Epstein-Barr-Virus-Driven Cardiolipin Synthesis Sustains Metabolic Remodeling During B-cell Lymphomagenesis

Epstein-Barr Virus (EBV) is associated with a range of B-cell malignancies, including Burkitt, Hodgkin, post-transplant, and AIDS-related lymphomas. Studies highlight EBV’s transformative capability to induce oncometabolism in B-cells to support energy, biosynthetic precursors, and redox equivalents necessary for transition from quiescent to proliferation. Mitochondrial dysfunction presents an intrinsic barrier to EBV B-cell immortalization. Yet, how EBV maintains B-cell mitochondrial function and metabolic fluxes remains unclear. Here we show that EBV boosts cardiolipin(CL) biosynthesis, essential for mitochondrial cristae biogenesis, via EBNA2-induced CL enzyme transactivation. Pharmaceutical and CRISPR genetic analyses underscore the essentiality of CL biosynthesis in EBV-transformed B-cells. Metabolomic and isotopic tracing highlight CL’s role in sustaining respiration, one-carbon metabolism, and aspartate synthesis, all vital for EBV-transformed B-cells. Targeting CL biosynthesis destabilizes mitochondrial one-carbon enzymes, causing synthetic lethality when coupled with a SHMT1/2 inhibitor. We demonstrate EBV-induced CL metabolism as a therapeutic target, offering new strategies against EBV-associated B-cell malignancies.


Introduction
Epstein-Barr Virus (EBV), infecting over 95% of the adult population worldwide, is a ubiquitous pathogen associated with a spectrum of diseases ranging from the benign infectious mononucleosis to more than 200,000 cases of cancer annually 1 .Among these, EBV-positive Burkitt lymphoma (BL) represents the most prevalent childhood cancer in Africa.Furthermore, EBV has been implicated in a variety of other malignancies, including Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), nasopharyngeal carcinoma (NPC), and gastric carcinoma (GC) [2][3][4][5][6][7] .EBV immortalizes B-cells in vitro.
The transformative ability plays a crucial role in the pathogenesis, especially in immunocompromised individuals such as organ transplant recipients and AIDS patients, where the incidence of EBV-associated lymphomas is notably high 8,9 .
EBV actively modulates host metabolic pathways to support the survival and proliferation of transformed cells.This modulation is orchestrated through a complex interplay of viral-encoded oncogenes, including six EBV nuclear antigens (EBNA1, EBNA2, EBNA 3A-C, and EBNA-LP) and two latent membrane proteins (LMP1 and LMP2), along with various viral non-coding RNAs (ncRNAs).These viral factors initiate a cascade of metabolic reprogramming early in the infection process, leading to signi cant reorganization in B-cell architecture that favors cell growth and division.The early expression of EBNA2 and its primary target, MYC, in particular, activates multiple metabolic pathways including oxidative phosphorylation (Ox-Phos), one-carbon (1C) metabolism and sterol biosynthesis, further compounded by the expression of LMP1 and LMP2 which mimic activated CD40 and B-cell receptor pathways, respectively 10,11 .The ability of EBV to reprogram host metabolism extends deeply into mitochondrial functions [12][13][14][15][16][17][18][19][20] , where it ensures the continuous generation of ATP and metabolic precursors critical for the synthesis of essential macromolecules and regulation of redox balance.
Any perturbations in mitochondrial pathways, including Ox-Phos, the tricarboxylic acid (TCA) cycle, and the mitochondrial 1C metabolism, is detrimental to the EBV transformation process 12,17,21 .Metabolic stress signi cantly hinders the transformation of B cells by EBV; newly infected cells that do not undergo successful transformation are characterized by mitochondrial dysfunction 17 .However, the mechanisms through which EBV sustains mitochondrial function and metabolic uxes during these early transformation stages remain elusive.
Cardiolipin (CL) is a four-acyl chain lipid that exists exclusively in the inner mitochondrial membrane (IMM), which plays an important role in the mitochondrial function and constitutes about 20% of the total mass of phospholipids of IMM.CL is essential for shaping the mitochondrial cristae and binding to electron transport chain (ETC) complexes in the IMM 22,23 .
Defects in CL metabolism led to compromised respiration and disruptions in the TCA cycle [22][23][24] .CL also modulates apoptosis by controlling cytochrome c release 25 .Here we show how early EBV infection in B-cells exploits CL biosynthesis pathways to establish specialized mitochondria that sustain extensive metabolic network remodeling.Our ndings highlight a key metabolic vulnerability in EBV's transformation process and provide a foundational basis for developing synthetic lethal strategies to treat EBV-induced B-cell lymphoproliferative disorders.

Results
EBV upregulates CL biosynthesis and remodels B-cell mitochondrial ultrastructure A central hub for metabolic regulation in cells is the mitochondrion.Yet, how EBV regulates mitochondrial remodeling is not fully understood.To address this, we rst investigated how EBV infection alters the mitochondrial ultrastructure to support B-cell transformation.To capture dynamic change of the mitochondrial ultrastructure upon EBV infection, a transmission electron microscopy (TEM) was used to analyze human B-cells, either uninfected or infected with EBV and collected at days 4, 7, and 28 post-infection (DPI).The infected cells appeared to be much larger in size with substantially increased cytosolic space (Fig. 1a).After EBV infection, there was a drastic increase in the number of mitochondria per cell, which was con rmed by a signi cant increase in the MitoTracker green signal and mtDNA abundance (Fig. 1a-b, Extended Data Fig. 1a).Analyzing a published RNAseq dataset of B-cells infected with EBV reveals a signi cant increase in the transcription of genes involved in the ETC 26 .2DPI cells exhibited the highest transcriptional changes in these ETC genes (Extended Data Fig. 1c).Gene ontology analysis of differentially expressed metabolic genes at 2DPI reveals terms including cellular respiration and respiratory chain complex assembly (Extended Data Fig. 1c).We therefore employed blue native polyacrylamide gel electrophoresis (BN-PAGE) to visualize mitochondrial respirasome assembly during EBV infection in B-cells.Compared to uninfected human resting B-cells, ETC supercomplexes in EBV-infected cells were readily detected from 20 µg of mitochondrial proteins starting from 2 DPI and onwards (Fig. 1c).From 10DPI, an additional increase of complex I and III 2 + IV + V supercomplexes was observed (Fig. 1c).Consistently, increases in mitochondrial membrane potential, basal and maximal oxygen consumption rates, as well as ATP production, were signi cantly elevated from 2 DPI (Fig. 1d-e, Extended Data Fig. 1d).These ndings suggest that EBV strongly regulates mitochondrial biogenesis, ETC supercomplex assembly, and respiration at the early stage of infection.
