Dowd, J.M., Fortunato, E.A., 2009. The presence of p53 influences the expression of multiple human cytomegalovirus genes at early times postinfection. Journal of Virology 83, 4316–4325. Kashanchi, F., Araujo, J., Doniger, J., Muralidhar, S., Hoch, R., Khleif, S., Mendelson, E., Thompson, J., Azumi, N., Brady, J.N., Luppi, M., Torelli, G., Rosenthal, L.J., 1997. Human herpesvirus 6 (HHV-6) ORF-1 transactivating gene exhibits malignant transforming activity and its protein binds to p53. Oncogene 14, 359–367. Kofod-Olsen, E., Møller, J.M., Schleimann, M.H., Bundgaard, B., Bak, R.O., Øster, B., Mikkelsen, J.G., Hupp, T., Höllsberg, P., 2013. Inhibition of p53-dependent, but not p53-independent, cell death by U19 protein from human herpesvirus 6B. PLoS One 8, e59223. Kofod-Olsen, E., Ross-Hansen, K., Mikkelsen, J.G., Höllsberg, P., 2008. Human herpesvirus 6B U19 protein is a PML-regulated transcriptional activator that localizes to nuclear foci in a PML-independent manner. Journal of General Virology 89, 106–116. Kovacs, A., Weber, M.L., Burns, L.J., Jacob, H.S., Vercellotti, G.M., 1996. Cytoplasmic sequestration of p53 in cytomegalovirus-infected human endothelial cells. American Journal of Pathology 149, 1531–1539. Kubbutat, M.H., Ludwig, R.L., Ashcroft, M., Vousden, K.H., 1998. Regulation of Mdm2-directed degradation by the C terminus of p53. Molecular and Cellular Biology 18, 5690–5698. Kussie, P.H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A.J., Pavletich, N.P., 1996. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 274, 948–953. Lacroix, A., Collot-Teixeira, S., Mardivirin, L., Jaccard, A., Petit, B., Piguet, C., Sturtz, F., Preux, P.M., Bordessoule, D., Ranger-Rogez, S., 2010. Involvement of human herpesvirus-6 variant B in classic Hodgkin's lymphoma via DR7 oncoprotein. Clinical Cancer Research 16, 4711–4721. Lacroix, A., Jaccard, A., Rouzioux, C., Piguet, C., Petit, B., Bordessoule, D., RangerRogez, S., 2007. HHV-6 and EBV DNA quantitation in lymph nodes of 86 patients with Hodgkin's lymphoma. Journal of Medical Virology 79, 1349–1356. Li, M., Brooks, C.L., Wu-Baer, F., Chen, D., Baer, R., Gu, W., 2003. Mono- versus polyubiquitination: differential control of p53 fate by Mdm2. Science 302, 1972–1975. Lilley, C.E., Chaurushiya, M.S., Weitzman, M.D., 2010. Chromatin at the intersection of viral infection and DNA damage. Biochimica et Biophysica Acta 1799, 319–327. Okuno, T., Takahashi, K., Balachandra, K., Shiraki, K., Yamanishi, K., Takahashi, M., Baba, K., 1989. Seroepidemiology of human herpesvirus 6 infection in normal children and adults. Journal of Clinical Microbiology 27, 651–653. Schleimann, M.H., Møller, J.M., Kofod-Olsen, E., Höllsberg, P., 2009. Direct Repeat 6 from human herpesvirus-6B encodes a nuclear protein that forms a complex with the viral DNA processivity factor p41. PLoS ONE 4, e7457.

