Epstein-Barr virus protein EBNA-LP engages YY1 through leucine-rich motifs to promote naïve B cell transformation

Epstein-Barr Virus (EBV) is associated with numerous cancers including B cell lymphomas. In vitro, EBV transforms primary B cells into immortalized Lymphoblastoid Cell Lines (LCLs) which serves as a model to study the role of viral proteins in EBV malignancies. EBV induced cellular transformation is driven by viral proteins including EBV-Nuclear Antigens (EBNAs). EBNA-LP is important for the transformation of naïve but not memory B cells. While EBNA-LP was thought to promote gene activation by EBNA2, EBNA-LP Knock Out (LPKO) virus-infected cells express EBNA2-activated genes efficiently. Therefore, a gap in knowledge exists as to what roles EBNA-LP plays in naïve B cell transformation. We developed a trans-complementation assay wherein transfection with wild-type EBNA-LP rescues the transformation of peripheral blood- and cord blood-derived naïve B cells by LPKO virus. Despite EBNA-LP phosphorylation sites being important in EBNA2 co-activation; neither phospho-mutant nor phospho-mimetic EBNA-LP was defective in rescuing naïve B cell outgrowth. However, we identified conserved leucine-rich motifs in EBNA-LP that were required for transformation of adult naïve and cord blood B cells. Because cellular PPAR-γ coactivator (PGC) proteins use leucine-rich motifs to engage transcription factors including YY1, a key regulator of DNA looping and metabolism, we examined the role of EBNA-LP in engaging cellular transcription factors. We found a significant overlap between EBNA-LP and YY1 in ChIP-Seq data and confirmed their biochemical association in LCLs by endogenous co-immunoprecipitation. Moreover, we found that the EBNA-LP leucine-rich motifs were required for YY1 interaction in LCLs. Finally, we used Cas9 to knockout YY1 in primary total B cells and naïve B cells prior to EBV infection and found YY1 to be essential for EBV-mediated transformation. We propose that EBNA-LP engages YY1 through conserved leucine-rich motifs to promote EBV transformation of naïve B cells.


Introduction
Epstein-Barr virus (EBV) is a gamma-herpesvirus that infectsand permanently resides innearly all individuals by adulthood.EBV infects resting B cells, which leads to B cell activation followed by a life-long latent infection in memory B cells [1].While infection is typically asymptomatic, EBV is associated with numerous malignancies including B cell lymphomas such as Hodgkin's Lymphoma, Burkitt Lymphoma, and lymphoproliferative disease in immunocompromised individuals [2].EBV-induced cellular proliferation is largely driven by the activity of viral proteins.