Along with the substantial increase in mitochondrial numbers following EBV infection, we observed a large boost in mitochondrial cristae biogenesis, as indicated by both the increased number per mitochondrion and extended length of cristae (Fig. 1a, 1f-g).Cristae are distinguished by their membrane invagination structures that project into the mitochondrial matrix.These IMM folds greatly expand the mitochondrial inner membrane's surface area 27 .CL plays a crucial role in shaping the curvature of cristae and exhibits a strong a nity for numerous mitochondrial supercomplexes, including the respirasome 24 .Our untargeted lipidomic analysis using Liquid chromatography-mass spectrometry (LC/MS) revealed a remarkable elevation in various CL species in EBV-infected B-cells at 7 or 10 DPI compared to uninfected resting B-cells (Fig. 1h).Based on these observations, we hypothesize that the upregulation of CL biosynthesis acts as a contributing factor in the mitochondrial remodeling induced by EBV, thereby facilitating key metabolic pathways driven by EBV.
The CL biosynthetic pathway involves a cascade of enzymatic reactions, including key enzymes such as protein tyrosine phosphatase mitochondrial 1 (PTPMT1) and cardiolipin synthase 1 (CRLS1) 22 .These enzymes work sequentially to convert phosphatidic acid into nascent CL (Fig. 1i).EBV robustly upregulated CRLS1 transcription and translation by 2 DPI, coinciding with the heightened expression of EBNA2 and MYC (Fig. 1j, Extended Data Fig. 1e).PTPMT1 protein was slightly increased at 2 DPI, and largely enhanced from 7 DPI, coinciding with the increase in LMP1 levels (Fig. 1j).By contrast, the PTPMT1 mRNA was modestly increased by 2 DPI and remained at a similar level as uninfected cells after 7DPI suggesting the post-transcriptional regulation of its expression, potentially regulated by LMP1 26 (Extended Data Fig. 1e).
Recognizing EBNA2 as a key viral regulator in B cell metabolism 12,28 , our study focused on its impact in initiating CL biosynthesis.We infected primary human B cells with either the B95.8 strain, or UV-inactivated B95.8 or the non-transforming P3HR-1 strain of EBV 29,30 (Fig. 1k).To maintain consistent infection levels across samples, we adjusted for input viral genome copy numbers, basing these adjustments on quantitative PCR analyses of the virus stocks (Extended Data Fig. 1f).Notably, the P3HR-1 strain lacks EBNA2 and most EBNA-LP open reading frames making it an ideal tool virus to study EBNA2 and/or EBNA-LP function 31 .The immunoblot revealed that only the B95.8 strain, but neither the P3HR-1 nor UV-inactivated B95.8, was capable of triggering the expression of CRLS1 and PTPMT1 by 2DPI.This observation was made while ensuring equal levels of infection, as evidenced by comparable post-infection intracellular viral loads (Extended Data Fig. 1g).These ndings indicate that EBV EBNA2 and/or EBNA-LP, rather than a broad response to EBV infection, are crucial for triggering cardiolipin biosynthesis.
In an effort to further understand the potential roles of EBNA2 in the activation of CRLS1, the rate limiting enzyme in CL biosynthesis, we utilized public available resources including ENCODE GM12878 LCL ChIP-seq dataset 32 , EBV EBNA2 ChIPseq dataset 33 and long-range chromatin interaction analysis 34 .We discovered that EBNA2, along with its host targets MYC, co-occupy the promoter of CRLS1 (Fig. 1l).The 2-2-3 LCL cell line expresses an engineered EBNA2, where EBNA2 is fused to a modi ed estrogen receptor ligand-binding domain.Therefore, EBNA2 transcriptional activity can be regulated by the presence of 4-hydroxytamoxifen (4HT).With 4HT, EBNA2-4HT is stabilized and translocated to the nucleus and regulates transcription 35 .When EBNA2 activity is conditionally shut down by withdrawing 4HT for a period of 48 hours, there is a noticeable reduction in the levels of CRLS1 and PTPMT1 (Fig. 1m).
It's well-established that EBNA2 and MYC collaboratively stimulate EBV target metabolic genes necessary for B-cell transformation 33,34 .ChIP-seq in LCLs showed that MYC and MAX, which form a heterodimer complex binding to E-box sites, co-occupied the LCL CRLS1 promoter (Fig. 1l).Next, we investigated MYC's role in EBV-induced CL metabolism.Using the P493-6 B cell line, an LCL with a 4HT-inducible EBNA2 cassette and a tetracycline-regulated MYC cassette, we found that merely activating MYC by withdrawing doxycycline could not induce CRLS1 expression, whereas activation of EBNA2 by adding 4HT was su cient to induce CRLS1 expression (Fig. 1m).This suggests that CRLS1 is an EBNA2-speci c target.
We also found EBNA2 and MYC in an upstream enhancer (about 110kbp away), marked by H3K27ac, H3K4me1, and EP300 co-ocupancy, linked to the CRLS1 promoter by a chromatin long range loop (Fig. 1l, rectangle).Using CRISPR interference (CRISPRi) on GM12878 LCL cells, we were able to disrupt the CRLS1 enhancer.GM12878 LCL expressing dCas9-repressor and CRLS1 enhancer targeted sgRNAs showed signi cantly reduced CRLS1 expression at both mRNA and protein level (Extended Data Fig. 1h-i).Overall, our data reveals an EBV-speci c transcriptional regulation mechanism for rate-limiting CL metabolic enzymes orchestrated by viral oncoprotein EBNA2.CL biosynthesis is critical for EBV B-cell transformation and LCL survival.
