Kaposi’s sarcoma-associated herpesvirus latency-associated nuclear antigen dysregulates expression of MCL-1 by targeting FBW7

Primary effusion lymphoma (PEL) is an aggressive B cell lymphoma that is etiologically linked to Kaposi’s sarcoma-associated herpesvirus (KSHV). Despite standard multi-chemotherapy treatment, PEL continues to cause high mortality. Thus, new strategies to control PEL are needed urgently. Here, we show that a phosphodegron motif within the KSHV protein, latency-associated nuclear antigen (LANA), specifically interacts with E3 ubiquitin ligase FBW7, thereby competitively inhibiting the binding of the anti-apoptotic protein MCL-1 to FBW7. Consequently, LANA-FBW7 interaction enhances the stability of MCL-1 by preventing its proteasome-mediated degradation, which inhibits caspase-3-mediated apoptosis in PEL cells. Importantly, MCL-1 inhibitors markedly suppress colony formation on soft agar and tumor growth of KSHV+PEL/BCBL-1 in a xenograft mouse model. These results strongly support the conclusion that high levels of MCL-1 expression enable the oncogenesis of PEL cells and thus, MCL-1 could be a potential drug target for KSHV-associated PEL. This work also unravels a mechanism by which an oncogenic virus perturbs a key component of the ubiquitination pathway to induce tumorigenesis.

Introduction derived mouse xenograft model. Taken together, these findings shed new light on the crucial role of MCL-1 in PEL transformation and identify MCL-1 as a potential therapeutic target for KSHV-associated PEL.

KSHV LANA interacts specifically with FBW7 in a phosphorylationdependent manner
Normally, FBW7 substrates harbor the CPD motif that contains a threonine or serine residue for phosphorylation in the "0" position and serine or glutamic acid residue in the "+4" position, in brief T/S-X-X-X-S/E [4]. While investigating the function of LANA, we identified two consensus CPD motifs in its N-terminal region (Fig 1A) that appear to be recognized by FBW7. To validate whether LANA interacts with FBW7 through its CPD motifs, we first carried out immunoprecipitation (IP) experiments to examine the interaction between LANA and FBW7. Notably, we found that LANA interacted with FBW7 (Fig 1B) but not with other F-box family proteins (Fig 1C). Since substrate phosphorylation is critical for being recognized by FBW7 [2][3][4], we examined whether LANA-FBW7 interaction occurs in a phosphorylationdependent manner. The results showed that LANA dephosphorylated by λ-phosphatase no longer interacted with FBW7 (Fig 1D), suggesting that phosphorylation of LANA is necessary for interaction with FBW7.
To further identify the significant phosphorylation sites that are responsible for LANA-FBW7 interaction, we performed IP assays after generating three different phosphorylation-dead LANA mutants: LANA T177A (designated as LANA-P1), LANA S219A/S223A (designated as LANA-P2/P3), and LANA T117A/S219A/S223A (designated as LANA-P1/P2/P3). Only LANA-P2/P3 bound efficiently to FBW7, suggesting that phosphorylation at threonine 117 (P1) is critical for the LANA-FBW7 interaction (Fig 1E). To eliminate the possibility that different intracellular localization of LANA-P1 from LANA-WT could affect the FBW7-LA-NA-P1 interaction, we performed immunofluorescence assays and found that LANA-P1, similar to LANA, was also localized in the nucleus as was FBW7 (Figs 1F and S1). Taken together, these results suggest that phosphorylation of the first CPD motif within LANA is essential for its interaction with FBW7.