EBV infection of B cells leads to the expression of the viral latency program which includes EBV Nuclear
Antigens (EBNAs) and Latent Membrane Protein (LMPs).EBNAs are transcriptional regulators which include EBNA1, 2, 3A, 3B, 3C, and -Leader Protein (LP).Other than EBNA1, the EBNA proteins do not directly bind DNA, but rather regulate transcription by engaging with cellular DNA sequence-specific binding proteins.EBNA2 and EBNA-LP are the initial viral latency proteins expressed, being detected as early as 12 hours post infection from the repeated viral W promoter (Wp) [3][4][5].Once EBNA2 and EBNA-LP are expressed, the C promoter (Cp) becomes transcriptionally active from which promoter EBNAs are then predominantly transcribed, followed by the LMP proteins which take 1-2 weeks to reach the level sustained in LCLs [5,6].In vitro, infected cells initially increase in size, followed by a phase of cellular hyperproliferation beginning 3-4 days post infection, followed by a slower period of cellular outgrowth [7].
Restriction by innate cellular barriers including metabolic stress and DNA damage ultimately leads to cellular arrest of many infected cells [8].However, cells that overcome these barriers in vitro are transformed into immortalized lymphoblastoid cell lines (LCLs), serving as a model to study the role of viral proteins in primary B cell infection and EBV-associated malignancies.
EBNA2 is an important regulator of both viral and cellular genes and is required at all stages of primary infection in vitro [9].Importantly, EBNA2 engages several host transcription factors including RBPJ and EBF1 to induce high levels of the oncogene Myc, among other cellular genes [10][11][12].While less is known about the role of EBNA-LP in EBV infection and cellular transformation, several studies have contributed to our knowledge of EBNA-LP activity in infected B cells.The main function of EBNA-LP has been ascribed to co-activating EBNA2 activity at both viral and host genes.In transfection experiments performed outside of the context of primary infection, EBNA-LP increases EBNA2 activation of the Cp, LMP1 promoter, RBPJ-binding sites, and cellular genes including HES1 [13][14][15].However, studies identifying which domains of EBNA-LP are responsible for this activity have found conflicting results [13][14][15].EBNA-LP is also hypothesized to promote EBNA2 activity by removing transcriptional repressors including NCoR and HA95 from EBNA2 targeted sites [15][16][17].EBNA-LP enriched sites on chromatin are also associated with several cellular transcription factors and DNA looping factors [15][16][17][18].Furthermore, upon B cell infection and in LCLs, EBNA-LP localizes to the subnuclear foci called PML bodies, which are implicated in restricting viral gene expression [19,20].However, the significance of these many EBNA-LP functions in the context of primary infection are still unclear.
The N-terminal part of EBNA-LP is composed of variable numbers of repeats of a 66 amino acid domain (encoded by W1 and W2 exons within the major internal repeat of the EBV genome, which we will call the W domain), and its C-terminus (called the Y domain, as it derives from exons Y1 and Y2) contains 45 amino acids.Circulating viruses encode at least 4 repeated W domains [21].Alternative Wp usage and splicing leads to expression of multiple EBNA-LP isoforms with variable numbers of W domains. Across the W and Y domains, there are five evolutionarily conserved regions (CR) between EBV EBNA-LP and its homologs in primate lymphocryptovirus (LCV) [22].One study found that all three conserved regions in the W domain (CR1, CR2, CR3) are required to enhance the activation of genes by EBNA2, while motifs within CR1 and CR2 contain a bi-partite nuclear localization signal [22][23][24].The Y domain contains two additional conserved regions (CR4 and CR5) [22].Importantly, the significance of the conserved regions has not been studied in the context of primary infection.
EBNA-LP is essential in transforming cord blood-derived B cells which are exclusively naïve, and is significantly more important for transformation of sorted adult peripheral blood naïve B cells, defined as IgD+/CD27, than adult peripheral blood memory B cells [32].A similar study in adenoid tissue-derived naïve B cells found EBNA-LP was critical during early stages of infection for cell division and survival [9].In the absence of the EBNA-LP Y domain, the efficiency of cellular transformation of infected B cells is significantly reduced [33,34].While the function of the Y domain is unknown, leucines in the Y domain CR4 have been implicated as the site of interaction with the transcription factor Estrogen Related Receptor Alpha (ERR) [35].Intriguingly, ERR is a transcription factor that is co-activated by the Peroxisome PPAR- Coactivator (PGC) family of proteins [36].The PGC proteins, like many other transcriptional co-activators, utilize their leucine-rich motifs (LRMS) typically of the sequence LXXLL to bind transcription factors and then recruit additional co-activators to promote transcription [36][37][38].We therefore sought to determine whether the LRMs within EBNA-LP could be important motifs functionally mimicking the LRMs in cellular PGC co-activators of transcription.
Despite the proposed role of EBNA-LP in assisting EBNA2-mediated transcriptional activation, genetic studies with an EBNA-LP knock out (LPKO) virus indicates EBNA-LP is in fact not required for EBNA2 to activate host genes including Myc, nor for EBNA2 recruitment to cellular chromatin [32], but rather may constrain excessive EBNA2-mediated activation of cellular genes.Therefore, we sought to uncover other important roles of EBNA-LP in EBV-infected B cells in a physiologically relevant context.To do so, we used a trans-complementation assay to define the importance of conserved regions and posttranslational modifications of EBNA-LP in the transformation of naïve B cells by EBV.