Collectively, these data suggest that the survival of EBV-transformed B-cells and BLs depends on a functional CL biosynthetic pathway.
Impaired CL biosynthesis disrupts mitochondrial respiration in EBV-transformed B-cells.
EBV-driven B-cell transformation relies on functional Ox-Phos 12,28 .Selectively inhibiting the electron transport chain halts EBV-driven B-cell transformation 12 .Given the essential role of CL in maintaining mitochondrial function, we rst investigated whether disrupting CL biosynthesis impacts mitochondrial respiration in cells newly infected with EBV.As expected, treatment with AD signi cantly reduced the B-cell mitochondrial membrane potential, as measured by TMRM, at 4 DPI (Fig. 3a, Extended Data Fig. 3a-b).Along with this, the oxygen consumption rates (OCRs) of both basal and maximal respiration, as well as ATP production, were signi cantly reduced (Fig. 3b-c).Notably, this inhibition of Ox-Phos is not attributable to decreased mitochondrial biogenesis; the level of MitoTracker Green was signi cantly increased after AD treatment (Fig. 3d, Extended Data Fig. 3c-d).
Given CL contributes to mitochondrial cristae, we hypothesize that AD treatment may disrupt cristae structure in EBV newly infected B-cells.We therefore performed TEM analysis on 4DPI cells that were treated with DMSO or AD for 24h (Fig. 3e, Extended Data Fig. 3e).In DMSO treated cells, the cristae are observed as shelf-like invaginations extending into the mitochondrial matrix (Fig. 3e, Extended Data Fig. 3e).They generally display a lamellar shape but sometimes appear to be short sphere which is probably due to ongoing cristae remodeling (asterisks).By contrast, in AD treated cells (Fig. 3e), the number of "empty" mitochondria lacking cristae was greatly increased (purple arrows).In some mitochondria, we observed a highly disorganized cristae structure which is usually located at one side of the mitochondria (orange arrows) and multiple long and distorted tube-like structures originate from it (orange arrows).Besides the defects in mitochondria, we also observed an increased number of intracellular vesicles, endoplasmic reticulum, and Golgi apparatus in AD treated cells (green arrows).
CL stabilizes the electron transport chain (ETC) supercomplexes located on the cristae 22,27,36,38 .We then investigated whether PTPMT1 de ciency destabilizes the respiration supercomplexes in EBV-transformed LCLs.We knocked out PTPMT1 in GM12878 cells and subsequently tested the abundance of ETC proteins from whole cell lysates using an Ox-Phos antibody cocktail.Overall, SDHB in complex II, UQCRC2 in complex III, COX II in complex IV, and ATP5A in complex V remained stable in PTPMT1 KO GM12878 LCLs.Interestingly, we observed a noticeable downregulation of NDUFB8 in complex I in PTPMT1 KO cells expressing two of the PTPMT1 sgRNAs(Fig.3f).We further investigated whether PTPMT1 de ciency might impact the assembly of ETC supercomplexes.Therefore, we isolated mitochondria from control and PTPMT1 KO GM12878 LCLs and performed BN-PAGE and then immunoblotting for ETC complexes.In this experiment, immunoblotting with the anti-Ox-Phos antibody cocktail revealed an additional unknown band with a size of around 550 kDa, as indicated by an arrow (Fig. 3g).Other than this, we did not observe any obvious differences between the control and PTPMT1 KO cells using anti-Ox-Phos antibody cocktail.However, when using individual antibodies targeting individual ETC complex, we found a striking reduction of SDHA in complex II, suggesting that PTPMT1 de ciency may disrupt SDHA assembly in complex II in EBV LCLs(Fig.3g).
Impaired CL biosynthesis leads to NADPH de ciency and oxidative stress in EBV-transformed B-cells.
To elucidate the metabolic impacts of AD treatment on newly EBV-infected B-cells, we further employed an LC/MS based untargeted metabolomic analysis.We used Piericidin A (PierA) as a comparative control, which is known for its selective inhibition of complex I, thereby hindering mitochondrial respiration (Fig. 4a).Freshly isolated human primary B cells were infected with EBV B95.8 at a multiplicity of infection (MOI) of 1.At 4DPI, the cells were treated with either DMSO, AD, or PierA for an additional 24 hours.Subsequent LC/MS-based metabolomic analyses of these samples unveiled signi cant variances in intracellular metabolites across cells treated with DMSO, AD, or PierA (Extended Data Figs. 4 and 5a).Relative to DMSO-treated cells, AD treatment led to an upregulation of metabolites within the B-cell malate aspartate shuttle, the metabolites associated with sterol synthesis, as well as folate, methionine, betaine, and nicotinamide metabolism (Fig. 4ce).PierA treatment, on the other hand, signi cantly downregulated metabolites in pyrimidine metabolism and the TCA cycle, while upregulating those in purine metabolism and contributing to the Warburg effect (Extended Data Fig. 5b-c).
Despite some overall correlation in the metabolic pro les between AD and PierA-treated cells, we identi ed notable exceptions: levels of citrate, succinate, itaconate, orotate, and glucosamine were increased in AD-treated cells but were signi cantly decreased in cells treated with PierA (Extended Data Fig. 5b, 5d).
Notably, AD treatment signi cantly reduced NADPH levels by 7-fold and increased NADP + by 1.5-fold, leading to a marked decrease in the NADPH/NAD + ratio (Fig. 4f).This change disrupts cellular redox balance and impairs biosynthetic pathways, including sterol synthesis and folate metabolism (Fig. 4c).We observed increased mitochondrial ROS in ADtreated B-cells, indicating elevated oxidative stress (Fig. 4g).Glutathione (GSH) supplementation reduced AD-induced cell mortality effectively, whereas N-acetyl-cysteine (NAC) was less effective (Fig. 4h).Unlike PierA treatment, AD did not affect NADH and NAD + levels, suggesting a targeted disruption of anabolic processes with little impact on catabolic processes like TCA cycle.This speci city indicates CL's role in modulating anabolic and oxidative stress responses.