KSHV LANA-FBW7 interaction alters expression of MCL-1 but not LANA
Given that phosphorylation of substrate is required for FBW7 to recognize and induce substrate ubiquitination [7], we investigated whether LANA-FBW7 interaction affects the stability of LANA. Cells were transfected with FBW7 along with LANA or LANA-P1, followed by exposure to cycloheximide (CHX) to block newly synthesized proteins. Intriguingly, the interaction of LANA with FBW7 did not downregulate LANA expression (Fig 2A), indicating that LANA is not a substrate of FBW7, even though FBW7 binds phosphorylated LANA.
Mounting evidence suggests that KSHV LANA deregulates expression of several FBW7 substrates, including c-Myc, Notch-1, and cyclin E, in BCBL-1 cells infected with KSHV [27][28][29]. Therefore, we speculated that LANA-FBW7 interaction potentially induces the stabilization of one of known FBW7 substrates via targeting. To examine this, we generated tetracycline-inducible TREx/BJAB cell lines that ectopically expressed Au-tagged LANA or LANA-P1. These cells were treated with doxycycline (Doxy) together with CHX for various periods of time and then harvested for immunoblot analysis with designated antibodies. Under these conditions, the half-life of MCL-1 was much longer in the presence of LANA-WT than in the presence of the empty vector or LANA-P1 (Fig 2B). In contrast, both LANA-WT and LANA-P1 elevated the intracellular domain of Notch-1 (ICN) expression (Fig 2B). We also found that LANA expression did not change the protein levels of other FBW7's substrates such as mTOR and cyclin E indicating that LANA-FBW7 interaction specifically alters MCL-1 expression (Fig 2B). We note that a previous study showed that the interaction of the C-terminal region of LANA with SEL10 (also known as FBW7) induced the stabilization of ICN [27]. In consistent with this study [27], our results showed that both LANA and LANA-P1 accumulated ICN, while LANA, but not LANA-P1, stabilized MCL-1 (Fig 2B). Our data suggested that MCL-1 is stabilized primarily through the binding of phosphorylated LANA to FBW7. Further analysis of the mechanism by which LANA increases the stability of MCL-1 via interaction with FBW7 revealed that MCL-1 was degraded rapidly when co-expressed with FBW7 (Fig 2C, compared lanes 1-4 with lanes 5-8). However, the half-life of MCL-1 was greatly increased in the presence of LANA but not in the presence of LANA-P1 (Fig 2C, lanes 9-12, compared the upper and lower panels). Collectively, these results indicate that LANA-FBW7 interaction stabilizes MCL-1.

KSHV LANA stabilizes MCL-1 via competitive interaction with FBW7, leading to reduced ubiquitination of MCL-1
Since the stability of MCL-1 protein is determined primarily by the interaction with E3 ubiquitin ligase FBW7 through phosphodegron sequences of MCL-1 [7,30], we hypothesized that LANA might compete with MCL-1 for binding to FBW7, thereby increasing MCL-1 protein levels by blocking its ubiquitin-proteasome-dependent degradation. To address this, we performed competitive protein-binding assays with cells transiently expressing different combinations of LANA-WT, LANA-P1, MCL-1, and FBW7 (Fig 3A). The results showed that as LANA levels increased, FBW7 binding to MCL-1 prominently decreased, but the interaction of FBW7 with MCL-1 was not affected by expression of LANA-P1 (Fig 3A, lanes 8-11). Next, we investigated whether sequestration of FBW7 by LANA affected FBW7-mediated ubiquitination of MCL-1. Consistent with a previous report [7], we found that ubiquitination of MCL-1 absolutely relies on the presence of FBW7 (Fig 3B upper panel, lane 4). Importantly, LANA-WT dramatically decreased MCL-1 ubiquitination (Fig 3B upper  . This result indicates that MCL-1 was mainly modified by K48-linked ubiquitin chain as shown by previous report [31], and LANA suppresses K48-linked ubiquitination of MCL-1, ultimately leading to the stabilization of MCL-1 (Fig 3B).
MCL-1 is considered as a mitochondrial protein [5], while LANA and FBW7 are localized in the nucleus [13,32]. To further understand how nuclear proteins LANA and FBW7 regulate mitochondrial protein MCL-1, their subcellular localization in mitochondria and nucleus was explored using tetracycline-inducible TREx/BJAB cell lines that ectopically expressed Autagged LANA or LANA-P1 (Fig 3C). MCL-1 was predominantly in the mitochondria as 293T cells were co-transfected with Au/LANA and Flag/FBW7. Cell lysates were treated with or without phosphatase, followed by IP with an anti-Flag antibody and IB with an anti-Au antibody. (E) 293T cells were transfected with the indicated combinations of phosphorylation-dead mutant plasmids followed by IP and IB. (F) Vero cells transiently expressing wild-type (WT) LANA or LANA-P1 together with Flag/FBW7, respectively, were fixed, stained with an anti-Au antibody (red) and an anti-Flag antibody (green), and viewed under a confocal microscope. Nuclei (blue) were stained with DAPI. https://doi.org/10.1371/journal.ppat.1009179.g001