Complementation with Wild-Type EBNA-LP
In order to assay the consequences of EBNA-LP mutations, we developed a trans-complementation assay.
First, we tested whether outgrowth of adult naïve B cells infected with EBNA-LP Knock Out (LPKO) virus could be rescued by expression of exogenous, wild type EBNA-LP.We created a complementation vector encoding FLAG-tdTomato, a P2A cleavage site, wild type FLAG-EBNA-LP including four of the repeated W domains codon optimized to improve plasmid stability (S1 Table ), and the EBV oriP and EBNA1 to allow episomal persistence of the vector (S1A-B Fig) .Naïve B cells derived from adult peripheral blood mononuclear cells (PBMCs) were isolated by immunomagnetic selection for IgD+/CD27-cells, although purity varied by donor ranging from 92.2-97.6% with the majority of contaminating cells coming from IgD-/CD27-B cells, highlighting the challenges of isolating pure naïve B cell from adult PBMCs (S2A Fig) .For each replicate, isolated cells were transfected with the complementation vector encoding EBNA-LP, with vector encoding only FLAG-tdTomato, or left untransfected (Fig 1A).Cells were then infected with LPKO or WT virus [32] at a titer determined to infect almost 100% of cells.Outgrowth of tdTomato positive cells, indicative of maintenance of the episomal vector, was then assayed by flow cytometry every seven days post transfection, with compensation for GFP produced by the virus (S2B Fig) .While the electroporation of primary B cells resulted in killing of some transfected cells, the similarity in total proliferating cells between untransfected cells and cells transfected with only vector for WT-infected cells indicated that the failure of LPKOinfected cells transfected with vector to proliferate was not a result of electroporation, but rather reflects the importance of EBNA-LP early during infection in peripheral blood-derived naïve B cells (S2C, S2D Fig) .In all three adult blood donors, trans-complementation of naïve B cells with EBNA-LP that were infected with LPKO virus led to outgrowth of tdTomato positive cells over time similar to WT virus infected cells transfected with the tdTomato vector lacking EBNA-LP (Fig 1B).Of note, there was a high degree of variability in outgrowth efficiency between replicates both within and between donors.LPKO infected cells transfected with vector encoding only tdTomato consistently failed to expand tdTomato positive cells, whereas trans-complementation with EBNA-LP rescued outgrowth and successfully generated tdTomato+ LCLs (Fig 1C -1E).As expected, WT virus transformed untransfected naïve B cells into tdTomato negative LCLs in each replicate across three donors (Fig 1E).WT infected cells transfected with vector also generated LCLs and retained tdTomato expression, albeit only in a small proportion on average fewer than 3%of cells (S2E Fig) .As the purified naïve B cell populations still contain contaminating unconventional memory B cells (IgD-/CD27-), which do not require EBNA-LP for outgrowth and cellular transformation [32,39]  retained EBNA-LP expression at levels comparable to WT LCLs (Fig. 1F).The ability of this trans-complementation assay to consistently rescue transformation of LPKO infected naïve B cells and retain EBNA-LP expression confirms this assay can also be used to assess the role of EBNA-LP mutants in the context of primary infection.

Phosphorylation of EBNA-LP is not Required for Naïve B Cell Transformation
Phosphorylation of EBNA-LP is considered essential for EBNA-LP-mediated coactivation of EBNA2 [23,24,29].Therefore, we sought to uncover whether phosphorylation is required for EBNA-LP-mediated transformation of naïve B cells.All three predicted phosphorylated serines (S34, S36, and S63) are conserved across EBV Type 1 and Type 2 strains, while S36 is also conserved across EBV strains and in strains of the non-human primate lymphocryptovirus (LCV), the closest common ancestor to EBV [22,24]  While phosphorylation of S36 has previously been confirmed, this is the first study to validate S34 and S63 as phosphorylation sites on EBNA-LP by mass spectrometry.
We then generated both a FLAG-tagged phospho-mutant (S3A) in which S34, S36, and S63 were modified to alanine, and a phospho-mimetic (S3E) in which S34, S36, and S63 were modified to glutamic acid.Wild Type, S3A, and S3E EBNA-LP were transfected into 293T cells, and FLAG immunoprecipitations to enrich for EBNA-LP were performed in triplicate for each construct prior to analysis by mass spectrometry using Tandem Mass Tag (TMT) labelling to quantify the signal of identified peptides across samples.Only wild type EBNA-LP was phosphorylated, with peptides containing phospho-residue S63, but no phosphorylated peptides were detected from either EBNA-LP-S3A or -S3E (Fig 2D ), although no peptides covering S34 and S36 were identified in this experiment.This confirms S34, S36, and S63 as the only sites of phosphorylation on EBNA-LP, further validating that other highly conserved serines including S61 are not phosphorylated.
We then tested whether EBNA-LP-S3A or -S3E could rescue LPKO virus infected naïve B cells.Despite the previously inferred importance of EBNA-LP phosphorylation sites, we found that, surprisingly, trans-complementation with either the phospho-mutant or phospho-mimetic could rescue LPKO infected naïve B cells to the same degree

Adult Total B Cell Infection
We next sought to confirm that the EBNA-LP LRM mutant had wild type level expression.While the LRM mutant was unable to generate tdTomato positive LCLs from infected naïve B cells, LRM mutant transfected LPKO infections of total B cells yielded LCLspresumably from transformation of memory B cells.Importantly, wild type and LRM mutant EBNA-LP were expressed at similar levels in these LCLs (Fig 5A).Furthermore, the LRM mutant was visible both in sub-nuclear puncta and diffuse within the nuclei of trans-complemented LPKO LCLs similar to wild type EBNA-LP (Fig 5B).These results suggest mutation of the LRMs result in loss of an interaction between EBNA-LP and a cellular protein that is important for naïve B cell transformation, rather than loss of protein expression or misfolding.