The PPP is the major cytosolic pathway generating NADPH.We further tested if PPP plays a potential role in replenishing NADPH in AD-treated cells.We therefore knocked out Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the PPP.Interestingly, our immunoblotting revealed an unexpected negative correlation between G6PD and PTPMT1 in GM12878 LCL (Fig. 4i), suggesting potential feedback between CL biosynthesis and PPP.We found G6PD KO itself did not affect cell growth in GM12878 LCL.When combined with AD treatment, G6PD KO led to an evident synthetic lethality (Fig. 4j).Collectively, these results suggest that inhibiting CL decreases NADPH levels and leads to cell death dependent on oxidative stress, which can be mitigated by the exogenous supplementation of GSH.
Impaired CL biosynthesis disrupts mitochondrial 1C metabolism in EBV-transformed B-cells 1C metabolism catalyzes the transfer of 1C units across various metabolites.A signi cant part of this process occurs in mitochondria, where key enzymes like SHMT2 and MTHFD2 catalyze reactions using folate as a one-carbon carrier 12 .EBV B-cell transformation activates and heavily relies on this metabolic pathway, which primarily contributes to NADPH, as well as purine and thymidylate synthesis (Fig. 5a) 12,39 .We found AD treatment in newly infected B-cells leads to an accumulation of intracellular serine and folate, while downstream product 5-methyl-THF is signi cantly reduced (Fig. 5b, Extended Data Fig. 4) suggesting the disruption in 1C metabolism.Interestingly, in cells treated with AD, there was a signi cant reduction in the protein levels of SHMT2 and MTHFD2, key enzymes in the mitochondrial 1C pathway (Fig. 5c).
The conversion of 10-formyl-THF to THF and CO 2 by ALDH1L2 contributes to the pool of NADPH (Fig. 5a).Interestingly, ALDH1L2 protein was reduced in both AD-and PierA-treated cells (Fig. 5c).By contrast, the cytosolic SHMT1 and mitochondrial outer membrane protein TOMM20 were not affected by AD treatment (Fig. 5c).Quantitative RT-PCR results showed that AD treatment, in fact, slightly increased the transcription of MTHFD2, SHMT2, and ALDH1L2 (Fig. 5d).
Additionally, the destabilization of SHMT2 and ALDH1L2 proteins was observed in PTPMT1 knockout GM12878 LCLs, whereas MTHFD2 levels remained unchanged (Fig. 5e).These data suggest that CL inhibition may destabilize the mitochondrial 1C enzymes.Therefore, we posited that targeting CL biosynthesis could potentially synergize with pharmaceutical inhibition of 1C metabolism to achieve synthetic lethality.SHIN1, a highly selective small molecular inhibitor that simultaneously blocks SHMT1 and SHMT2 40 , has been shown to impair EBV B-cell transformation 12 .
Importantly, we found that SHIN1 exhibits a striking synthetic lethal effect when combined with AD treatment in GM12878 and GM15892 LCLs.Similar effects were observed in Mutu I and Daudi BLs (Fig. 5f).
Impaired CL biosynthesis leads to dysfunction of GOT2-driven aspartate biosynthesis in EBV-transformed B-cells.levels of aspartate and ornithine (Fig. 6a, Extended Data Fig. 4).Aspartate plays a critical role in the growth and proliferation of cancer cells, serving not only as an amino acid for protein synthesis but also as a key precursor for the de novo synthesis of purines and pyrimidines 41,42 .During the hyperproliferative phase of EBV B-cell transformation, typically occurring between 3 to 7 DPI, the infected B-cells divide approximately every 8 hours.This rapid division places a high demand on the synthesis of purines, pyrimidines, and proteins.Given that aspartate biosynthesis is closely regulated by the mitochondrial electron transport chain (ETC) 43,44 , this decrease in aspartate could be a consequence of AD's inhibition of oxidative phosphorylation (Ox-Phos), as shown in in Extended Data Fig. 5a-d..
While human plasma has low aspartate levels, certain cancer cells can uptake aspartate from their surroundings through the expression of aspartate membrane transporters 41,45 .SLC1A3, the primary transporter of aspartate 41 , exhibits minimal to negligible transcription levels during EBV transformation in primary human B cells, particularly when compared to more common amino acid transporters like SLC1A5 (Extended Data Fig. 6a) 26 .Notably, SLC1A3 expression was shut down once the newly infected B-cells fully transformed into LCL (Extended Data Fig. 6b, 28DPI).This implicates that de novo synthesis of aspartate might be indispensable in sustaining rapid cellular proliferation.
Aspartate biosynthesis involves the enzymes glutamate oxaloacetate transaminase 1 (GOT1) and 2 (GOT2), which function in the cytosol and mitochondria, respectively.EBV infection highly regulates B-cell GOT1 and GOT2 (Extended Data Fig. 6a-b) 12,26 .In cells competent in mitochondrial respiration, aspartate synthesis primarily occurs in the mitochondria, where GOT2 catalyzes its conversion from glutamate and oxaloacetate (Fig. 6b).We therefore investigated aspartate biosynthesis in EBV B-cell transformation.We infected primary B cells with EBV for 2 hours to ensure equal viral entry.Cells were then labeled with CFSE and treated with DMSO or Aminooxyacetic acid (AOA), a dual inhibitor of GOT1 and GOT2.EBV-driven cell proliferation was assessed by CFSE intensity at 5DPI using ow cytometry.Our ndings indicate that pharmaceutical inhibition of aspartate synthesis halted the B-cells proliferation induced by EBV (Fig. 6b).To further dissect the roles of GOT1 and GOT2 in EBV-transformed B-cells, we next employed CRISPR/Cas9 to knock out GOT1 or GOT2 individually in GM12878 LCL.Expressing two independent GOT1 sgRNAs consistently led to about a 30% reduction in GM12878 LCL viability.Interestingly, AD treatment synergized with GOT1 de ciency, which led to over 70% reduction in LCL viability within 48h post-treatment (Fig. 6c-d).By comparison, we found that GM12878 LCL survival is highly dependent on GOT2.Successful GOT2 KO by expressing GOT2 sgRNA #2 and #3 completely diminished GM12878 growth in comparison to cells expressing control sgRNA or GOT2 sgRNA #1, which failed to KO GOT2 (Fig. 6e-f).These data suggest that EBV transformation and LCL survival are highly dependent on functional mitochondrial aspartate biosynthesis.Importantly, we further found GOT2 but not GOT1 protein was markedly reduced in PTPMT1 KO GM12878 LCL (Fig. 6g).Our data suggest inhibiting CL biosynthesis may lead to GOT2 de ciency in EBV + LCLs.
