Epstein-Barr Virus Viral Protein Lmp1 Increases Rap1

EPSTEIN-BARR VIRUS VIRAL PROTEIN LMP1 INCREASES RAP1

SUMOYLATION, ENHANCING TELOMERE MAINTENANCE DURING

TUMORIGENESIS

WYATT TYLER CRAMBLET

B.S., Biology, Gordon State College, 2016

A Thesis Submitted to the Faculty

of Mercer University School of Medicine

in Partial Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE IN BIOMEDICAL SCIENCE

Macon, GA

2018 EPSTEIN-BARR VIRUS VIRAL PROTEIN LMP1 INCREASES RAP1

SUMOYLATION, ENHANCING TELOMERE MAINTENANCE DURING

TUMORIGENESIS

WYATT TYLER CRAMBLET

Approved:

______Date ______Gretchen L. Bentz, Ph.D. Faculty Advisor for Thesis

______Date ______Richard O. McCann, Ph.D. Thesis Committee Member

______Date ______Laura Silo-Suh, Ph.D. Thesis Committee Member

______Date ______Jon Shuman, Ph.D. Thesis Committee Member

______Date ______Jean Sumner, MD Dean, Mercer University School of Medicine

ACKNOWLEDGEMENTS

I would like to thank the National Institutes of Health for providing the funding for this project and Mercer University School of Medicine for the opportunity for discovery and knowledge this experience has provided me.

My utmost respect and gratitude goes to Dr. Gretchen Bentz. Along the course of my research Dr. Bentz has always provided what I required to be successful: materials, focus, experience, and understanding. Her guidance at both the lab and the writing desk is what has allowed me to accomplish the greatest achievement of my academic career.

I thank Dr. Richard McCann and Dr. Christy Bridges who have been the

Directors of the Master of Science Biomedical Sciences Program. Their leadership has been invaluable to my peers and me for becoming the professionals we set out to be. Along with Dr. Bentz and Dr. McCann I would like to thank the other members of my Graduate Thesis Committee, Dr. Jon Shuman and Dr. Laura Silo-

Suh. Their insights have helped me to hone my knowledge from the amorphous mass that it once was in the classroom into a tool that will sustain me for the rest of my life. My thanks to our laboratory technician Angela Lowrey, whose expertise and friendship were greatly appreciated in our success, and ever more so when success eluded us. I offer my thanks and support to the undergraduates Ashley

iii Ray, Sekhar Tummala, Robert Roach, Supreet Raina, and Shakti Biswas whom

I’ve worked with and I wish you all well in your careers. And to all the professors and staff of Mercer University School of Medicine who have either instructed me in the classroom or provided me the classroom in the first place you have my thanks.

My friend and mentor Dr. Phillip Jen, you shown a light on the path my life would follow at a time when I was lost. For all that I have done and all that I shall do my thanks to you for reminding me of my strength.

Finally, Peter and Cathey, Dad and Mom, my drive and my support. You’ve succeeded in raising a man who is proud of his life and whose life has been a success. For all your love, you have mine.

iv TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS……………………………………………………………...iii

ABSTRACT…………………………………………………………………………...…vi

CHAPTER

1. INTRODUCTION TO THE STUDY……………………………………………1

2. MATERIALS AND METHODOLOGY…………………………………………9

3. RESULTS………………………………………………………………...…….16

4. DISCUSSION…………………………………………………………..………32

REFERENCES…………………………………………………………………………37

v ABSTRACT

WYATT TYLER CRAMBLET

EPSTEIN-BARR VIRUS VIRAL PROTEIN LMP1 INCREASES RAP1 SUMOYLATION, ENHANCING TELOMERE MAINTENANCE DURING TUMORIGENESIS

Under the direction of GRETCHEN L. BENTZ, Ph.D.

Cancer remains the second leading cause of death in the USA, and there are multiple avenues of study to decipher how to combat the disease [1]. One approach in cancer treatments is to attack the tumor’s ability to replicate indefinitely. Normally healthy cells can only replicate a finite number of times before reaching their senescence limit due to loss of telomere DNA. During tumorigenesis, cells must gain the ability of maintaining chromosomal telomeres through continuous cycles of replication. Two mechanisms by which telomeres can be maintained are through activation of the telomerase complex and alternate lengthening of telomeres (ALT), a telomerase-independent mechanism that uses homologous recombination to maintain telomere length. Epstein-Barr Virus (EBV), a ubiquitous ϒ-herpesvirus, establishes a life-long latent infection within the host, and latent EBV infections are linked to the development of several epithelial and lymphoid malignancies. The principal viral oncoprotein is latent membrane protein-

vi 1 (LMP1). Early during the establishment of latency, EBV relies on ALT for maintaining telomeres [2]. However, telomerase-dependent telomere maintenance is observed in EBV-associated lymphomas.

Understanding how and when the switch from ALT/telomerase- independent to telomerase-dependent telomere maintenance occurs could open new paths to the prevention or treatment of EBV-associated lymphomas. We focused on the Human Repressor Activator Protein 1 (RAP1) because it is involved in both telomerase-independent and telomerase-dependent telomere maintenance. We hypothesize that LMP1 CTAR3 induces the SUMOylation of

RAP1, which contributes to LMP1-mediated oncogenesis through the maintenance of telomeres initially by ALT processes before switching to telomerase-dependent telomere elongation.

Here we show conditions for telomerase-independent telomere maintenance improve when LMP1 induces the SUMOylation of PML and increases formation of

PML nuclear bodies. We also show LMP1 blocks the interaction of PML with RAP1 while also increasing RAP1 association with SUMO proteins and telomerase activity. RAP1 point mutants that prevent the increased association of RAP1 with

SUMO proteins also demonstrated a decrease in telomerase activity in conjuncture with increased association with PML. This data confirms that LMP1 contributes to maintaining telomeres, which not only aids EBV latency but also tumorigenesis.

vii

By better understanding how LMP1 affects telomere maintenance via its interaction with RAP1 we may discover novel therapies to prevent LMP1-mediated tumorigenesis.

viii

CHAPTER 1

INTRODUCTION TO THE STUDY

The Herpesviridae family of viruses is ubiquitous throughout the world, with few individuals who do not have one or more herpes viral infections. This family of viruses is characterized by its linear, double-stranded genome surrounded by an icosahedral capsid, tegument consisting of viral and cellular proteins, and an envelope derived from the host cell. There are three herpesvirus subfamilies that are grouped based on viral characteristics, pathogenesis, and disease manifestation [40].

Epstein-Barr Virus (EBV), or human herpesvirus-4, is a gamma-herpesvirus that can cause lytic, persistent, latent, and immortalizing infections. As a herpesvirus, EBV infects over 90% of the world’s population. The virus is transmitted in saliva, which can contain cell-free virus, as well as EBV-infected cells, leading to lytic infection of oral epithelial cells [41]. Virions produced by the oral epithelium can infect circulating B-lymphocytes in the oral epithelium or in lymphoid organs [3].

While a primary EBV infection is usually asymptomatic, it can manifest as infectious mononucleosis, which is characterized by fatigue, fever, pharyngitis, rash and swollen lymph nodes of the neck [4]. During primary infection of B- lymphocytes, the linear viral genome circularizes via its terminal repeats and establishes a latent infection that persists for the lifetime of the host. Latent EBV

1 infection is associated with several malignancies including Burkitt’s lymphoma,

Hodgkin lymphoma, post-transplant lymphoproliferative disease, and AIDS- associated CNS lymphomas [5]. The principal viral oncoprotein in the majority of

EBV-associated lymphomas is latent membrane protein-1 (LMP1).