Association with YY1
Given the role of leucine-rich motifs in coactivation by PGC family members, we used publicly available ChIP-Seq data from LCLs to identify cellular transcription factors whose binding sites on the genome had significant overlap with those of EBNA-LP.We found that YY1, a transcription factor co-activated by PGC proteins, shared many sites with EBNA-LP in agreement with previous work [18,43,44]  As the interaction interface between EBNA-LP and YY1 is unknown, we investigated whether the leucinerich motifs were indeed important.Using the trans-complemented LPKO LCLs derived from total B cells, reciprocal co-immunoprecipitations showed that wild type, but not LRM mutant EBNA-LP pulled down YY1 (Fig 6D ), and conversely that YY1 co-immunoprecipitation pulled down wild type EBNA-LP, but not the LRM mutant protein (Fig 6E).Therefore, these LRM motifs in EBNA-LP that are required for transformation of naïve B cells are also required for interaction with the cellular transcription factor YY1, indicating a novel function for EBNA-LP in cellular transformation during EBV infection.
An essential role for YY1 in EBV infection and B cell transformation has not previously been described.
Therefore, we used a Cas9 RNP-based knockout approach to assess the importance of YY1 in EBV-infected B cell outgrowth.The non-essential cell surface protein CD46 was used as a control and proxy for gene targeting as previously described by others [45].We found that YY1 knockout in either total B cells (