We further tested whether adding exogenous aspartate could rescue AD-induced cell death in GM12878 LCLs.Since EBV + LCLs only express minimal level of SLC1A3, we rst created a stable GM12878 LCL expressing SLC1A3 (Fig. 6h).A control cell line expressing GFP was also established.Those cells were treated with DMSO or AD. 1 mM of aspartate was used to rescue the AD effects on cell growth defects.We found exogenous aspartate was barely able to rescue the cells in the GFP group by 48 hours post-AD treatment.However, this rescue effect was enhanced in the SLC1A3-expressing cells (Fig. 6i).
Similarly, exogenous aspartate was able to partially rescue the growth defects induced by PTPMT1 KO in LCLs (Fig. 6j).These data suggest that CL inhibition induced growth defects are associated with aspartate de ciency.
Two alternative cytosolic pathways related to pyruvate can compensate for aspartate biosynthesis when mitochondrial respiration is disrupted: (1) MDH1 converts malate to oxaloacetate using NAD + as an electron acceptor, with GOT1 catalyzing reverse transamination to produce aspartate.Pyruvate may enhance this pathway by converting NADH to NAD + via lactate dehydrogenase (LDH).(2) Pyruvate carboxylase (PC) can convert pyruvate to oxaloacetate.Increased LDHA and PC expressions have been noted in EBV transformation, as shown by RNAseq data (Extended Data Fig. 6) 26 .We therefore examined if exogenous pyruvate could restore AD-induced aspartate de ciency.Yet, adding exogenous pyruvate did not prevent cell death in GM12878 LCLs with AD (Fig. 6k), indicating neither PC nor MDH1 pathways could compensate for aspartate biosynthesis in EBV + LCLs under CL inhibition.
We further investigated the carbon source supporting aspartate biosynthesis during EBV B-cell transformation.Our metabolomic analysis of newly infected B-cells revealed that AD treatment generally increased TCA cycle metabolite levels, contrary to PierA treatment, which shut down the upper TCA cycle (Fig. 6l).Notably, a signi cant increase in glutamine and citrate was observed in AD-treated cells, suggesting the possibility that citrate might produce oxaloacetate via ATP-citrate lyase (ACLY) in the cytosol.Given that citrate is primarily generated through glutaminolysis and reductive carboxylation in cells with impaired respiration, glutamine utilization might also contribute to aspartate maintenance in AD-treated newly infected B-cells.To explore this hypothesis, we conducted an isotopic tracing study using glutamine labeled with uniformly distributed 13-carbon atoms (U-13C glutamine).We collected newly transformed B-cells at 4DPI and subsequently treated them with either DMSO or AD for 24 hours.Then, we introduced U-13C glutamine to these cells for an additional 8-hour period to facilitate 13C integration.In this experiment, the oxidation of glutamine results in the generation of M + 4 labeled compounds, whereas its reductive carboxylation leads to the formation of M + 3 labeled compounds (Fig. 6m).Notably, after 8 hours, about 82% of the malate and 69% of the aspartate derived from glutamine were labeled in DMSO-treated cells (Fig. 6m), suggesting that glutamine is the major carbon source supporting aspartate biosynthesis in newly infected B-cells.With AD treatment, there was not only an increase in malate abundance but also in the proportion of glutaminederived malate, reaching about 85% (Fig. 6n).Treatment with AD led to a 20% increase in the production of malate from glutamine oxidation (M + 4) and a concurrent decrease in M + 3 malate produced via glutamine's reductive carboxylation (Fig. 6n-o).Although there was a signi cant overall reduction in aspartate derived from glutamine, the proportion of M + 4 aspartate showed a modest increase (Fig. 6n-o).The overall M + 4 aspartate was much higher than M + 3 aspartate (Fig. 6o).Aligned with the isotopic tracing data, glutamine restriction sensitized GM12878 cells to AD treatment (Fig. 6p).
Collectively, these data indicate that glutamine serves as a primary carbon source for aspartate biosynthesis and CL inhibition destabilizes GOT2 which results in aspartate de ciency in EBV-transformed B-cells.

Discussion
Shortly after entry, EBV actively initiates aerobic glycolysis in B cells through the translational activation of all glycolytic enzymes and quickly downregulates TXNIP, a strong GLUT1 negative regulator.Glucose consumption and lactate release are evident at 2 DPI and peak at 4 DPI 12 .EBV early infection also boosts key fatty acid synthesis enzymes, ACACA and FASN, to convert acetyl-CoA derived from glycolysis into palmitate, facilitating lipogenesis 13 .Here, we show that EBV concurrently activates CL biosynthesis within 4 days post-infection.We speculate that the initial increase in ux of glycerol-3-phosphate from glycolysis and acyl groups from lipogenesis may boost CL biosynthesis resulting in mitochondrial remodeling.Previous research has shown signi cant growth defects in newly infected EBV cells when glucose in the culture medium is replaced with galactose.How this substitution affects dynamic mitochondrial CL biosynthesis remains to be further addressed.