LMP1 is a 386 amino acid integral membrane viral protein that belongs to the tumor necrosis factor receptor family and mimics CD40 with the exception that it is constitutively active and its activation is ligand independent [6]. LMP1 consists of a short 24-amino-acid cytoplasmic N-terminal domain (regulates

LMP1 turnover), six transmembrane domains (required for oligomerization of

LMP1 and its constitutive activity), and a 200-amino-acid cytoplasmic C-terminal domain, which contains three C-terminal activating regions (CTAR1, CTAR2,

CTAR3). Most LMP1-mediated signal transduction events are mediated via the extensively characterized CTAR1 and CTAR2 and involve the induction of protein posttranslational modification, such as phosphorylation and ubiquitination, of host cellular proteins and viral proteins regulating their function [42, 43].

Not much is known about the role of CTAR3, which falls between CTAR1 and CTAR2, in LMP1-induced signaling. Bentz et al. identified the first function for

CTAR3 in modulating a third major posttranslational modification, SUMOylation or covalent modification by Small Ubiquitin-like Modifier [7]. Specifically, CTAR3 hijacks Ubc9, increasing protein SUMOylation and contributing to EBV latency.

Overall, protein SUMOylation is important to the cellular changes occurring during latent EBV infections [7, 8, 44].

Protein SUMOylation is vital to normal cellular function and development

[9]. There are four known small ubiquitin-like modifier (SUMO) isoforms in humans and are designated numerically SUMO-1 through SUMO-4. SUMO-1 is approximately 50% similar to SUMO-2 and SUMO-3, which only differ from each other by three N-terminal residues and are often identified as SUMO-2/3 [45].

SUMO-4 is less commonly expressed within cells and appears to be linked to extreme cellular stress [10]. Each SUMO isoform begins as an inactive pro- peptide that must be cleaved at its C-terminal domain by SENP enzymes in order to expose a di-glycine motif. The C-terminal glycine residue is activated in an ATP- dependent mechanism by the SUMO-activating enzyme (a dimer of SAE1 and

SAE2). A thioester bond is formed between SAE2 and the SUMO terminal glycine residue [11]. The primed SUMO protein is transferred to Ubc9, which identifies the consensus SUMOylation motif KxD/E; ( is a large hydrophobic residue) within a target protein and conjugates the SUMO protein to the lysine residue of the motif

[12, 13]. SUMO-2/3 contains a SUMOylation motif, resulting in the formation of poly-SUMO chains that are capped by SUMO-1 [14]. However, the exact function of poly-SUMOylation is unknown. SUMOylation of a target protein can result in a variety of effects that normally can be assigned to one of three common mechanisms [9]. First, SUMOylation can block the binding site on a target protein from being available to interact with other proteins or DNA [46]. Second,

SUMOylation can act as a scaffold, aiding other proteins in interacting with

SUMOylated substrates [47]. Third, SUMO can induce a conformational change in the target protein exposing or hiding active sites or regulatory domains [48].

Furthermore, SUMO can compete with ubiquitin for modification of the lysine residue(s) within the target protein, regulating protein turnover, and stability.

Dysregulation of cellular SUMOylation processes can affect cell development, cell death, and tumorigenesis.

As described above, latent EBV infection is associated with the development of different B-cell lymphomas, which we propose is partly due to its ability to hijack SUMOylation processes in transformed B-cells. Understanding how LMP1 dysregulates SUMOylation processes is of great importance. Bentz et al. found that suppression of cellular SUMOylation or deletion of LMP1 CTAR3 enhances viral reactivation and lytic replication [8]. Specifically, LMP1 induces the SUMOylation of the transcriptional co-repressor KAP1, increasing its co- repressor activity on host and viral lytic promoters. While KAP-1-mediated repression of the EBV lytic promoters aids the maintenance of latency [8], we were interested in investigating how LMP1-induced SUMOylation affected viral and cellular genomic integrity, in particular the maintenance of cellular telomeres.

Human chromosomes are linear and have a 5’→3’ requirement for replicating DNA. Due to this requirement one strand of DNA is referred to as the lagging strand because its replication must be regularly reinitiated from seeding

RNA primers, resulting in segments of DNA (Okazaki fragments) that are later ligated together to form a continuous strand of replicated DNA. Following removal of the RNA primer, a short single-stranded overhang remains at the end of the chromosome, and this overhang will be cleaved and lost if replication is not completed. These ends are fully replicated due to the presence of telomeres,

4 which are short and repeating segments of DNA (TTAGGG in humans [15]) that function to protect the coding DNA from exonucleases and DNA damage repair enzymes and overcome the inability to fully replicate the lagging strand. As each short overhang of telomeric DNA is typically lost during replication, a separate

DNA polymerizing enzyme complex (telomerase) uses an integrated RNA template and human telomerase reverse transcriptase (hTERT) to continuously synthesize more telomeric DNA on the overhanging strand [16]. Without the maintenance of telomeric DNA cells continually shorten their replicated chromosomes instituting a senescence limit on replication.

For EBV to maintain latency in host B-cells it must overcome the senescence limit or promote lytic replication. Because, lytic EBV replication increases as cellular telomerase activity decreases [17], EBV likely possesses some means of ensuring telomeric maintenance in infected B-cells in order to ensure viral latency. Early following the establishment of latency in B-cells telomerase activity is relatively unaffected compared to non-transformed cells

[49], but as latently infected B-cells proliferate, cells significantly increase telomerase activity, resulting in their immortalization [18]. This increase in telomerase activity is likely due in part to increased translocation of hTERT from the cytoplasm to the nucleus via its enhanced binding to NF-ϰB, which is induced by LMP1 [19]. As mentioned above, increased telomerase activity does not appear to be involved in telomere maintenance early following the establishment of latent EBV infections [49]. Instead, alternate lengthening of telomeres (ALT),

5 which maintains telomere length via homologous recombination, is used for telomere maintenance [2].

ALT often results in heterogeneous lengths of telomeres dependent upon the location of strand invasion during homologous recombination, and it relies on the DNA damage repair (DDR) enzymes instead of the telomerase complex [20].

However, for ALT enzymes to carry out their respective functions they must be able to bypass Shelterin complexes, which shield telomeric DNA from being manipulated [50].

Shelterin complexes bind to telomeric DNA and protect the chromosome from degradation [50]. During initial stages of tumorigenesis, Shelterin complexes can be dysregulated, leading to telomere shortening [21]. One protein important in regulation and dysregulation of the Shelterin complexes is human Repressor

Activator Protein 1 (RAP1), which is also known as Telomeric Repeat-Binding

Factor 2(TRF2)-Interacting Protein. RAP1 is a 399 amino acid or 44.2 kDa size protein that interacts with nuclear and cytoplasmic proteins [22-24]. Cytoplasmic

RAP1 acts as an IKK adaptor protein to regulate NF-ϰB activation [22], and nuclear RAP1 augments the binding of TRF2 to telomeric DNA [23, 24]. While

RAP1 directly interacts with TRF2 and not DNA [23], the RAP1/TRF2 interaction decreases the binding affinity of TRF2 for double-stranded DNA and increases its specificity for telomeric DNA and the single-strand/double-strand junction [24].

RAP1 also aids the localization of the Shelterin complex to telomeres where it protects the telomeres while also positively and negatively regulates telomere maintenance [25]. RAP1 increases telomere elongation in a telomerase-

6 dependent manner [26] and inhibits the ALT pathway by decreasing the rate of recruitment of DDR enzymes to telomeres and decreasing homologous recombination [27]. In addition to being a component of the Shelterin complex,

RAP1 interacts with promyelocytic leukemia proteins (PML), which is a common target for SUMOylation and is involved in ALT processes [31].

The pml gene is approximately 53 kilobases in size and consists of 10 exons that are subject to shuffling through alternative splicing, yielding multiple

PML isoforms that can be up to 882 amino acids or approximately 97.5 kDa in size [28]. The different PML isoforms have multiple functions including tumor suppression and anti-viral responses [51, 52]. PML is normally found in nuclear bodies that consist of over 100 proteins that can be SUMOylated or contain

SUMO-binding motifs. The SUMOylation of PML is vital to the formation of PML nuclear bodies, and any disruption of host SUMOylation processes drastically alters PML nuclear body formation [29].