Discussion
EBV viral proteins aid in maintaining life-long, latently infected B cells by facilitating the proliferation and survival of infected cells.When the virus-host balance is disturbed, such as in the absence of a functional immune system, the characteristics of these viral proteins can contribute to oncogenesis as modeled by in vitro transformation of primary B cells by EBV.While EBNA-LP is essential for the transformation of naïve B cells by EBV, it has primarily been studied outside of the context of infection.As EBNA-LP is encoded in the viral genome across multiple repeat units of the major internal repeat, making mutations to the EBNA-LP coding sequence in the viral genome is particularly challenging and time consuming.Thus, we have developed a trans-complementation system by which the well-conserved regions of EBNA-LP could be assessed in the context of primary B cell infection.
Excitingly, we could reliably transfect resting primary B cells with an episomal construct that was maintained upon EBV infection and outgrowth into LCLs.This assay could therefore be used to screen a large number of EBNA-LP mutants in the context of EBV infection of primary B cells, beyond those we present here.This transcomplementation strategy presents a system by which it is possible to screen important functions of EBNA-LP so that the more precise but technically challenging and time-consuming approach of constructing mutant viruses can be more intelligently targeted.It also opens up the possibility of trans-complementing with proteins from other viruses that target the same biological processes, and can be adapted for trans-complementing EBV mutants more generally.Therefore, by pairing infection of knock-out viruses with trans-complementation of mutant viral proteins, this assay could be applied to identify key features of other viral proteins in EBV transformation of primary B cells.
Unexpectedlygiven its importance for the perceived main function of EBNA-LP phosphorylation in EBNA2 co-activationwe found that EBNA-LP phosphorylation does not contribute detectably to the outgrowth of LCLs.As such, our work highlights both the limitations of studying viral proteins outside of the context of primary infection and suggests EBNA-LP has numerous roles in the survival and transformation of infected naïve B cells.Instead, we identified highly conserved leucine-rich motifs in both the W domain CR1 and Y domain CR4 that are required to rescue LPKO infected naïve B cells derived from adult peripheral blood and cord blood.While the presence of small numbers of antigen-experienced B cellsboth conventional and double negative (IgD-/CD27-) memory B cellsin our adult peripheral blood naïve B cell fractions led to outgrowth of some tdTomato negative LCLs, our results indicate that the leucine-rich motifs in EBNA-LP play an important role in EBV-mediated transformation.
We found the conserved leucine-rich motifs in EBNA-LP mediate interaction with YY1.YY1 is both a transcription factor and DNA looping factor that promotes interactions between enhancer and promoter regions [46].YY1 is important in B cells as a transcriptional regulator of the germinal center reaction [47].In the absence of YY1, naïve B cells are unable to differentiate into germinal center B cells or plasma cells [48][49][50].The requirement for YY1 may in part be due to regulation of apoptosis, as YY1 knock out in pro-B cells and germinal center B cells causes cells to be more prone to apoptosis [48,50].YY1 deficient naïve B cells also display reduced proliferation upon stimulation compared to controls [48].YY1 has also been implicated in transcriptional regulation of genes involved in oxidation phosphorylation in B cells [48,49,51], upregulation of which is required to avoid cellular arrest upon EBV infection [8].As EBV-infected cells undergo B cell activation and differentiation in a manner that mimics the remodeling of antigen-experienced B cells through the germinal center [52,53], YY1 may therefore be required for survival and effective cellular remodeling during EBV infection and transformation.Furthermore, the unique importance for EBNA-LP in naïve and not memory B cell transformation upon EBV infection, suggests basal differences between naïve and memory B cells exist that require different strategies for viral transformation.Naïve B cells, for example, may require additional viral assistance in avoiding cell death compared to memory B cells, as naïve B cells express lower levels of antiapoptotic proteins and are more likely to undergo apoptosis in both the presence and absence of antigen stimulation than memory B cells [54].Memory B cells also proliferate more rapidly upon antigen activation than naïve B cells [55,56] as naïve B cells express elevated levels of negative cell cycle regulators [55].Memory cells are also more predominantly in the cell cycle phase G1 compared to naïve B cells [57], suggesting memory B cells are better primed to enter cell division while naïve B cells may have greater intrinsic barriers to EBV induction of proliferation.Further, naïve B cell activation by antigen increases both glycolysis and oxidative phosphorylation [58] and requires mitochondrial remodeling [59].While little is known about the metabolic differences between resting naïve and memory B cells, memory T cells have increased mitochondrial mass compared to naïve T cells, and also have a greater capacity for both oxidative phosphorylation and glycolysis upon activation suggesting the transition from naïve to memory in immune cells alters metabolic capacity long term [60].Therefore, EBNA-LP association with YY1 may be important in naïve B cells to overcome barriers to transformation including apoptosis, initiation of proliferation, and cellular metabolism which may be innately more restrictive in naïve B cells than memory B cells.Additional functions of EBNA-LP including disruption of anti-viral PML nuclear bodies may also be important for elucidating the requirement for EBNA-LP in naïve B cell infection and outgrowth.
B cell activation and differentiation also requires a large degree of chromatin remodeling, which YY1, as a chromatin looping factor, may contribute to.During the transition from naïve B cells to germinal center B cells, roughly 95% of chromatin compartments are remodeled, primarily to a more active chromatin state, followed by 73% of remodeled regions returning to their original states in memory B cells [61].EBV infection largely mimics this chromatin remodeling process [62], however, whether differences in naïve and memory B cell chromatin architecture includes YY1 motif accessibility or YY1-regulated DNA loops remains to be studied, but could provide insight as to further differences in naïve B cell dependence on EBNA-LP and YY1 association.
As a mechanism for cellular gene regulation, YY1 has also been implicated in the formation of "EBV superenhancers" on host chromatin, which are defined as regions of accessible chromatin where multiple EBNAs and cellular transcription factors associate leading to increased expression of target genes [63]."EBV super-enhancers" target genes important in infected cell survival and proliferation including Myc [63].Therefore, EBNA-LP association with YY1 for genome rearrangement may in fact be a mechanism by which EBNA-LP stabilizes induction of EBNA2 associated genes, including both important cellular genes and viral genes.YY1 is able to alter the structure and epigenetic marks of numerous DNA virus genomes resulting in both activation and repression of viral genes [64].
These viruses include herpesviruses such as Human Cytomegalovirus and Kaposi's Sarcoma-Associated Herpesvirus, small DNA tumor viruses including Human Papilloma Virus, and many others [64][65][66][67][68].In the EBV genome, YY1 binds and represses the promoters of the immediate early genes BZLF1 and BRLF1, preventing induction of the productive replication cycle [69][70][71].There is also a YY1 binding site upstream of the immediate-early latency promoter Wp [72].This region including the YY1 binding site is important for transcription from both the Cp and initial Wp, and deletion hinders transformation of infected cells [73].Therefore, YY1 binding to the viral genome may contribute to inducing adequate expression of viral latency proteins through regulation of viral Wp and Cp promoter usage during early infection and transformation.Whether YY1 occurrence at these cellular and viral genomic sites is EBNA-LP dependent requires further investigation.sequence in each W domain was identical, but the DNA sequence in each W domain was unique in order to avoid recombination of repeated W domains and facilitate downstream cloning.S3A and S3E EBNA-LP genes were synthesized with the S34, S36, and S63 in each W domain modified to alanine or glutamic acid respectively.For the LRM mutant, EBNA-LP cDNA was first synthesized such that L25, L28, and L29 in each W domain were modified to alanine.QuikChange XL Site-Directed Mutagenesis (Agilent, 200517) was then used to modify L13, L17, and L18 in the Y domain to alanine.
EBNA-LP constructs were first cloned into pSG5-FLAG-Gateway (a gift from Eric Johannsen) for expression in 293T cells by amplifying with primers encoding attB1/attB2 recombination sites and performing recombination by gateway cloning (ThermoFisher 11789020, and 11791020).For trans-complementation assays, FLAG-tdTomato-P2A was  to infect almost all B cells and an optimal ratio for outgrowth, similar to previous reports [9].Every seven days, cell outgrowth was quantified by flow cytometry.Media was added during outgrowth upon increased cell density.