MYC is a master regulator of metabolism 46 .EBNA2 and MYC jointly activate glycolysis, Ox-Phos, 1C metabolism, and lipogenesis during EBV B-cell transformation 28 .Despite ChIP-seq analysis in LCLs revealing MYC co-occupancy at the promoters of CRLS1, our data suggest that EBNA2 may independently initiate CRLS1 expression in LCLs.We speculate that EBNA2 plays a dominant role in CRLS1 expression, with this effect potentially being further augmented by MYC.Additionally, it's noteworthy that EBNA2-driven PTPMT1 activation begins at 2 DPI and increases signi cantly after 7 DPI in newly infected cells.This aligns with the onset of LMP1, which mimics CD40 and activates key signaling pathways including NF-κB, MAPK, and PI3K/AKT 28,47,48 .LMP1 notably upregulates GLUT1 via canonical NF-κB and PI3K/AKT pathways 49 and enhances glycolysis by inducing HIF1α through MAPK signaling 19 .We speculate that PTPMT1 could be a critical LMP1 target, whose activation could better utilize LMP1-induced glycolysis to facilitate mitochondrial remodeling and nalize the transformation.
CL contributes to the structural integrity and functionality of mitochondrial cristae.While there is ongoing debate regarding CL's cone-shaped structure's direct contribution to cristae curvature, it interacts with numerous mitochondrial protein complexes to support cristae formation.The Mitochondrial Contact Site and Cristae Organizing System (MICOS) play crucial roles in shaping and maintaining cristae structure 50 .Notably, EBV transcriptionally upregulates MICOS genes, including MIC60, during B-cell transformation 26 .The loss of CL is associated with MICOS disruption, leading to cristae structure loss and mitochondrial dysfunction, as observed in broblasts from Barth Syndrome patients 50 .How EBV-driven CL biosynthesis coordinates with MICOS to support cristae biogenesis remains to be investigated.Furthermore, the dimerization of F1Fo-ATP synthase and the integration of complex II into the respirasome are crucial for cristae curvature formation.CL is known to bind to and stabilize all ETC complexes and ATP synthase (Complex V), modulating their stability and function [51][52][53][54][55] .Our ndings indicate that PTPMT1 de ciency results in a highly disordered tubular structure in the mitochondria of newly infected B-cells.We are tempted to speculate that disruptions in the assembly and integration of protein complexes into the cristae may contribute to the observed disorder.Supporting this hypothesis, our BN-PAGE data revealed a de ciency in Complex II, where the assembly of the SDHA subunit was disrupted due to PTPMT1 de ciency in EBV-transformed B-cells.
We discovered that blocking CL biosynthesis leads to the destabilization of mitochondrial 1C enzymes targeted by EBV, resulting in a signi cant reduction of NADPH production.This nding reveals a previously unrecognized role of CL in maintaining mitochondrial 1C metabolism.As mentioned above, CL's ability to bind to and interact with a wide variety of mitochondrial protein complexes was well documented.The interaction between CL and proteins involves strong hydrophilic interactions facilitating the binding between its negative charged glycerol head group and various amino acid residues of the protein 23 .We speculate that during EBV B-cell transformation, CL in the IMM probably binds to mitochondrial 1C enzymes, which may promote 1C enzymes to form metabolons to meet the increased demand for nucleotides and NADPH.Therefore, further biochemical and functional studies are essential to thoroughly understand the interactions between CL and mitochondrial 1C enzymes and to elucidate how these interactions contribute to EBV-induced lymphomagenesis.
Our research has, for the rst time, demonstrated that aspartate, crucial for protein and nucleotide synthesis during EBVdriven cell proliferation, is predominantly synthesized in mitochondria from glutamate and oxaloacetate by GOT2.This pathway represents a signi cant metabolic vulnerability in EBV-transformed B-cells.Notably, besides maintaining respiration, CL plays a vital role in stabilizing GOT2 throughout the transformation process, thereby ensuring sustainable aspartate synthesis.Interestingly, while some of the most aggressive cancers, characterized by compromised respiration, depend on GOT1-driven pathways to compensate for aspartate synthesis, our ndings from pyruvate rescue experiments and isotopic tracing suggest that EBV-transformed B-cells lack the ability to utilize these major alternative pathways.This implies that directly targeting GOT2 could be considered as an effective strategy for treating lymphomas associated with EBV B-cell transformation.Furthermore, our data reveals that in the initial stages of B-cell transformation, glutamine oxidation via the TCA cycle, is the primary source of aspartate synthesis.The combination of glutamine restriction and AD treatment displayed synthetic lethality, underscoring a potential therapeutic vulnerability.These insights suggest that cotargeting CL biosynthesis and glutaminolysis may offer innovative approaches to induce aspartate depletion, presenting a promising avenue for the treatment of EBV-associated lymphomas.AD, a bis-biguanide compound commonly utilized as an oral antibiotic and for preventing gingivitis 56 , has been found to be particularly effective against the EBV + nasopharyngeal carcinoma cell line C666-1, but not in non-transformed cell types such as GM05757, HNEpC, or NIH/3T3 57 .Our ndings underscore that AD signi cantly suppresses EBV B-cell transformation.Importantly, it exhibits a pronounced synthetic lethal effect when used in conjunction with SHIN1, a SHMT inhibitor in EBV-transformed B-cells and related B-cell lymphomas.Given the rapid clearance of SHIN1 in vivo, it would be of interest to test SHMT antagonist SHIN2, a second generation SHIN1 derivative with improved in vivo pharmacokinetic properties in EBV lymphoma mouse xenografts models 14,58 .
In conclusion, our ndings reveal a key role of CL biosynthesis in generating EBV-specialized mitochondria to meet the energetic, biosynthetic, and redox demands during B-cell lymphomagenesis.Targeting CL biosynthesis and employing CLrelated synergistic combinations could serve as a promising new therapeutic approach for treating EBV-associated cancers.