PLOS PATHOGENS
Deregulation of the MCL-1 by KSHV LANA

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Deregulation of the MCL-1 by KSHV LANA expected (Fig 3C). Interestingly, we first found that expression of LANA in TREx/BJAB cells significantly increased the nuclear localization of FBW7 compared to vector or LANA-P1 expression (Fig 3C lanes 5-7). More importantly, LANA expression reduced the mitochondria localization of FBW7 (Fig 3C, compared lanes 1 and 3 with lane 2), while highly elevated the expression of MCL-1 (Fig 3C, compared lanes 1 and 3 with lane 2). Consequently, these data indicate that LANA effectively sequesters FBW7 into the nucleus and impairs the FBW7-mediated ubiquitination of MCL-1 in the mitochondria, thereby resulting in accumulation of MCL-1.

Stabilized MCL-1 by LANA inhibits caspase-3-mediated apoptosis
As MCL-1 plays a pivotal role in caspase-3-mediated apoptosis [5,6], we next determined the impact of LANA-mediated stabilization of MCL-1 on the cellular apoptotic response. Three stable TREx/BJAB cell lines expressing empty vector, LANA, or LANA-P1 were treated with Doxy in the presence or absence of etoposide. Apoptosis was then examined by flow cytometry after propidium iodide (PI) and Annexin-V staining, or by immunoblotting with a caspase-3 specific antibody. As shown in Fig 4A, etoposide induced apoptosis of TREx/BJAB-vector (63.1 ± 3.48%) and TREx/BJAB-Au-LANA-P1 cells (67.4 ± 3.17%), but had little effect on TREx/BJAB-Au-LANA cells (21.9 ± 3.98%). Additionally, we observed that cleaved caspase-3 levels were greatly diminished in LANA-expressing cells than compared to vector and LANA-P1-expressing cells (Fig 4B). It has been reported that etoposide destabilizes MCL-1 by targeting FBW7 in a GSK-3β-mediated phosphorylation-dependent manner [33,34]. Consistent with these reports, we found that etoposide decreased endogenous MCL-1 level markedly in vector-and LANA-P1-expressing cells, whereas LANA protected MCL-1 from etoposideinduced degradation (Fig 4B). This result further supports the data presented in Fig 3B showing that LANA reduced ubiquitination of MCL-1 via interaction with FBW7. Moreover, endogenous FBW7 IP assay showed that LANA efficiently competes with FBW7 for MCL-1 interaction both in the presence and absence of etoposide (Fig 4C). Taken together, these results demonstrate that LANA inhibits caspase-3-mediated apoptosis by increasing the stability of MCL-1 in a FBW7-dependent manner.

MCL-1 is highly accumulated upon KSHV infection via LANA-FBW7 interaction-dependent manner
In order to examine the effect of LANA-mediated stabilization of MCL-1 in the context of the KSHV infection, we first generated a LANA-P1 mutant KSHV by replacing Theronine at amino acid 177 in LANA encoded in KSHV BAC16 to Alanine (rKSHV-BAC16-LANA-P1) via "scarless" mutagenesis [35]. To rule out the possibility of second-site mutations, we also constructed a revertant clone in which the wild-type (WT) LANA sequence was restored (rKSHV-BAC16-Rev) (Fig 5A). After validating the recombinant constructs by Nhe I restriction enzyme digestion and DNA sequencing (Fig 5A), we produced infectious virus using iSLK cell lines carrying WT, LANA-P1, and Rev KSHV BAC16 clones (S2A Fig) [35]. We then determined the effect of LANA-P1 mutant on the viral gene expression as well as production of infectious virus. To this end, we induced lytic reactivation of KSHV in iSLK cells, harboring followed by IB with the indicated antibodies. (A, upper and bottom right) Time course of expression was determined by semi-quantification of IB bands. (B) TREx/BJAB LANA/Au and TREx/BJAB LANA-P1/Au cells were treated with doxycycline (Doxy, 1 μg/ml) together with CHX (20 μg/ml) for the indicated periods. Equal amounts of total protein were analyzed by IB with the indicated antibodies. (C) Cells were transfected with the MCL-1 constructs together with the FBW7 and LANA or LANA-P1 constructs. Cells were then treated with CHX (20 μg/ml) for the indicated periods, followed by IB with the designated antibodies. https://doi.org/10.1371/journal.ppat.1009179.g002

PLOS PATHOGENS
Deregulation of the MCL-1 by KSHV LANA We found that MCL-1 is highly accumulated in both BJAB-rKSHV-BAC16 and BJAB-rKSHV-BAC16-Rev cells, but not in BJAB-rKSHV-BAC16-LANA-P1 ( Fig  5B). In addition, we observed that MCL-1 stabilized via rKSHV-infection markedly increased cells proliferation (Fig 5C), and dramatically reduced apoptosis measured by PI staining (Fig  5D). Collectively, our results demonstrate that KSHV LANA appears to be a critical viral protein required for MCL-1 stabilization during KSHV infection.