PML nuclear bodies also include the DDR pathway proteins responsible for homologous recombination during ALT [53]. One hallmark of the ALT pathway is the inclusion of chromosomal and extra-chromosomal telomeric DNA within PML nuclear bodies forming ALT-associated PML Bodies (APB) [30]. These APBs are required for canonical ALT processes and are closely related to the amount of

SUMO-1 indirectly bound to telomeric regions [32], possibly due to the

SUMOylation of members of the Shelterin complex [31].

LMP1-expression increases the formation of PML nuclear bodies, in part due to LMP1-mediated increased levels of PML [33]. Treatment of latent EBV

7 infected cells with arsenic trioxide, an inhibitor of protein SUMOylation, disrupts

PML nuclear body formation, induces lytic replication, and upregulates the expression of the immediate-early viral protein Zta [33]. Newly established lymphoblastoid cell lines (LCLs; one month) show a significant decrease in

Shelterin complex proteins and RNA levels compared to well established LCLs

(five years) from the same donor [2]. Even when new EBV-transformed LCLs have normal TRF2 levels, the Shelterin complex fails to co-localize with telomeric

DNA [2]. It is likely that LMP1 upregulates PML nuclear body or APB formation and downregulates expression of Shelterin complex proteins early in EBV infection, resulting in activation of ALT [2, 30]. However, as latency progresses,

Shelterin complex expression levels return to normal, yet TRF2 remains relatively unassociated with telomeric DNA [2], which may be due to oxidative stress resulting in unprotected telomeric DNA [54]. In addition to its role in telomere maintenance, TERT is capable of triggering reduction of reactive oxygen species, which would allow TRF2 to localize to telomeric DNA [55]. However, the mechanism involved in the switch from ALT to telomerase-dependent maintenance of telomeres during EBV latency is unknown. We propose that

LMP1-mediated regulation of SUMOylation processes contribute to LMP1- induced tumorigenesis.

We hypothesize that LMP1 CTAR3 induces the SUMOylation of RAP1, which contributes to LMP1-mediated oncogenesis through the maintenance of telomeres initially by ALT processes before switching to telomerase-dependent telomere elongation.

CHAPTER 2

MATERIALS AND METHODOLOGY

Images in this document are presented in color.

Cell Lines

Human embryonic kidney (HEK) 293 and 293T cells were cultured in DMEM

(Corning) with 10% heat-inactivated fetal bovine serum (FBS; Corning) and antibiotics/antimycotics (Corning) at 37°C with 5% CO2. EBV-WT and EBV-Δ3 expressing HEK 293, a gift from Wolfgang Hammerschmidt (Munich, Germany) were cultured in DMEM with 10% FBS, antibiotics/antimycotics, and hygromycin B

[34]. EBV-transformed LCLs were cultured in RPMI (Corning) with 10% FBS and antibiotics/antimycotics.

Plasmids

FLAG-LMP1, FLAG-LMP1 PQAA, FLAG-LMP1 YIID were gifts from Dr. Nancy

Raab-Traub [35]. Flag-LMP1Δ3 has been described previously [34, 35]. GFP-RAP1 was a gift from Dr. Mark Swanson. Myc-SUMO-1/2/3 was purchased from

Addgene. Plasmids were transformed into Quickchange XL Ecoli cells, and plasmid DNA was extracted using the QIAGEN Plasmid Plus Maxi Kit (QIAGEN).

DNA yield was measured using the NanoDrop 2000c (ThermoFisherScientific).

Transfections

3 x 106 HEK 293 cells or 2 x 106 HEK 293T cells were plated in DMEM supplemented with 10% FBS 24 hours prior to transfection with DNA using PEI.

Amounts of plasmid DNA used were 1ug Flag- LMP1 (WT and mutants), 2ug GFP-

RAP1 (WT and mutants), 3ug myc- SUMO1/2/3, pcDNA empty vector as needed.

DNA, 1mL TransfectagroTM, and 12uL PEI (2.0 ug/uL) were combined in 1.5mL microcentrifuge tubes, and gently pulsed to mix. After 15 minutes incubation at room temperature transfection tube contents were added dropwise to pre-labeled

100mm culture dish. 48 hours posttransfection, cells were harvested.

Immunofluorescence Microscopy

Cells were transfected, as described above, in 6-well dishes maintaining relative volumes of reagents, plasmids, and cells. 48 hours post-transfection cells were fixed in 4% paraformaldehyde and then washed with PBS. Cells were permeabilized in 1% Triton X-100 and washed with PBS. Cells were blocked in

3% Bovine Serum Albumin for 1 hour. 0.5ug of primary antibodies were added to required wells and incubated at 4°C overnight. The following morning cells were washed with PBS and fluorescently labeled secondary antibodies were added to incubate for 1 hour at room temperature. After final incubation, coverslips were mounted on glass slides utilizing SlowFade® Gold antifade reagent with DAPI

(Invitrogen). Images were captured at 60X magnification using a Nikon A1 laser confocal microscope.

Protein Turnover

Cells were transfected in triplicate with pcDNA control vector or Flag-tag-

LMP1. 24 hours posttransfection, replicates were treated with DMSO (vehicle control), Cycloheximide (35uM CHX), or MG132 (75uM). 48 hours posttransfection whole cell lysates were harvested. Stability over time was examined under similar transfection conditions, with CHX added every 2 hours for 8 hours prior to harvesting.

Western Blot

Western blot analyses were performed using Mini-PROTEAN® TGXTM precast gels (Bio-Rad) and Tris/Glycine/SDS running buffer (Bio-Rad). Gels were transferred to PVDF membranes using the Trans-Blot®TurboTM Transfer System

(Bio-Rad). Membranes were blocked for 1 hour in 5% blotting grade powdered milk (Bio-Rad) in TBS-Tween 20, pH 7.4 (Affymetrix). Membranes were incubated with primary antibodies (in 5% milk) overnight at 4°C. Membranes were washed three times in TBST. If secondary antibodies were required, membranes were incubated with secondary antibodies (in 5% milk) for one hour followed by three washes in TBST. Membranes were incubated with ClarityTM Western ECL Blotting

Substrates (Bio-Rad) for 60 seconds and analyzed using the ChemiDocTM Imaging

System (Bio-Rad). Densitometric data was generated from western blots using

Image Lab™ (Bio-Rad).

Native Immunoprecipitations (nIPs)

48 hours posttransfection, cells were harvested. 10% of cells were collected for whole cell lysates (Laemmli sample buffer added and samples denatured). The remaining sample was resuspended in 500uL RIPA lysis buffer (20mM Tris pH 7.5,

150mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 1mM EDTA) supplemented with proteinase inhibitors, NEM, DNase I, Dnase I buffer, and benzonase. Samples were freeze-thawed four times. Additional RIPA lysis buffer supplemented with proteinase inhibitors and NEM (500uL) was added to each sample. Cell lysates were collected, antibodies were added (concentrations recommended by the manufacturer), and lysates were incubated for 2 hours at

4°C in an end-over-end rocker. 30uL PierceTM Protein G Magnetic Beads

(ThermoFisherScientific) were added to each lysate sample and incubated overnight at 4°C in an end-over-end rocker. Beads were washed four times with

RIPA lysis buffer, and beads were resuspended in 40µL 3x Laemmli buffer and denatured for 10 minutes.