Flow Cytometry
Flow cytometry was used to assess purity of isolated primary cells and to quantify outgrowth of trans-complemented naïve B cells.For sample preparation following isolation, cells were washed once in FACS Buffer (PBS with 2% FBS), stained with indicated antibodies (Table 1) for 15 minutes in the dark at room temperature, then rinsed again with FACS buffer.For samples in the trans-complement assays, samples were prepared and collected in a 96-well Vbottom plate.First, samples were evenly resuspended, and a known volume of each sample was moved to the plate.
Samples were then washed once in FACS buffer and resuspended in 100 uL of FACS buffer prior to collection.
Samples were collected using the BD FACSCanto-II with a high throughput sampler to collect precise sample volumes.Analysis was performed using FlowJo software (FlowJo, LLC).
Cells were washed one time with PBS before pelleting.Pellets were resuspended in 1x RIPA buffer with protease and phosphatase inhibitors added.After shaking samples at 4C at 600 rpm, samples were centrifuged for 10 minutes at 14,000xg.The lysates were then quantified using Bradford assay and diluted to 1x LDS.Samples were loaded on a 4-12% Bis-Tris gel (ThermoFisher, NP0321BOX) and run in MOPS Running Buffer (ThermoFisher, NP0001) followed by transfer to PVDF membranes using a TransBlot Turbo Transfer System at 25 V, 1.3 A, for 12 min (Bio-Rad).Total protein was quantified using total protein stain (LI-COR, 926-11011) and blocked in 5% milk in 1x TBST.
Blot was incubated overnight with primary antibody in 1x TBST with 5% BSA at 4C.Blots were then washed in 1x TBST, incubated in secondary antibody for 1 hour at room temperature in 5% milk, and washed three times for 10 minutes.Blots were incubated with HRP substrate and imaged on the LI-COR Odyssey XF Imager.

Immunofluorescence
Immunofluorescence was performed as described previously [8].Slides were mounted in VECTASHIELD HardSet Antifade Mounting medium with DAPI (H-1500-10).Slides were imaged using the Olympus IX81 microscope using a 60X oil objective.

ChIP-Seq Analysis
Analysis of YY1 and EBNA-LP ChIP-Seq data was performed using HOMER [76].The functions makeTagDirectory and findPeaks were used to call YY1 peaks.EBNA-LP called peaks were obtained as previously described [18].The function mergePeaks with a distance of 200 base pairs was used to identify overlapping and uniquely bound sites between YY1 and EBNA-LP.The function annotatePeaks.pl was then used to assign overlapping peaks to genomic regions.YY1 ChIP-Seq in GM12878 data was obtained from ENCODE (Experiment ENCSR000BNP).