LANA-mediated stabilization of MCL-1 is essential for survival of KSHVassociated PEL cells
Since LANA, a master regulator of KSHV latency, is highly expressed in all latently infected tumor cells, we examined the relative expression levels of MCL-1 in KSHV-positive PEL cell lines compared to a KSHV-negative cell line. Strikingly, we detected higher expression of MCL-1 in KSHV + BCBL-1, BC-3 cells, and KSHV + EBV + BC-1 cells than in KSHV − BJAB cells (Fig 6A). Consistent with this, endogenous MCL-1 ubiquitination was significantly lower in BCBL-1 cells than in BJAB cells upon MG132 treatment, indicating that high levels of MCL-1 protein correlates inversely with ubiquitination of endogenous MCL-1 (Fig 6B). In addition, we further verified that LANA competes with MCL-1 for FBW7 binding via endogenous FBW7 IP in BCBL-1 cells (S3 Fig). Furthermore, siRNA-mediated depletion of MCL-1 in KSHV + PEL cell lines, such as BCBL-1, BC-3 and BC-1, but not in KSHV -BJAB cells, increased cell death, suggesting that KSHV + PEL cell lines require MCL-1 for survival (Figs 6C and S4A). Thus, elevated expression of MCL-1 in PEL cells prompted us to assess the therapeutic potential of MCL-1 inhibitors AT-101 and A-1210477. AT-101 is a pan-BCL-2 inhibitor with anti-MCL-1 activity [36,37], while A-1210477 is a selective MCL-1 small-molecule inhibitor [37,38]. Treatment of BJAB and PEL cells with AT-101 or A-1210477 suppressed cell growth and induced cell death in KSHV + PEL cells but had very marginal effect on BJAB cells (Figs 6D, S4B, S4C and S4D). Taken together, these data indicate that LANA-mediated stabilization of MCL-1 via sequestering FBW7 from MCL-1 is essential for the survival of PEL cells.

MCL-1 promotes tumorigenesis of KSHV + BCBL-1 cells
Next, to evaluate the anti-tumor activity of MCL-1 inhibitors in KSHV + BCBL-1 cells ex vivo, we performed soft agar colony assays in the absence or presence of MCL-1 inhibitors (AT-101

PLOS PATHOGENS
Deregulation of the MCL-1 by KSHV LANA or A-1210477), followed by crystal violet staining to count colonies. Notably, both co-treatment of BCBL-1 cells with MCL-1 inhibitors and treatment of cells with MCL-1 inhibitors after colony formation led to marked inhibition of colony formation (Fig 7 upper panel and  S5A Fig upper panel), whereas BJAB cells successively formed colonies in soft agar in the presence or absence of MCL-1 inhibitors (Fig 7 bottom panel and S5A Fig bottom panel). To confirm the direct effect of MCL-1 on KSHV-associated transformation, we depleted MCL-1 from BCBL-1 and BJAB cells using siRNA. As expected, depletion of MCL-1 considerably reduced the efficacy of colony formation by BCBL-1 cells, but not by BJAB cells (S5B Fig). Collectively, these results suggest that MCL-1 plays a crucial role in the tumorigenic properties of BCBL-1 cells.