Denatured Immunoprecipitations (dIPs)

48 hours posttransfection cells were harvested. 10% of cells were collected for whole cell lysates (Laemmli sample buffer added and samples denatured). The remaining samples were resuspended in 175uL pre-boiled denaturing lysis buffer

(Tris HCL pH 7.5, 0.48% βME), pulse vortexed, and boiled for 10 minutes. 1 mL

RIPA lysis buffer supplemented with proteinase inhibitors and NEM was added to

12 each sample. Supernatant fluids were collected, antibodies were added

(concentrations recommended by the manufacturer), and lysates were incubated for 2 hours at 4°C in an end-over-end rocker. 30uL PierceTM Protein G Magnetic

Beads (ThermoFisherScientific) were added to each lysate sample and incubated overnight at 4°C in an end-over-end rocker. Beads were washed four times with

RIPA lysis buffer, and beads were resuspended in 40uL 3x Laemmli buffer and denatured for 10 minutes.

Cell Compartments

Cells were harvested and the Subcellular Protein Fractionation Kit for

Cultured Cells (ThermoFisherScientific) was used according to manufacturer’s guidelines to isolate specific cellular compartments. As each fraction was separated from the cell lysates, 3x Laemmli buffer was added to the extracted fraction for a final Laemmli buffer concentration of 1x. Samples were boiled for 10 minutes.

Site-Directed Mutagenesis

SUMOplotTM (Abgent) was used to identify potential sites of SUMOylation in RAP1 (Table B1). Using NCBI, forward and reverse primers, to change the lysine within the SUMO motif to arginine were constructed as shown in Table B1.

Using these primers and the Stratagene QuikChange II Site-Directed Mutagenesis

Kit (Agilent Technologies), site-specific point-mutations were generated in the

GFP-RAP1 expression vectors. Mutants were verified by sequencing (Eurofins

Genomics).

Table B1. Probability of lysine SUMOylation at target sites for site- directed mutagenesis. Potential sites of SUMOylation within RAP1 were identified and primers were constructed to mutate target lysines residues.

Site Probability Score Primer (K) Primer (R)

K114 (AKPG) .62 GAC ACC GGC TCG GAA GAC ACC GGC TCG GAA GCA GCA AAG CCC GGG GCC AGG CCC GGG GCC CTG

CTG GCC GAG GCC GAG

K240 (VKEE) .93 GAA GAA GAG TAT GTG GAA GAA GAG TAT GTG AGG AAG GAA GAA ATC CAG GAA GAA ATC CAG GAG AAT

GAG AAT G G

Chromatin Fraction

48 hours posttransfection, cells and whole cell lysates were collected [56].

Cells were resuspended in 200uL CSK buffer (10mM PIPES, 100mM NaCl,

300mM sucrose, 3mM MgCl2, 1mM EGTA, 1mM DTT, 0.1% Triton X-100) supplemented with proteinase inhibitors. Samples were centrifuged for 5 minutes at 4000rpm and buffer was removed from pellets. Samples were washed twice using 1mL CSK buffer. Insoluble pellets/chromatin fractions were resuspended in

60uL SDS buffer and samples were boiled for 10 minutes.

Telomerase Activity

48 hours posttransfection cells and whole cell lysates were harvested. Cell numbers were determined and 1 x 106 cells were collected, lysed, extracts collected, and PCR amplification conducted using the Millipore TRAPeze®

Telomerase Detection Kit (Millipore Sigma) according to manufacturer’s guidelines. PCR products were analyzed by PAGE on 12% Mini-PROTEAN®

TGXTM precast gels (Bio-Rad) and incubated with EtBr solution for 20 minutes at room temperature. Gels were then washed with ddH2O for 30 minutes and analyzed using the ChemiDocTM Imaging System (Bio-Rad).

CHAPTER 3

RESULTS

LMP1 Increases PML Nuclear Body Formation in a CTAR3-dependent Manner.

LMP1 induces the SUMOylation of cellular proteins, modulating LMP1- mediated signaling, aiding the maintenance of latency, and contributing to the oncogenic potential of the protein, which raised the question of whether LMP1- mediated protein SUMOylation affects genomic integrity in latently infected cells

[7, 8, 44]. This question led to our current investigations into telomere maintenance. Because LMP1 expression increases the formation of PML nuclear bodies [33] and promotes ALT at early times post-transformation [2], we first examined if LMP1-induced SUMOylation affected PML nuclear body formation.

Immunofluorescence microscopy of EBV-WT or EBV-Δ3 293 cells (for the formation of PML nuclear bodies in endogenous conditions) and 293 cells transiently transfected with vector control, Flag-LMP1, or Flag-LMP1 Δ3 (for the formation of PML nuclear bodies when LMP1 is exogenously expressed) showed that LMP1 expression coincided with an increase in PML nuclear body formation, which was abrogated by the loss of CTAR3 (Figure A1a). Quantitation of the number of PML nuclear bodies per cell confirmed that LMP1 expression coincided with a significant (P < 0.05) increase in the number of PML nuclear

16 bodies per cell and that this increase occurred in a CTAR3-dependent manner

(Figure A1b).

LMP1 Induces the SUMOylation of PML in a CTAR3-dependent Manner.

Because PML nuclear body formation is tightly regulated by cellular

SUMOylation processes, we next investigated if PML was SUMOylated by LMP1.

Using EBV-negative BL41 cells (no EBV) infected with EBV-WT or P3HR-1 (a defective EBV virus that is unable to express LMP1 due to loss of function EBNA2)

[60-62], denaturing immunoprecipitations were performed to pull down all proteins covalently modified by SUMO-1. Western blot analyses revealed that PML was

SUMOylated during EBV-WT transformation, and that loss of expression of LMP1 resulted in lower levels of PML SUMOylation (Figure A2a). These findings suggest that PML is SUMOylated in a LMP1-dependent manner.

Using EBV-transformed LCLs that were transformed with wild-type EBV

(EBV WT) or EBV lacking LMP1 CTAR3 (EBV Δ3), denaturing immunoprecipitations were performed to pull down all proteins covalently modified by SUMO-1. Western blot analyses revealed that PML was SUMOylated during latent EBV infections, and deletion of CTAR3 resulted in lower levels of

SUMOylated PML (Figure A2b). These findings provide additional support demonstrating that LMP1 expression increases PML nuclear body formation and show that LMP1 induces the SUMOylation of PML in a LMP1-dependent and a

CTAR3-dependent manner.

LMP1 inhibits the RAP-1/PML Interaction.

Telomeric DNA associates with PML nuclear bodies during the formation of

ALT-associated PML Bodies (APB) [30], which also incorporates members of the

Shelterin complex. Specifically, PML interacts with TRF1, which associates with

TRF2 and RAP1 [36]. Therefore, we next examined the indirect interaction of PML with RAP1. Native immunoprecipitations to pull-down all proteins that interact with

RAP1 revealed that in control-expressing cells RAP1 associated with endogenous

PML (Figure A3a). Expression of LMP1 coincided with loss of the RAP1/PML interaction (Figure A3a).

Use of select LMP1 mutants (PQAA has a non-functional CTAR1, YII2 has a non-functional CTAR2, and Δ3 lacks CTAR3) revealed inactivation of CTAR1 or

CTAR2 as well as deletion of CTAR3 did not abrogate LMP1-mediated inhibition of the RAP1/PML interaction (Figure A3b). These findings suggest that none of the

C-terminal activating regions alone influence the RAP1/PML interaction, but it does not rule out the possibility that multiple CTARs are involved.

RAP1 Association with SUMO-1 Increases in the Presence of LMP1.

To identify additional mechanisms by which LMP1-induced SUMOylation may affect the switch from ALT to telomerase-dependent telomere maintenance, we investigated if the Shelterin complex protein RAP1 was SUMOylated during

LMP1 transfection. Native immunoprecipitations to pull-down myc-SUMO1- associated proteins revealed that RAP1 only interacted with myc-SUMO1 when

LMP1 was expressed (Figure A4). In addition, slower migrating forms of RAP1, suggestive of posttranslational modification of RAP1, were only detected when

LMP1 was co-expressed (Figure A4). These data suggest that LMP1 expression increases the modification of RAP1 and the association of RAP1 with SUMOylated proteins.