Shangary, S., Wang, S., 2009. Small-molecule inhibitors of the MDM2-p53 protein– protein interaction to reactivate p53 function: a novel approach for cancer therapy. Annual Review of Pharmacology and Toxicology 49, 223–241. Shieh, S.Y., Ahn, J., Tamai, K., Taya, Y., Prives, C., 2000. The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes & Development 14, 289–300. Shimizu, H., Burch, L.R., Smith, A.J., Dornan, D., Wallace, M., Ball, K.L., Hupp, T.R., 2002. The conformationally flexible S9-S10 linker region in the core domain of p53 contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo. Journal of Biological Chemistry 277, 28446–28458. Takemoto, M., Koike, M., Mori, Y., Yonemoto, S., Sasamoto, Y., Kondo, K., Uchiyama, Y., Yamanishi, K., 2005. Human herpesvirus 6 open reading frame U14 protein and cellular p53 interact with each other and are contained in the virion. Journal of Virology 79, 13037–13046. Takemoto, M., Mori, Y., Ueda, K., Kondo, K., Yamanishi, K., 2004. Productive human herpesvirus 6 infection causes aberrant accumulation of p53 and prevents apoptosis. Journal of General Virology 85, 869–879. Tsao, E.H., Kellam, P., Sin, C.S., Rasaiyaah, J., Griffiths, P.D., Clark, D.A., 2009. Microarray-based determination of the lytic cascade of human herpesvirus 6B. The Journal of General Virology 90, 2581–2591. Turcanova, V.L., Bundgaard, B., Höllsberg, P., 2009. Human herpesvirus-6B induces expression of the human endogenous retrovirus K18-encoded superantigen. Journal of Clinical Virology 46, 15–19. Vassilev, L.T., Vu, B.T., Graves, B., Carvajal, D., Podlaski, F., Filipovic, Z., Kong, N., Kammlott, U., Lukacs, C., Klein, C., Fotouhi, N., Liu, E.A., 2004. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303, 844–848. Vousden, K.H., Lane, D.P., 2007. p53 in health and disease. Nature Reviews Molecular Cell Biology 8, 275–283. Vousden, K.H., Prives, C., 2009. Blinded by the light: the growing complexity of p53. Cell 137, 413–431. Wallace, M., Worrall, E., Pettersson, S., Hupp, T.R., Ball, K.L., 2006. Dual-site regulation of MDM2 E3-ubiquitin ligase activity. Molecular Cell 23, 251–263. Wang, J., Belcher, J.D., Marker, P.H., Wilcken, D.E., Vercellotti, G.M., Wang, X.L., 2001. Cytomegalovirus inhibits p53 nuclear localization signal function. Journal of Molecular Medicine (Berlin) 78, 642–647. Wang, J., Marker, P.H., Belcher, J.D., Wilcken, D.E., Burns, L.J., Vercellotti, G.M., Wang, X.L., 2000. Human cytomegalovirus immediate early proteins upregulate endothelial p53 function. FEBS Letters 474, 213–216. Ward, K.N., Gray, J.J., Fotheringham, M.W., Sheldon, M.J., 1993. IgG antibodies to human herpesvirus-6 in young children: changes in avidity of antibody correlate with time after infection. Journal of Medical Virology 39, 131–138. Yamanishi, K., Okuno, T., Shiraki, K., Takahashi, M., Kondo, T., Asano, Y., Kurata, T., 1988. Identification of human herpesvirus-6 as a causal agent for exanthem subitum. Lancet 1, 1065–1067. Zhang, Z., Evers, D.L., McCarville, J.F., Dantonel, J.C., Huong, S.M., Huang, E.S., 2006. Evidence that the human cytomegalovirus IE2-86 protein binds mdm2 and facilitates mdm2 degradation. Journal of Virology 80, 3833–3843. Øster, B., Bundgaard, B., Höllsberg, P., 2005. Human herpesvirus 6B induces cell cycle arrest concomitant with p53 phosphorylation and accumulation in T cells. Journal of Virology 79, 1961–1965. Øster, B., Höllsberg, P., 2002. Viral gene expression patterns in human herpesvirus 6B-infected T cells. Journal of Virology 76, 7578–7586. Øster, B., Kofod-Olsen, E., Bundgaard, B., Höllsberg, P., 2008. Restriction of human herpesvirus 6B replication by p53. Journal of General Virology 89, 1106–1113.