MCL-1 is a potential candidate drug target in KSHV + PEL
To further evaluate the in vivo anti-tumor activity of MCL-1 inhibitors for PEL, we utilized NOD/SCID xenograft mice injected intraperitoneally with BCBL-1 cells, as described previously [39,40]. Two days after injection, mice began receiving a weekly intraperitoneal injection of either DMSO as a delivery vehicle or AT-101 lasting for 6 weeks. We observed abdominal distention in DMSO-treated mice but not in AT-101-treated mice (Fig 8A). Given that abdominal distention reflects the development of ascites, we collected and measured the volume of ascites produced in each mouse. On average, 1.77 ml ascites were collected from DMSO-treated mice versus 0.12 ml from AT-101-treated mice (Fig 8B). The volume of ascites correlated with the increase in body weight (Fig 8C), verifying that weight gain was attributable to tumor establishment. Previous studies reported that mice injected with KSHV + PEL cells, including BCBL-1 cells, exhibited notable splenomegaly when compared with normal NOD/SCID mice due to infiltration of anaplastic cells [41]. Notably, the size of the spleen in AT-101-treated mice was obviously reduced compared to the DMSO-treated control mice (Fig 8D). KSHV + PEL cells, including BCBL-1 cells isolated originally from patients typically expressed CD45 and CD38, but are usually negative for B-cell markers such as CD19, CD20, CD79, and PAX5 [42][43][44]. We also verified the expression of both CD45 and CD38 on the surface of ascites cells of our xenograft model by using FACS analysis, validating our in vivo system for studying tumorigenesis by PEL cells (S6 Fig). These results conclusively confirmed the anti-tumor activity of the MCL-1 inhibitor, AT-101, thereby identifying MCL-1 as a potential therapeutic target for treatment of KSHV + PEL.

Discussion
KSHV-associated PEL has an unfavorable prognosis and treatment options for the patients are highly limited [45]. This calls for an urgent need to understand its mechanism of progression to establish rational for developing alternative target-based therapeutics. Very recently, an attempt has been made at using genome-wide loss-of-function CRISPR screens to verify MCL-1 as a critical host gene in PEL [10]. Moreover, tissue microarray technology revealed high expression of MCL-1 in tissue specimens from KSHV-associated malignancies [10,11]. Nevertheless, the molecular mechanism of increased levels of MCL-1 in PEL and its function in KSHV-associated pathogenesis have yet to be understood. Here, we showed that KSHV LANA interacts specifically with FBW7, which competes for MCL-1 binding and reduces KSHV-infected BJAB were harvested and equal amounts of cell lysates were used for IB with the indicated antibodies. (C) BJAB-rKSHV-BAC16-WT, BJAB-rKSHV-BAC16-LANA-P1, and BJAB-rKSHV-BAC16-Rev cells were counted every 12 h. Error bars represent the SEM for three independent experiments. (D) After treatment with etoposide (50 μM), Cells were then stained with PI and carried out the FACS analysis. Data represent the mean ± SEM and P-values were calculated by Student's t-test. NS, p > 0.05; � , p < 0.05; N = 3. https://doi.org/10.1371/journal.ppat.1009179.g005

PLOS PATHOGENS
Deregulation of the MCL-1 by KSHV LANA Alternative splicing of human FBW7 gene produces three isoforms (α, β, and γ), which show different subcellular localizations given that FBW7 harbors both a nuclear localization signal (NLS) and a nuclear export signal (NES) [46,47]. FBW7-α, which is mainly localized to the nucleus, is thought to carry out most of FBW7 functions even though specific actions for other isoforms have also been described [47]. Recently, Song et. al., reported that FBW7 translocates from the nucleus to cytoplasm when cells are infected with VSV to stabilize RIG-I [48]. Moreover, phosphorylation of FBW7 at specific residues can cause the exclusion of FBW7

PLOS PATHOGENS
Deregulation of the MCL-1 by KSHV LANA from the nucleus resulting in its mislocalization, which can impede the role of FBW7 in degradation of specific substrates [47,49,50]. Since most of the FBW7 substrates are proto-oncogenes, relocalization of FBW7 is also likely to derive transformation. In accordance with this, our study shows that the interaction of LANA with FBW7 elevated the presence of FBW7 in the nucleus while decreased FBW7 level in the mitochondria, which correlated with increased protein expression of MCL-1. Thus, our results suggest that LANA-mediated mislocalization of FBW7 leads to the stabilization of MCL-1.
Additional studies will be necessary to understand the mechanism by which LANA-FBW7 interaction can specifically alter MCL-1 protein expression while not affecting the expression of other FBW7's substrates. There is one possibility that may explain how LANA specifically sequesters FBW7 from MCL-1. FBW7 has been shown to bind to conserved CPD motifs in FBW7's substrates, which have different binding affinities depending on what specific residues are phosphorylated in CPD. High-binding affinity CPDs include two phosphorylated residues (both "0" position and "+4" position), while low-affinity CPDs contain a negatively charged residue in the second site ("+4" position, such as glutamic acid (E)) [4,51,52]. As shown in Fig   Fig 8. MCL-1