LMP1 CTAR2 and CTAR3 Induce the Interaction of RAP1 with SUMO1.

To determine which of the EBV LMP1 CTAR regions is responsible for the detected increase in the RAP1/SUMO interaction, we used our select Flag-LMP1 constructs (WT, PQAA, YIID, Δ3). Native immunoprecipitations to pull-down all proteins interacting with SUMO-1 confirmed that LMP1 expression coincided with an increase in RAP1 association with SUMO-1 (Figure A5). Loss of a functional

CTAR1 domain did not alter this LMP1-mediated increase. Instead, loss of a functional CTAR2 domain and deletion of CTAR3 resulted in a marked decrease in RAP1 association with SUMO when compared with LMP1-WT expression

(Figure A5). These findings suggest that both CTAR2 and CTAR3 are vital to

LMP1-mediated changes in RAP1. In addition, these findings raised the possibility that RAP1 may be SUMOylated during EBV latency.

RAP1 K240 is Necessary for the LMP1-mediated RAP1/SUMO1 Interaction.

The finding that slower migrating forms of RAP1 were detected when LMP1 was expressed led us to propose that LMP1 induces the SUMOylation of RAP1.

Analysis of the RAP1 sequence using SUMOplot® revealed multiple lysine residues with a high probability of being SUMOylated (Table B1). Lysine residues in proposed SUMOylation motifs were mutated to arginine. Native immunoprecipitations confirmed that LMP1 expression coincided with increased association of RAP1 with SUMO1 (Figure A6). Mutation of RAP1 K114 or RAP1

K249 to arginine yielded detectable decreases in LMP1-induced RAP1/SUMO-1 association (Figure A6). These findings suggest that both lysine residues (and

SUMOylation motifs) are involved in the LMP1-induced RAP1/SUMO interaction.

LMP1 Expression Does Not Affect RAP1 Turnover.

SUMOylation of a target protein can alter a protein’s stability (by competition with K48-linked ubiquitination of lysine residues), protein localization, protein- protein interactions, and protein-DNA interactions [57-59], so we examined how

LMP1 could affect RAP1 biology. To examine the ability of LMP1 to alter RAP1 stability, transfected cells were treated with DMSO (vehicle control), cyclohexamide (CHX 35uM; to inhibit translation), or MG132 (75uM; to inhibit the proteasome) for 18 hours prior to harvesting whole cell lysates. Western blot analyses revealed that no significant change in RAP1 turnover was seen within

LMP1-expressing cells when compared with control-expressing cells (Figure A7).

In addition, inhibition of the proteasome with MG132 revealed no differences in the targeting of RAP1 for degradation by the proteasome when comparing LMP1- expressing cells with control-expressing cells. Densitometric analysis of repeat experiments confirmed these results, which suggest that LMP1-induced

SUMOylation of RAP1 does not affect the stability RAP1.

To confirm these results, a time-course of DMSO and cyclohexamide treatments were performed. Data again showed no difference in RAP1 stability when comparing LMP1-expressing cells with control-expressing cells (Figure A8).

These findings suggest that LMP1-induced modification of RAP1 does not compete with ubiquitin, resulting in increased stability and decreased turnover.

LMP1 Expression Does Not Affect RAP1 Localization.

Next, we examined if expression of LMP1 altered the intracellular localization of RAP1. Whole cell lysates, cytoplasmic extracts, membrane fractions, nuclear extracts, and chromatin fraction extracts were harvested, and results showed that the localization of RAP1 did not change with the expression of

LMP1 (Figure A9), which suggests that LMP1-induced modification of RAP1 does not affect its localization.

Cytoplasmic Nuclear Chromatin Extract Envelope Fraction

A A A A A D A D D I I I Q I 3 Q I 3 Q I 3 l l l P Y Δ P P Y Δ o o Y Δ o r r r t 1 1 1 1 t 1 1 1 1 t 1 1 1 1 n P P P P n P P P P n P P P P o M M M M o M M M M o M M M M C L L L L C L L L L C L L L L RAP1

Loading controls GAPDH PARP Histone H1 Figure A9: RAP1 localization is unaffected by LMP1. 293 cells were transfected with control- or LMP1-expression constructs and different cell compartments were harvested. Western Blot analyses were performed to detect endogenous RAP1 levels. GAPDH, PARP, and Histone H1 were used as loading controls.

LMP1 Expression Does Affect the Ability of RAP1 to Interact with Chromatin.

Nuclear RAP1 indirectly associates with telomeric DNA [24], so we examined the effect of LMP1 on the ability of RAP1 to associate with cellular chromatin. Isolation of chromatin fractions revealed that LMP1 expression coincided with over a two-fold increase in the chromatin association of RAP1 when compared with control-expressing cells (Figure A10a). Interestingly, inactivation or deletion of each LMP1 CTAR region only minimally affected the RAP1/chromatin association. These findings suggest that the three CTARs cooperate to regulate the association of RAP1 with the cellular chromatin.

A 3.5

)

o 3

e 2.5

a 2

n 1.5

a o 1

h 0.5

( 0 Control LMP1 LMP1 PQAA LMP1 YIID LMP1 dCTAR3

B 3.5 Control

)

n LMP1

o 3

e 2.5

a 2

n 1.5

a o 1

h 0.5

( 0 RAP1 WT RAP1 K240R RAP1 K114R Figure A10: LMP1 increases RAP1 association with cellular chromatin in a CTAR2-dependent manner yet independent on RAP1 K240 or RAP1 K114. 293 cells were transfected as indicated and chromatin fractions and whole cell lysates were harvested. Western blot analyses were performed to detect A) endogenous or B) exogenous RAP1 levels and densitometry was performed. Results are shown as mean ± standard deviation of experiments performed in triplicate.

To determine if LMP1 targeted RAP1 K240 or RAP1 K114 in order to increase its association with chromatin, we used our generated RAP1 mutants.

Data show that neither lysine residue is required for LMP1-induced

RAP1/chromatin association (Figure A10b). Together these findings suggest that the ability of LMP1 to affect the RAP1/SUMO interaction does not alter the ability of RAP1 to associate with chromatin.

LMP1 Targets RAP1 K114 to Disrupt RAP1/PML Interaction.

Because neither RAP1 K240 nor K114 are necessary for LMP1-induced

RAP1 association with chromatin, we were interested in determining if either RAP1 lysine residue was targeted by LMP1 to inhibit the RAP1/PML interaction, which would inhibit ALT telomere maintenance. Confirming our earlier results, expression of LMP1 coincided with loss of the RAP1/PML interaction. Use of RAP1 point mutants (K240R and K114R) revealed that mutation of RAP1 K240 did not restore the RAP1/PML interaction (Figure A11).

However, mutation of RAP1 K114 appeared to partially abrogate LMP1- mediated inhibition of the RAP1/PML interaction (Figure A11). These findings suggest that RAP1 K114 is likely the principal residue targeted by LMP1 that leads

28 to the loss of interaction with PML, and possibly the eventual switch from ALT to telomerase processes for telomere maintenance.

LMP1 Expression Increases Telomerase-dependent Telomere Maintenance.

While the ALT pathway is activated in newly EBV-infected cells, this coincides with reduced expression of some members of the Shelterin complexes

[2]. However, levels of RAP1 and other members of the Shelterin complex increase at later points following transformation, which corresponds with increased telomerase activity [2, 18]. Because EBV-mediated tumorigenesis occurs many years following transformation, we propose that the switch in expression of members of the ALT pathway (telomerase-independent) to telomerase-dependent telomere maintenance will augment tumorigenesis.