Declarations conditional P493-6 LCLs, a gift from Dr. Ben Gewurz, contain a conditional EBNA2-HT allele and an exogenous Tet-OFF MYC allele.Cells were washed three times with PBS, then seeded at 0.3 million/mL in RPMI-1640 media with 10% doxycycline-free FBS, and treated under various conditions for 48h as speci ed.To culture P493-6 cells at a low MYC state, they were grown without 4-hydroxytamoxifen (4HT, SML1666, Sigma-Aldrich) and with 1 mM doxycycline (HY-N0565, MedChemExpress).For a high MYC BL-like state, both doxycycline and 4HT were removed.For a high EBNA2 LCL state, cells were treated with 1 µg/mL doxycycline and 1 µM 4HT.For a high MYC and EBNA2 state, cells received 1 µM 4HT but no doxycycline.HEK-293 T cell line was obtained from ATCC.All the BL, DLBCL, LCL, and PEL cell lines, as well as primary B cells, were cultured in RPMI 1640 medium (Gibco, Life Technologies) with 10% fetal bovine serum (FBS, Gibco) or with dialyzed FBS where indicated (Gibco).HEK-293 T cells were cultured in Dulbecco's Modi ed Eagle's Medium (DMEM, Gibco) with 10% FBS.To obtain the stable Streptococcus pyogenes Cas9 expression, cell lines were treated with lentiviral transduction and blasticidin (5 µg/mL, ant-bi-1, InvivoGen) selection.To select the transduced cells, puromycin (3 µg/mL, A11138-03, Gibco) or hygromycin (100 µg/mL, 10687010, ThermoFisher) were added to the post-infection cells.All Cells were cultured at 37° in 5% CO2 incubator.

Human Primary B cells isolation
Discarded, de-identi ed leukocyte fractions left over from platelet donations were obtained from the Brigham and Women's Hospital Blood Bank.Blood cells were collected from platelet donors following institutional guidelines.Since these were deidenti ed samples, the gender was unknown.Our studies on primary human blood cells were approved by the Tufts University Institutional Review Board (Tufts IRB: STUDY00004385).Primary human B cells were isolated by negative selection using RosetteSep Human B Cell Enrichment and EasySep Human B cell enrichment kits (Stem Cell Technologies), according to the manufacturers' protocols.B cell purity was con rmed by plasma membrane CD19 positivity through FACS.Cells were then cultured with RPMI 1640 with 10% FBS.

EBV production and concentration
The EBV B95-8 strain was generated from B95-8 cells engineered for inducible ZTA expression (a gift from Dr. Ben Gewurz).The activation of EBV lytic cycle was achieved by treating the cells with 1 µM of 4HT for 24 hours.Subsequently, the 4HT was removed, and the cells were cultured in RPMI medium supplemented with 10% FBS, devoid of 4HT, for an additional 96 hours.The viral supernatants obtained were then cleared of producer cells by passing through a 0.45 µm lter.The viral titer was assessed using a transformation assay.Similarly, the P3HR-1 strain of EBV was obtained from a P3HR-1 cell line that expresses 4HT-inducible ZTA-HT and RTA-HT alleles, generously provided by Dr. Ben Gewurz.The induction process involved treating P3HR1 ZHT/RHT cells with 1 µM of 4HT for a 24-hour period.Afterwards, the culture medium was replaced with fresh RPMI/FBS medium, and the cultures were allowed to incubate for 96 hours to collect the virus-rich supernatants.These supernatants were then ltered using a 0.45 µM lter for puri cation.The supernatant was transferred to an ultracentrifuge tube (326823, Beckman Coulter) and centrifuged at 25,000 rpm for 2 h at 4°C in an ultracentrifuge (OPTIMA XPN-100, Beckman Coulter).The viral pellet was resuspended and aliquoted in PBS with 2% dialyzed FBS, stored at − 80°C until infection.The genomic DNA of virus was quanti ed by PCR targeting the BALF5 gene from the extracted viral genome.This quanti cation was used to standardize the virus amounts for cell infection experiments.

Transmission electron microscopy (TEM)
A mixture of 2.5% paraformaldehyde, 5% glutaraldehyde, and 0.06% picric acid in 0.2 mol/L Cacodylate buffer was freshly prepared before use, then the above mixture was diluted 1:1 with dH2O.

Mitochondria isolation
The mitochondria were isolated following the manufacturer's protocols of Mitochondria Isolation Kit for Cultured Cells (89874, Thermo Fisher Scienti c).Speci cally, 20 million cells were harvested into a 2.0 mL microcentrifuge tube, centrifuged at 850 g for 2 minutes, the supernatant was removed and then the cell pellet was added with 800 µL of Mitochondria Isolation Reagent A containing EDTA-free protease inhibitor (cOmplete™, Millipore Sigma).Gently vortex and incubate on ice for exactly 2 minutes.10 µL of Mitochondria Isolation Reagent B was then added to the mixture and incubated on ice, fully vortexing the mixture at every minute.After 5 minutes, 800 µL of Mitochondria Isolation Reagent C containing EDTA-free protease inhibitor was included, gently inverted tube several times before centrifuging at 700 g for 10 minutes at 4°C.Transfer the supernatant to a new, 2.0 mL tube and centrifuge at 12,000 g for 15 minutes at 4°C.The obtained mitochondrial pellet was resuspended in 100 µL of Mitochondria Isolation Reagent C containing EDTA-free protease inhibitor.The protein content of mitochondria was further measured by Qubit 4 Fluorometer (Q33226, Thermo Fisher Scienti c) with Qubit Protein Assay kit (Q33212, Thermo Fisher Scienti c).
Sample preparation.The BN gel electrophoresis and Immunoblot was conducted as previously described 59 .The NativePAGE sample prep kit (BN2008, Thermo Fisher Scienti c) was used to make the mitochondrial Sample buffer cocktail.50 µg mitochondrial protein was mixed with 5 µL 4× NativePAGE sample buffer, 8 µL 5% digitonin, and 7 µL water in the kit.Then we incubated the solubilized mitochondria on ice for 20 min.After that, solubilized mitochondria were then centrifuged at 20,000 g for 10 min at 4°C. 15 µL supernatant was transferred into a new tube and fully mixed with 2 µL Coomassie G-250 sample additive in kit.