Cas9-RNP Transfection
TrueCut Cas9 protein-v2 (ThermoFisher, A36499), 10 pmol per target, was incubated with 30 pmol sgRNA (Gene Knockout Kit v2, Synthego) per target (Table 1).Isolated naïve and total B cells were washed in PBS and resuspended in Buffer T (Neon Transfection System).350,000 cells were transfected with Cas9/RNP complexes and infected with B95-8 virus as described above.Outgrowth was assessed by flow cytometry upon staining cells with anti-CD46 (Table 1).*Indicates conditions in which some replicates generated tdTomato negative LCLs.† Indicates a tdTomato negative LCL which grew out slower compared to those trans-complemented with wild type EBNA-LP.
(S2A Fig), tdTomato negative LCLs were observed following LPKO infection in both untransfected and cells trans-complemented with only tdTomato in some donors (Fig 1E, S2E Fig).Rescued LCLs (Fig 2A).We assessed phosphorylation of EBNA-LP by performing FLAG immunoprecipitation followed by mass spectrometry with trypsin digestion of our FLAG-tagged EBNA-LP encoding 4 W domains expressed in 293T cells.In total, 81 peptides were identified which covered most of the EBNA-LP protein (Fig 2B).Only two peptides spanning S34 and S36 were identified, likely because they are located in a region rich in arginine, the site of trypsin cleavage (Fig 2B) [40, 41].However, of these identified peptides, one contained phosphorylated S34 (32-HRSpPSPTR-39) (Fig 2C and S3A Fig) and the other phosphorylated S36, (34-SPSpPTRGGQEPR-45) suggesting phosphorylation is a highly abundant modification at S34 and S36 (Fig 2C and S3B Fig).Peptides spanning S63 were also identified (including 51-VLVQQEEEVVSGSpPSGPR-68) (S3C Fig) with phosphorylation at S63 observed in 6 out of 41 peptides (Fig 2C).
as wild-type EBNA-LP both 14 days post infection (Fig 2E) and 28-35 days post infection (Fig 2F).Both mutants also successfully generated tdTomato positive LCLs similar to wild type EBNA-LP (Fig 2G).Further, EBNA-LP-S3A and -S3E constructs were retained and expressed EBNA-LP protein in trans-complemented LCLs (Fig 2H).These findings suggest that the EBNA2 co-activation assay may not faithfully recapitulate the functions important for EBNA-LP in naïve B cell transformation.EBNA-LP Contains Conserved, Leucine-Rich Motifs that are Essential for Naïve B Cell Transformation Since the sites of EBNA-LP phosphorylation are non-essential for transformation of naïve B cells, we next sought to determine what other motifs in EBNA-LP could be important for mediating naïve B cell transformation.We identified highly conserved leucine-rich motifs (LRM) in both the repeated W domain CR1s and Y domain CR4 [22, 35] (Fig 3A).Because these motifs are reminiscent of the leucine-rich motifs in the PGC coactivator family, we hypothesized that these conserved regions could be important for mediating EBNA-LP interaction with cellular factors.We therefore generated constructs in which the leucines in LRMs of both the W domain CR1s and Y domain CR4 were mutated to alanine (LRM mutant) (Fig 3B).At 14 days post infection, there were significantly fewer tdTomato positive cells in the LPKO virus infected cells trans-complemented with the LRM Mutant compared to transcomplementation with wild type EBNA-LP (Fig 3C).At this early time point there was also a higher total number of cells in LPKO virus rescued by wild type EBNA-LP compared to the LRM Mutant (S4A Fig), further supporting the inability of the LRM mutant to rescue LPKO virus early during infection.Trans-complementation with wild type EBNA-LP continued to promote higher outgrowth of tdTomato positive cells compared to the LRM mutant 28-35 days post infection (Fig 3D), and the LRM mutant failed to generate tdTomato positive LCLs from LPKO infected adult naïve B cells (Fig 3E).We did, however, observe rare outgrowth of tdTomato negative LCLs in all donors as even a small level of contaminating memory cells could ultimately take over the culture during weeks of B cell outgrowth (Fig 3E, S4B and S4C Fig).As cord blood provides a purer naïve B cell population [42], we next assessed the ability of the LRM mutant to rescue LPKO infected cord blood B cells.Like adult naïve B cells, the cord blood B cells could support outgrowth of rescued, tdTomato-positive cells over time (Fig 4A and 4B) resulting in a higher total number of cells (S5A and S5B Fig) and establishment of tdTomato-positive LPKO LCLs when trans-complemented with wild type EBNA-LP but not with the LRM mutant (Fig 4C and S5B and S5D Fig).These findings confirm that the conserved leucine-rich motifs are important for EBNA-LP activity in naïve B cells.
Fig 6F) or naïve B cells (Fig 6G) significantly reduced EBV-induced proliferation compared to the CD46 knockout control.These data complement our biochemical data and mutagenesis supporting a role for EBNA-LP and YY1 in EBV transformation of naïve B cells.
cloned into pCEP4 vector by restriction digest cloning.FLAG-EBNA-LP constructs were then cloned into this vector using XhoI and BamHI restriction sites immediately following the P2A cleavage site (S1 Fig).Following P2A cleavage, EBNA-LP protein includes a proline (site of cleavage), FLAG epitope, linker including the attB1 recombination site from gateway cloning, and full-length EBNA-LP (S1 Fig).
Fig 1. Trans-complementation rescues LPKO virus infected naïve B cells. A. Schematic of experimental design.B. Outgrowth of tdTomato positive cells over time (n=3).WT infected cells are indicated with dashed lines, while LPKO infected cells are solid lines.C. Total tdTomato positive cells in each condition at 14 days post-infection (n=3).P values from unpaired t-test.Mean and standard deviation are plotted.D. Total tdTomato positive cells at the final time point for each donor, 28 or 35 days post-infection (n=3).E. Number of