To determine if LMP1-mediated targeting of RAP1 affected telomerase- dependent telomere maintenance, TRAPeze® Telomerase Detection Kit was used. Densitometric analysis of elongated telomeres indicated that LMP1 expression coincided with increased telomerase activity when compared with control-expressing cells (Figure A12a). Inactivation or deletion of each LMP1

CTAR region significantly decreased cellular telomerase activity, which again suggests that there is cooperation between the three CTARs in regulating the maintenance of telomeres. Performing similar assays with our RAP1 point mutants

(K240R and K114R), data showed that mutation of either lysine residue decreased telomerase activity in control-expressing cells. However, only mutation of RAP1

K240R appeared to completely abrogate any effect of LMP1 on telomerase activity

(Figure A12b). These data suggest that LMP1 targets RAP1 K240, contributing to increased telomerase activity during latent EBV infections in a CTAR1/2/3- dependent manner.

A LMP1 LMP1 LMP1 LMP1 5000 control WT PQAA YIID dCTAR3

e 0

T -5000

C -10000 RAP1 WT RAP1 RAP1 5000 Control B K240R K114R

v LMP1

i t 0

r -5000

e -10000

g -15000

C -20000

Figure A12: LMP1 increases telomerase activity in a CTAR1/2/3- dependent manner that was also dependent on RAP1 K240. 293 cells were transfected as indicated and TRAP assays were performed on cell lysates 48 hours post-transfection. Samples were run on 12% polyacrylamide cells and washed with ethidium bromide. Telomere bands were visualized and densitometric analysis of elongated telomeres was performed. Results are shown as mean ± standard deviation of experiments performed in triplicate.

Together, these findings provide additional support that LMP1 can promote both ALT and telomerase-dependent telomere maintenance. First LMP1 induces the SUMOylation of PML and the formation of PML nuclear bodies where ALT occurs. Second, LMP1 inhibits the interaction of PML with a major member of the

Shelterin complex (RAP1), increasing the association of RAP1 with SUMOylated proteins and increasing telomerase activity. However, exactly mechanisms of how

LMP1 mediates the switch from ALT to telomerase-dependent telomere maintenance remains unknown.

CHAPTER 4

DISCUSSION

At early times following the EBV-mediated immortalization of B-cells, telomeres are maintained by ALT maintenance of telomeres [2]; however, increased telomerase-dependent telomere maintenance and telomerase activity are detected in EBV-positive lymphomas and at later times after immortalization of

B-cells [18]. It has been shown that a telomerase-positive cell can switch to using

ALT mechanisms for telomere maintenance [37], so understanding how EBV contributes to telomere maintenance via ALT and telomerase-dependent processes could identify mechanisms by which a vital component of tumorigenesis can be targeted. Here we show the following: 1) LMP1 increases SUMOylation of

PML and PML nuclear body formation, which is involved in ALT telomere maintenance [30]; 2) LMP1 interrupts the PML/RAP1 interaction, which could inhibit ALT telomere maintenance; 3) LMP1 increases the association of RAP1 with SUMOylated proteins; 4) LMP1 increases the association of RAP1 with chromatin; and 5) LMP1 increases telomerase activity. Together, these findings lead us to propose that LMP1 may target RAP1 to inhibit ALT and promote telomerase-dependent telomere maintenance in EBV LMP1-positive lymphomas.

Our studies found that the formation of PML nuclear bodies increases when

LMP1 is expressed in a CTAR3-dependent manner. This increase is likely due to the LMP1 CTAR3-mediated SUMOylation of PML detected in

32 latently infected LCLs. Given the importance of PML in the formation of APBs, which are a common factor of ALT, an increase in PML nuclear body formations could potentially lead to increased ALT activity. However, here we also show that

LMP1 disrupts the normal association between RAP1 and PML, which is also involved in ALT-mediated telomere maintenance. The loss of the RAP1/PML interaction could potentially decrease the overall affinity for PML to nucleate near

Shelterin complexes. If other Shelterin complex proteins, such as TRF1 or TRF2, also decrease their affinities for interacting with PML in the presence of LMP1, the likelihood of APB formation along the telomeres becomes increasingly unlikely while the general increase in PML nuclear body formation is maintained. A loss of

APB formation along the telomeres would provide further counterweight to favor telomerase-dependent telomere maintenance over time and would help to account for why telomerase-dependent telomere maintenance is detected in EBV-positive lymphomas [18]. However, the possibility of both ALT/APB and telomerase- dependent maintenance occurring concomitantly during EBV latency still exists because both modes of telomere maintenance is detected in other cancers, including breast cancer [38].

Interestingly, while deletion of CTAR3 abrogated LMP1-induced PML nuclear body formation, inactivation or deletion of any one of the three CTARs did not restore the RAP1/PML interaction. Because loss of functional CTAR2 and

CTAR3 abrogated LMP1-mediated increased association of RAP1 with

SUMOylated proteins, we propose that the ability of LMP1 to affect the RAP1/PML interaction is independent of LMP1-mediated dysregulation of cellular

SUMOylation processes. LMP1 CTAR1 and CTAR2 induce numerous different posttranslational modifications in order to induce its downstream signal transduction events [57-59], with phosphorylation, regulatory ubiquitination, and degradative ubiquitination being three main modifications. Therefore, it is likely that multiple LMP1-induced posttranslational modifications contribute to the abrogation of the RAP1/PML interaction.

Additional support for the thought that more than one posttranslational modifications are involved in regulating the RAP1/PML interaction is our finding that both RAP1 K240 and K114 are targeted by LMP1 to increase the association of RAP1 with SUMOylated proteins. However, only mutation of RAP1 K114 partially restored the RAP1/PML interaction in the presence of LMP1. These data highlight the importance of both RAP1 lysine residues and suggest that LMP1 differentially modifies each to modulate telomere maintenance.

Although we found that no one CTAR region alone was responsible for the loss of PML/RAP1 interactions, we still wanted to determine how LMP1 could decrease RAP1 availability for PML. Our data suggests that turnover of RAP1 and its localization are unaffected by LMP1 and induced SUMOylation. Instead, LMP1 expression promoted increased association of RAP1 with cellular chromatin.

While LMP1 CTAR2 seemed to be the major contributor to LMP1-induced

RAP1/chromatin association, CTAR1 and CTAR3 also contribute to this association. Again, the ability of LMP1 to increase the association of RAP1 with chromatin was independent of LMP1-dysregulation of cellular SUMOylation processes and independent of RAP1 K240 and K114. Because RAP1 associates

34 indirectly with DNA through TRF2, an increase in RAP1 association with chromatin necessitates the presence of TRF2 and possibly other members of the Shelterin complex on the chromatin. Continued buildup of the Shelterin complexes along the chromatin over time would impede ALT functions [27]. These findings, together with the observations that increased RAP1 and telomerase expression promotes telomerase-dependent lengthening of telomeres [26], suggests that LMP1 may gradually favor telomerase-dependent telomere maintenance over ALT by increasing the accumulation of RAP1 along chromatin and away from APBs.

In Saccharomyces cerevisiae, SUMOylation of RAP1 allows for the recruitment of telomerase inhibitors [39]. Although the human orthologs to these inhibitors have not been identified, we wanted to determine if LMP1-increased

RAP1 association with SUMOylated proteins and the chromatin impedes telomerase activity. Interestingly, LMP1 expression coincided with increased telomerase activity. While LMP1 is known to increase telomerase activity, we now show that loss of CTAR1, CTAR2, or CTAR3 significantly inhibited cellular telomerase activity, leaving the exact mechanism by which LMP1 induces these changes in unknown.

Both RAP1 K240 and K114 appear to be vital for telomerase activity because mutation of either residue significantly decreased telomerase activity in control-expressing cells. However, LMP1 expression still increased telomerase activity in cells expressing RAP1 K114R, suggesting that LMP1 does not target

RAP1 K114 to increase telomerase activity. Mutation of RAP1 K240 abrogated the ability of LMP1 to increase telomerase activity. Therefore, we propose that the

35 ability of LMP1 to modulate cellular SUMOylation processes targets RAP1, specifically RAP1 K240, inhibiting ALT, increasing telomerase activity, and contributing to the switch from ALT to telomerase-dependent telomere maintenance.