PLOS PATHOGENS
Deregulation of the MCL-1 by KSHV LANA 1A, LANA contains a negatively charged residue at the second site within the decoy CPD motif, suggesting that LANA has a low affinity CPD that acts as a weak binding substrate of FBW7. Strikingly, the majority of FBW7's substrates have two phosphorylation sites in their CPD motifs except MCL-1 [2,4,53,54]. Thus, we hypothesize that both LANA and MCL-1 having a low-affinity CPD, LANA can compete with MCL-1 for FBW7 binding but not with proteins that have high-affinity CPD (e.g. c-Myc), which can bind stronger to FBW7. Additional studies will be required to test this hypothesis.
Although LANA-FBW7 interaction did not dysregulate the expression of other FBW7's substrates, it is noteworthy that LANA employed other strategies to upregulate them. In the case of c-Myc, LANA interacts with glycogen synthase kinase 3 beta (GSK-3β), which interferes with the phosphorylation of c-Myc that can block the binding of FBW7 to its CPD motif, leading to the stabilization of c-Myc [29]. Indeed, mounting data reported that GSK-3β phosphorylates the central threonine ("0" position) of the CPD in substrates containing a priming site (like c-Myc, cyclin E, and Jun), resulting in timely degradation of several substrates of FBW7. These results suggest that LANA has evolved to dysregulate the tumor suppressor function of FBW7-related signaling pathway by targeting either FBW7 or its upstream factor, like GSK-3β. Numerous studies have shown that LANA has various properties that could clearly contribute to KSHV-mediated tumorigenesis, including inhibiting G1-cell cycle arrest [28], stimulating S-phase entry via upregulation of cyclin D1 and β-catenin [55], or suppressing p53-mediated apoptosis [56]. These results indicate that the KSHV LANA specifically targets either protein-protein interactions or post-translational modifications, like ubiquitination, which can contribute to KSHV-associated pathogenesis, further emphasizing the important role of LANA as a viral oncogenic factor.
In summary, our studies provide experimental evidence for the role of LANA in governing MCL-1-mediated intrinsic apoptotic pathways via regulation of one UPS component, FBW7, within the FBW7-MCL-1 axis to contribute to KSHV pathogenesis. Notably, MCL-1 plays an oncogenic role in regulating the survival and tumorigenicity of PEL cells but not in Burkitt's lymphoma cells (e.g., BJAB). We also supply a mechanistic explanation for why MCL-1 is enriched in PEL, as well as in Kaposi's sarcoma (KS). These findings indicate that high expression of MCL-1 in PEL cells supports its oncogenic role, making it a potential drug target. In particular, we demonstrate for the first time that inhibiting MCL-1 function markedly represses PEL-induced tumor growth in xenograft models. Taken together, these results increase our understanding of how KSHV utilizes the cellular ubiquitin system to promote KSHV-associated pathogenesis.

Ethics statements
Animal experiments were approved by the Seoul National University Institutional Animal Care (SNU-190506-5-2) and Use Committee and performed following its guideline.

Generation of recombinant KSHV clones
The LANA mutant (P1) KSHV and its revertant were generated by bacterial artificial chromosome (BAC)-based homologous recombination using the KSHV clone BAC16 in the E. coli strain GS1783, as previously described [35,59]. The recombinant BAC16 clones were verified by Sanger sequencing and Nhe I restriction enzyme digestion followed by pulsed-field gel electrophoresis analysis. Primers used for making LANA mutant P1, in which at amino acid 177 Threonine was changed to Alanine, are the following: LANA T177A bacF: 5' CTCCACACTC ACCCACGGCTCCTCCACCCGAGCCTCCCTCCAAGTCGTCACCAGACTCTTAGGATG ACGACGATAAGTAGGG, LANA bacR: 5' ACGCAGGGTAGACGGAGCTAAAGAGTCT GGTGACGACTTGGAGGGAGGCTCGGGTGGAGGAACCAATTAACCAATTCTGATTA G. The primer used for making the revertant (Rev) from the P1 mutant, in which at amino acid 177 Alanine was changed back to Threonine, along with LANA bacR is the following:

Establishment and characterization of iSLK-BAC16 cell lines
The iSLK-BAC16 WT, LANA-P1, and Rev cell lines were established as previously described (Golas G, Virology, 2020). Briefly, iSLK cells were transfected with BAC16 DNA using FuGENE HD (Promega). Two days after transfection, we started the selection of iSLK cells carrying BAC16 using 1 mg/ml hygromycin B. The GFP + /hygromycin B resistant iSLK-BAC16 cell lines were established after 3 weeks of hygromycin B selection. For virus reactivation, the cells were treated with 1 μg/ml Doxy and 1 mM NaB. Cells were harvested for immunoblot analysis upon 72 hours after chemical treatment. At 5 days post-induction, the cell culture supernatant was collected, filtered through 0.45 μm SFCA syringe filter and purified for virion-associated DNA. Supernatant was treated with proteinase K followed by phenol-chloroform extraction of virion-associated DNA, which was then measured by KSHV DNA specific real-time quantitative PCR. Based on a standard curve using serial dilution of BAC16 DNA, we calculated the amount of virus produced by iSLK-BAC16 cell lines.

Generation of rKSHV-BAC16-infected BJAB cells
The iSLK-BAC16-WT, iSLK-BAC16-LANA-P1, or iSLK-BAC16-Rev cell lines were reactivated by 1 mM NaB together with 1 μg/ml Doxy for 3 days. Subsequently, BJAB cells were cocultured with three different reactivated iSLK-BAC16 cell lines. After 4 days, infected BJAB cells were separated from the adherent cell lines by gently shaking the tissue culture plates and transferred into the new plate. These collected cells were treated with 200 μg/ml hygromycin B. After three weeks of culture, polyclonal BJAB cells infected with rKSHV-BAC16 were designated as BJAB-rKSHV-BAC16 cells.

Subcellular fractionation assay
Cells were lysed in fractionation buffer (20 mM HEPES [pH 7.4], 10 mM KCl, 2 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM PMSF) and then passed through a 27 G needle (30x). The nuclear pellet was obtained after centrifugation at 720 g for 15 min. The supernatant was centrifuged at 12000 g for 15 min. The obtained mitochondria pellet was lysis in the buffer (150 mM NaCl, 0.5% NP40, 50 mM Tris [pH 8.0]). Both nucleus and mitochondria samples were mixed with SDS loading dye and were analyzed by immunoblotting.
siRNA Negative-control siRNA and siRNA for MCL-1 (AM51331-#120644) were purchased from Ambion. All siRNAs were used at a final concentration of 100 pmol and transfected into cells using the Neon transfection system according to the manufacturer's instructions (Invitrogen).

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Deregulation of the MCL-1 by KSHV LANA

Apoptosis assay
TREx/BJAB cells (5 x 10 5 cells/ml) stably expressing WT or mutant LANA were cultured for 24 h and then treated with 50 μM etoposide for up to 24 h. The detailed procedure was previously described [58].

Soft agar foci formation assay
BCBL-1 (2.5 x 10 5 cells/ml) or BJAB (2.0 x 10 5 cells/ml) cells were seeded in agar matrix in sixwell plates. After solidification, medium containing 10 μM MCL-1 inhibitor was added on top of the cell/agar matrix. After 2 weeks, colonies were stained with 0.05% crystal violet (Daejung) for 30 min and washed. The colonies were viewed using the EVOS-5000 imaging system (Invitrogen).

Xenograft models
BCBL-1 cells were washed three times with PBS and injected intraperitoneally into 6-week-old female NOD/SCID mice (Koatech, Gyunggi-do, Korea). Each group utilized 15 mice. AT-101 dissolved in DMSO was diluted in PBS and intraperitoneally administered to mice at a dose of 2.5 mg kg −1 once per week after 2 days from BCBL-1 cell inoculation. Inhibition of tumor growth was monitored daily for 28 days by comparing body weight between AT-101-and vehicle-treated mice. After euthanasia, peritoneal ascites was collected using a 5-ml syringe, and the spleen and solid tumor in the peritoneum were isolated. All data were analyze by GraphPad Prism 8.3 software and two-tailed unpaired t-test.

Statistics
Statistical analyses were performed using GraphPad Prism software. P-values were calculated by Student's t-test and p < 0.05 denoted statistical significance. Data are presented as the mean ± SD.