Figure 2 .
Figure 2. Phosphorylation of EBNA-LP is not required for EBV-infected naïve B cell transformation.A. Known phosphorylated serines in EBNA-LP W domain across human EBV strains (Type 1 is B95-8, Type 2 is AG876) and EBNA-LP homologs in primate LCV strains.Identity bar indicates conservation of amino acids, with green indicating highly conserved regions, yellow indicating less conserved, and red are poorly conserved regions.Colors of residues indicate amino acid properties.Phosphorylated serines are indicated by black boxes.Previously defined conserved regions (CR1, CR2, CR3) are underlined (22).B. Amino acid sequence coverage of FLAGtagged EBNA-LP (wild type) construct encoding 4 W domains expressed in 293T cells, followed by mass spectrometry of FLAG immunoprecipitation.Underlined and highlighted residues indicate peptides identified by mass spectrometry, with the number of peptides indicated below.Phosphorylated residues are red, with residue number above.C. Abundance of phosphorylated peptides for all identified phospho-serines out of the total peptides spanning that residue from FLAG immunoprecipitation in B. D. Intensity of Tandem Mass Tag (TMT) signal for peptides with S63 phosphorylation when FLAG-tagged EBNA-LP (wild type), the EBNA-LP S3A mutant, and EBNA-LP S3E mutant are expressed in 293T cells prior to FLAG immunoprecipitation, TMT labeling, and mass spectrometry (n = 3).E. Total tdTomato positive cells at 14 days post infection for each donor in LPKO virus infected, trans-complemented cells (n = 3).Significance determined by unpaired t-test.Mean and standard deviation are plotted.F. Total tdTomato positive cells at 28 or 35 days weeks post infection for each donor (n=3).G.Total tdTomato+ LCLs generated per condition.*Indicates conditions in which tdTomato negative LCLs were generated.H. Trans-complemented LPKO LCLs express wild type and mutant FLAG-tagged EBNA-LP.W indicates number of W domains in EBNA-LP protein expressed from virus or expression construct.M indicates molecular weight marker.

Figure 3 .
Figure 3. EBNA-LP contains Leucine-rich motifs which are important for adult naïve B cell transformation.A. Alignment of EBNA-LP W and Y domains across human EBV (Type 1 is B95-8, Type 2 is AG876) and LCV strains, with identified leucine-rich motifs highlighted in black boxes.Identity bar indicates conservation of amino acids, with green indicating highly conserved regions, yellow indicating less conserved, and red are poorly conserved regions.Colors of residues indicate amino acid properties.Previously defined conserved regions (CR1, CR2, CR3, CR4, CR5) are underlined (22).B. LRM Mutant construct generated with mutations in the leucine-rich domains has modified leucines to alanines.C. Total tdTomato positive cells at 14 days post infection for each donor in LPKO virus infected, trans-complemented cells (n = 3).D. Total tdTomato positive cells in adult naive B cells from three donors at 28 or 35 days post infection (n = 3).P values from unpaired t-test.Mean and standard deviation are plotted.E. Total tdTomato positive LCLs generated from trans-complemented LPKO infected adult naïve B cells.*Indicates conditions in which some replicates generated tdTomato negative LCLs.

Figure 4 .
Figure 4. EBNA-LP Leucine-rich motifs are important in transformation of cord blood B cells. A. Total tdTomato positive cells at 21 days post infection for each cord blood donor in LPKO virus infected, transcomplemented cells (n = 3).P values from unpaired t-test.Mean and standard deviation are plotted.B. Total tdTomato positive cells from trans-complemented cord blood B cells at 28 or 35 days post infection weeks (n = 3).C. Number of tdTomato positive LCLs generated from trans-complemented LPKO infected cord blood B cells.

Figure 5 .
Figure 5. EBNA-LP Leucine-rich mutant is expressed and localizes similar to wild-type EBNA-LP in LCLs derived from adult total B cell infection with LPKO virus.A. LCLs from Adult Donor 4, total B cells infected with LPKO virus retain wild type and LRM mutant EBNA-LP expression.B. Immunofluorescence of total B cell derived LCLs, Adult Donor 5 WT LCL, and Adult Donor 4 LPKO derived LCLs trans-complemented with wild type EBNA-LP, LRM mutant EBNA-LP, or vector only.Green = Anti-EBNA-LP (JF186), Blue = DAPI.

Figure 6 .
Figure 6.EBNA-LP associates with YY1 cellular transcription factor through leucine-rich motifs.A. EBNA-LP localized sites on chromatin overlap with YY1 in LCLs by publicly available ChIP-Seq data.B. Location on chromatin of overlapping peaks between YY1 and EBNA-LP.C. Endogenous co-immunoprecipitation

Table 1 . Reagents and materials used in methods.
Purified adult naïve or cord blood B cells were resuspended in Belzer UW Cold Storage Solution (BridgetoLife) at 480,000 cells/7 L and combined with 1.2 g of DNA in 5 L Buffer T (Neon Transfection Kit) for excess volume to avoid air bubbles.As 10 L tips were used for transfect, approximately 400,000 cells total were transfected per replicate.DNA was prepared by maxi prep (ZymoPURE, D4203), eluting DNA in Buffer T (Neon Transfection Kit).Cells were transfected using the Thermo Fisher Neon Transfection System at 2150 V, 20 ms, 1 pulse, 10 L tips and resuspended in 200 L RPMI with 20% FBS.One hour after transfection, cells were infected with LPKO or WT virus at 0.2 Raji Green Units per cell for 1 hour at 37 C, a ratio we previously determined by EBNA2 immunofluorescence