By understanding how each factor of EBV latency contributes to the switch from ALT to telomerase-dependent telomere maintenance we may be able to identify potential therapeutic targets to prevent this switch. Should EBV- association lymphomas not be compatible with ALT processes, preventing telomerase-dependent telomere maintenance could inhibit or significantly prevent tumorigenesis in latently infected cells. Currently, there are no therapies to inhibit

ALT telomere maintenance, although some cancers do switch to ALT processes following telomerase inhibition [37]. We have determined that EBV LMP1 does increase telomere maintenance in part due to its ability to induce cellular

SUMOylation processes and affect the function of RAP1. Furthermore, we have identified changes in RAP1 biology due to expression of LMP1 that could contribute to a switch from ALT to telomerase-dependent telomere maintenance.

Future elucidation into LMP1 induced SUMOylation of other Shelterin complex proteins could provide additional evidence of how EBV switches from ALT to telomerase-dependent telomere maintenance.

REFERENCES

1. Health, United States, 2016: With Chartbook on Long-term Trends in Health. National Center for Health Statistics 2017, 1:128.

2. Kamranvar, S., Chen, X., Masucci, M.: Telomere dysfunction and activation of alternative lengthening of telomeres in B-lymphocytes infected by Epstein-Barr virus. Oncogene 2013, 32: 5522-5530.

3. Stanfield, B., Luftig, M.: Recent advances in understanding Epstein- Barr virus [version 1; referees: 4 approved]. F1000Research 2017, 6: 386.

4. Epstein-Barr Virus and Infectious Mononucleosis. Centers for Disease Control and Prevention 2016. https://www.cdc.gov/epstein-barr/about- mono.html

5. The American Cancer Society medical and editorial content team: Viruses that can lead to cancer. American Cancer Society 2016. https://www.cancer.org/cancer/cancer-causes/infectious-agents/infections- that-can-lead-to-cancer/viruses.html

6. Adler, B., Schaadt, E., Kempkes, B., Zimber-Strobl, U., Baier, B., Bornkamm, G.: Control of Epstein-Barr virus reactivation by activated CD40 and viral latent membrane protein 1. Proceedings of the National Academy of Sciences of the United States of America 2001, 99(1): 437- 442.

7. Bentz, G., Whitehurst, C., Pagano, J.: Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) C-Terminal-Activating Region 3 Contributes to LMP1-Mediated Cellular Migration via Its Interaction with Ubc9. Journal of Virology 2001, 85(19): 10144-10153.

8. Bentz, G., Moss, C., Whitehurst, C., Moody, C., Pagano, J.: LMP-1 induced SUMOylation influences the maintenance of Epstein-Barr virus latency through KAP1. Journal of Virology 2015, 89: 7465-7477.

9. Wilkinson, K., Henley, J.: Mechanisms, regulation and consequences of protein SUMOylation. Biochemical Journal 2012, 428(2): 133-145.

10. Wei, W., Yang, P., Pang, J., Zhang, S., Wang, Y., Wang, M., Dong, Z., She, J., Wang, C.: A stress-dependent SUMO4 SUMOylation of its substrate proteins. Biochemical and Biophysical Research Communications 2008, 375(3): 454-459.

11. Gong, L., Li, B., Millas, S., Yeh, E.: Molecular cloning and characterization of human AOS1 and UBA2, components of the sentrin-activating enzyme complex. Federation of European Biochemical Societies Letters 1999, 448: 185-189.

12. Schwarz, S., Matuschewski, K., Liakopoulos, D., Scheffner, M., Jentsch, S.: The ubiquitin-like proteins SMT3 and SUMO-1 are conjugated by the UBC9 E2 enzyme. Proceedings of the National Academy of Sciences of the United States of America 1997, 95: 560-564.

13. Rodriguez, M., Dargemont, C., Hay, R.: SUMO-1 Conjugation in Vivo Requires Both a Consensus Modification Motif and Nuclear Targeting. The Journal of Biological Chemistry 2000, 276(16): 12654- 12659.

14. Tatham, M., Jaffray, E., Vaughan, O., Desterro, J., Botting, C., Naismith, J., Hay, R.: Polymeric Chains of SUMO-2 and SUMO-3 Are Conjugated to Protein Substrates by SAE1/SAE2 and Ubc9. The Journal of Biological Chemistry 2001, 276(38): 35368-35374.

15. Moyzis, R., Buckingham, J., Cram, L., Dani, M., Deaven, L., Jones, M., Meyne, J., Ratliff, R., Wu, J.: A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proceedings of the National Academy of Sciences of the United States of America 1988, 85: 6622-6626.

16. Morin, G.: The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 1989, 59(3): 521-529.

17. Giunco, S., Dolcetti, R., Keppel, S., Celeghin, A., Indraccolo, S., Dal Col, J., Mastorci, K., De Rossi, A.: hTERT Inhibition Triggers Epstein-Barr Virus Lytic Cycle and Apoptosis in Immortalized and Transformed B Cells: A Basis for New Therapies. Clinical Cancer Research 2013, 19(8): 2036-2047.

18. Kataoka, H., Tahara, H., Watanabe, T., Sugawara, M., Ide, T., Goto, M., Furuichi, Y., Sugimoto, M.: Immortalization of immunologically committed Epstein-Barr virus-transformed B-lymphoblastoid cell lines accompanied by a strong telomerase activity. Differentiation 1997, 62: 203-211.

19. Ding, L., Li, L., Yang, J., Tao, Y., Ye, M., Shi, Y., Tang, M., Yi, W., Li, X., Gong, J., Cao, Y.: Epstein-Barr virus encoded latent membrane protein 1 modulates nuclear translocation of telomerase reverse transcriptase protein by activating nuclear factor-ϰB p65 in human nasopharyngeal carcinoma cells. The International Journal of Biochemistry & Cell Biology 2005, 37: 1881-1889.

20. Henson, J., Neumann, A., Yeager, T., Reddel, R.: Alternative lengthening of telomeres in mammalian cells. Oncogene 2002, 21: 598-610.

21. Artandi, S., DePinho, R.: Telomeres and telomerase in cancer. Carcinogenesis 2010, 31(1): 9-18.

22. Teo, H., Ghosh, S., Luesch, H., Ghosh, A., Wong, E., Malik, N., Orth, A., de Jesus, P., Perry, A., Oliver, J., Tran, N., Speiser, L., Wong, M., Saez, E., Schultz, P., Chanda, S., Verma, I., Tergaonkar, V.: Telomere- independent Rap1 is an IKK adaptor and regulates NF-ϰB-dependent gene expression. Nature Cell Biology 2010, 12(8): 758-767.

23. Li, B., Oestreich, S., de Lange, T.: Identification of Human Rap1: Implications for Telomere Evolution. Cell 2000, 101: 471-483.

24. Janoušková, E., Nečasová, I., Pavloušková, J., Zimmermann, M., Hluchý, M., Marini, V., Nováková, M., Hofr, C.: Human Rap1 modulates TRF2 attraction to telomeric DNA. Nucleic Acids Research 2015, 43(5): 2691- 2700.

25. O’Conner, M., Safari, A., Liu, D., Qin, J., Songyan, Z.: The Human Rap1 Protein Complex and Modulation of Telomere Length. The Journal of Biological Chemistry 2004, 279(27): 28585-28591.

26. Li, B., de Lange, T.: Rap1 Affects the Length and Heterogeneity of Human Telomeres. Molecular Biology of the Cell 2003, 14: 5060-5068.

27. Xin, H., Liu, D., Songyan, Z.: The telosome/shelterin complex and its functions. Genome Biology 2008, 9: 232.

28. PML Gene, GeneCards, Weizmann Institute of Science 2017. http://www.genecards.org/cgi-bin/carddisp.pl?gene=PML

29. Shen, T., Lin, H., Scaglioni, P., Yung, T., Pandolfi, P.: The Mechanisms of PML-Nuclear Body Formation. Molecular Cell 2006, 24: 331-339.

30. Bechter, O., Zou, Y., Shay, J., Wright, W.: Homologous recombination in human telomerase-positive and ALT cells occurs with the same frequency. European Molecular Biology Organization Press 2003, 4(12): 1138-1143.

31. Chung, I., Osterwald, S., Deeg, K., Rippe, D.: PML body meets telomere. Nucleus 2012, 3(3): 263-275.

32. Chung, I., Leonhardt, H., Rippe, K.: De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation. Journal of Cell Science 2011, 124: 3603-3618.

33. Sides, M., Block, G., Shan, B., Esteves, K., Lin, Z., Flemington, E., Lasky, J.: Arsenic mediated disruption of promyelocytic leukemia protein nuclear bodies induces ganciclovir susceptibility in Epstein-Barr positive epithelial cells. Virology 2011, 416: 86-97.

34. Dirmeier, U., Neuhierl, B., Kilger, E., Resbach, G., Sandberg, M., Hammerschmidt, W.: Latent membrane protein 1 is critical for efficient growth transformation of human B cells by Epstein-Barr virus. Cancer Research 2003, 63: 2982-2989.

35. Miller, W., Mosialos, G., Kieff, E., Raab-Traub, B.: Epstein-Barr virus LMP1 induction of the epidermal growth factor receptor is mediated through TRAF signaling pathway distinct from NF-kappaB activation. Journal of Virology 1997, 71: 586-594.

36. Yu, J., Lan, J., Wang, C., Wu, Q., Zhu, Y., Lai, X., Sun, J., Jin, C., Huang, H.: PML3 interacts with TRF1 and is essential for ALT-associated PML bodies assembly in U2OS cells. Cancer Letters 2010, 291: 177- 186.

37. Hu, Y., Shi, G., Zhang, L., Li, F., Jiang, Y., Jiang, S., Ma, W., Zhao, Y., Songyang, Z., Huang, J.: Switch telomerase to ALT mechanism by inducing telomeric DNA damages and dysfunction of ATRX and DAXX. Scientific Reports 2016, 6: 32280.

38. Xu, B., Peng, M., Song, Q.: The co-expression of telomerase and ALT pathway in human breast cancer tissues. Tumor Biology 2014, 35: 4087-4093.

39. Hang, L., Liu, X., Cheung, I., Yang, Y., Zhao, X.: SUMOylation regulates telomere length homeostasis by targeting Cdc13. Nature Structural & Molecular Biology 2011, 18(8): 920-927.

40. Davison, A.: Herpesvirus systematics. Veterinary Microbiology 2010, 143: 52-69.

41. Hadinoto, V., Shapiro, M., Sun, C., Thorley-Lawson, D.: The Dynamics of EBV Shedding Implicates a Central Role for Epithelial Cells in Amplifying Viral Output. PLoS Pathogens 2009, 5(7): e1000496.

42. Gires, O., Kohlhuber, F., Kilger, E., Baumann, M., Kieser, A., Kaiser, C., Zeider, R., Scheffer, B., Ueffing, M., Hammerschmidt, W.: Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins. European Molecular Biology Organization Journal 1999, 18(11): 3064-3073.

43. Liu, X., Li, Y., Peng, S., Yu, X., Li, W., Shi, F., Luo, X., Tang, M., Tan, Z., Bode, A., Cao, Y.: Epstein-Barr virus encoded latent membrane protein 1 suppresses necroptosis through targeting RIPK1/3 ubiquitination. Cell Death and Disease 2018, 9: 53.

44. Bentz, G., Shackelford, J., Pagano, J.: Epstein-Barr virus latent membrane protein 1 regulates the function of interferon regulatory factor 7 by inducing its sumoylation. Journal of Virology 2012, 86: 12251-12261.

45. Tamitani, T., Kito, K., Nguyen, H., Fukuda-Kamitani, T., Yeh, E.: Characterization of a Second Member of the Sentrin Family of Ubiquitin-like Proteins. The Journal of Biological Chemistry 1998, 273(18): 11349-11353.

46. Pichler, A., Knipscheer, P., Oberhofer, E., van Dijk, W., Korner, R., Olsen, J., Jentsch, S., Melchior, F., Sixma, T.: SUMO modification of the ubiquitin-conjugating enzyme E2-25K. Nature Reviews Molecular Cell Biology 2007, 8: 947-956.

47. Mahajan, R., Delphin, C., Guan, T., Gerace, L., Melchior, F.: A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 1997, 88: 97-107.

48. Hardeland, U., Steinarcher, R., Jiricny, J., Schar, P.: Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover. European Molecular Biology Organization Journal 2002, 21: 1456-1464.

49. Tahara, H., Tokutake, Y., Maeda, S., Kataoka, H., Watanabe, T., Satoh, M., Matsumoto, T., Sugawara, M., Ide, T., Goto, M., Furuichi, Y., Sugimoto, M.: Abnormal telomere dynamics of B-lymphoblastoid cell strains from Werner’s syndrome patients transformed by Epstein- Barr virus. Oncogene 1997, 15: 1911-1920.

50. de Lange, T.: Shelterin: the protein complex that shapes and safeguards human telomeres. Genes & Development 2005, 19: 2100- 2110.

51. Salomoni P., Pandolfi, P.: The role of PML in tumor suppression. Cell 2002, 108: 165-170.

52. Everett, R., Chelbi-Alix, M.: PML and PML nuclear bodies: implications in antiviral defence. Biochimie 2007, 89(6-7): 819-830.

53. Nabetani A., Yokoyama, O., Ishikawa, F.: Localization of hRad9, hHus1, hRad1 and hRad17 and caffeine-sensitive DNA replication at the alternative lengthening of telomeres-associated promyelocytic leukemia body. The Journal of Biological Chemistry 2004, 279: 25849- 25857.

54. Opresko, P., Fan, J., Danzy, S., Wilson, D., Bohr, V.: Oxidative damage in telomeric DNA disrupts recognition by TRF1 and TRF2. Nucleic Acids Research 2005, 33(4): 1230-1239.

55. Indran, I., Hande, M., Pervaiz, S.: hTERT Overexpression Alleviates Intracellular ROS Production, Improves Mitochondrial Function, and Inhibits ROS-Mediated Apoptosis in Cancer Cells. Cancer Research 2011, 71(1): 266-276.

56. Izumi, M., Yatagai, F., Hanaoka, F.: Cell cycle-dependent proteolysis and phosphorylation of human Mcm10. The Journal of Biological Chemistry 2001, 276: 48526-48531.

57. Kerscher O., Felberbaum, R., Hochstrasser, M.: Modification of proteins by ubiquitin and ubiquitin-like proteins. Annual Review of Cell and Developmental Biology 2006, 22: 159-180.

58. Kroetz, M.: SUMO: a ubiquitin-like protein modifier. Yale Journal of Biology and Medicine 2005, 78: 197-201.

59. Kerscher, O.: SUMO junction—what’s your function? New insights through SUMO-interacting motifs. EMBO Reports 2007, 8: 550-555.

60. Ning, S., Hahn, A., Huye, L., Pagano, J.: Interferon regulatory factor 7 regulates expression of Epstein-Barr virus latent membrane protein 1: a regulatory circuit. Journal of Virology 2003, 77: 9359-9368.

61. Calender, A., Cordier, M., Billaud, M., Lenoir, G.: Modulation of cellular gene expression in B lymphoma cells following in vitro infection by Epstein-Barr virus (EBV). International Journal of Cancer 1990, 46: 658- 663.

62. Cherney, B., Sgadari, C., Kanegane, C., Wang, F., Tosato, G.: Expression of the Epstein-Barr virus protein LMP1 mediates tumor regression in vivo. Blood 1998, 91: 2491-2500.