Electrophoresis.Each well (NativePAGE 3-12% gradient gel, BN2011, BX10, Thermo Fisher Scienti c) was gently washed with 1 mL of dark blue 1X cathode buffer (BN2002, Thermo Fisher Scienti c).Subsequently, 15 µL of mitochondrial sample was loaded into the gel.The inner chamber was lled with 1X dark blue cathode buffer, and 600 mL of 1X running buffer (BN2002, Thermo Fisher Scienti c) was added to the outer chamber.Electrophoresis took place in an XCell SureLock Electrophoresis-Cell (EI0001, Novex) at a constant voltage of 150 V for 30 minutes, with the current limited to 15 mA.Following this, the buffer in the inner chamber was replaced with light blue buffer (created by mixing 20 mL of dark blue 1X cathode buffer with 180 mL of distilled water).Electrophoresis continued at 250 V for 60 minutes.
Immunoblot.The BN-PAGE gel was gently washed with water for 5 minutes to remove the cathode buffer.It was then placed in bicarbonate transfer buffer (10 mM NaHCO 3 , 3 mM Na 2 CO 3 ) for a 15-minute incubation.The PVDF membrane was activated by immersion in 100% methanol for 2 seconds and then washed with water for 5 minutes, followed by a 5minute incubation in bicarbonate transfer buffer.The transfer was carried out at a constant current of 300 mA for 1 hour in the cold room.Following the transfer, the membrane was rinsed with PBS and then xed with 8% acetic acid for 5 minutes.
The membrane was subsequently washed with water three times, each for 5 minutes.To remove the Coomassie blue, the membrane underwent shaking with methanol three times, each for 5 minutes.This was followed by a water wash, also three times for 5 minutes each.The membrane was incubated with 5% milk in PBS-Tween 20 (PBST) for blocking for 1 hour.The membrane was washed with PBST three times, each for 5 minutes, and then incubated with primary antibodies for the electron transport chain Complexes I, II, III, IV, and Ox-Phos overnight at 4°C.The next day, the membrane was washed with PBS three times, each for 5 minutes, followed by a 1-hour incubation with the secondary antibody and then washed three times with PBST for 5 minutes each.Chemiluminescent detection was performed on the membranes by LI-COR XF system.

Lipidomic pro ling analysis
The intracellular lipidomic pro ling was performed as described 60 .Newly isolated human B-cells were mock infected or infected with EBV at a MOI of 1 for 5 days.B-Cells were counted and pelleted at 1,200 rpm for 5 minutes at 4°C with an equal number of cells in each sample.Lipidomic pro ling was performed as described previously 60,61 .They were then resuspended in 200 µL of HPLC-grade water (270733, Sigma-Aldrich) and mixed vigorously with 2.5 mL of HPLC-grade methanol (A456, Fisher Scienti c) in glass tubes.Following this, 5 mL of methyl tert-butyl ether (MTBE, 1634-04-4, Supelco) was added, and the samples were agitated for 1 hour at room temperature.To separate phases, 1.5 mL of water was added, and after vigorous vortexing, the samples were centrifuged at 1000 x g for 10 minutes at room temperature.
The upper phase was then dried a speed vacuum concentrator (Savant SPD 1010, Thermo sher Scienti c) for 4h at RT and stored at − 80°C.
For analysis, samples were reconstituted in 35 µL of a 1:1 mixture of LCMS-grade isopropanol and methanol, and subjected to liquid chromatography-mass spectrometry (LC-MS) as previously outlined, employing a high-resolution hybrid QExactive HF Orbitrap mass spectrometer (Thermo Fisher Scienti c) set to data-dependent acquisition mode (Top 8) with the capability of switching between positive and negative ion polarities.Lipid species identi cation and quanti cation were performed using the LipidSearch 4.1.30software (Thermo Fisher Scienti c), leveraging an internal database comprising ≥ 20 major lipid classes and ≥ 80 subclasses.For verifying signal linearity, a pooled sample was created by combining 5 µL from each sample, which was then diluted with a 1:1 mixture of isopropanol and methanol to generate dilutions of 0.3x and 0.1x, alongside a blank.These dilutions underwent analysis, and for each lipid species within this series, the Pearson correlation coe cient between ion count and sample concentration was computed.Only lipids exhibiting a correlation coe cient (r) greater than 0.9 were retained for nal analysis.The abundance of individual lipid species was normalized against the total ion count of the sample.Using R, lipids were categorized by class, and the total ion intensity for each lipid class in each sample was calculated.

Intracellular metabolite pro ling
The intracellular metabolites pro ling was performed as described 62 .Newly infected B-cells collected at 4DPI were washed 3 times with PBS and counted.6 million cells were seeded into a T25 ask with 20 mL RPMI-1640 with 10% dialyzed FBS.
Cells were incubated with DMSO, 2uM AD, or 100nM ofPiericidin A (MedChemExpress, 2738-64-9) for 24h.The cells were counted and washed 3 times with pre-chilled PBS.The cell pellet was fully resuspended with 100uL PBS by vortex, the metabolism was quenched by adding 3.3 mL of dry ice-cold 80% aqueous methanol (A456, Fisher Scienti c), and kept at -80°C overnight.The lysate was centrifuged at 21,000 g for 15 min at 4°C.The supernatants were obtained and dried by a speed vacuum concentrator (Savant SPD 1010, Thermo sher Scienti c) for 4h at RT.Samples were re-suspended using 20 uL HPLC grade water for mass spectrometry.5-7 µL were injected and analyzed using a hybrid 6500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu) via selected reaction monitoring (SRM) of a total of 300 endogenous water soluble metabolites for steady-state analyses of samples and 150 endogenous metabolites for 13C/15N isotopomer ux tracing.Some metabolites were targeted in both positive and negative ion mode for a total of 311 SRM transitions using positive/negative ion polarity switching.ESI voltage was + 4950V in positive ion mode and − 4500V in negative ion mode.The dwell time was 3 ms per SRM transition and the total cycle time was 1.55 seconds.Approximately 9-12 data points were acquired per detected metabolite.Samples were delivered to the mass spectrometer via hydrophilic interaction chromatography (HILIC) using a 4.6 mm i.d x 10 cm Amide XBridge column (Waters) at 400 µL/min.Gradients were run starting from 85% buffer B (HPLC grade acetonitrile) to 42% B from 0-5 minutes; 42% B to 0% B from 5-16minutes; 0% B was held from 16-24 minutes; 0% B to 85% B from 24-25 minutes; 85% B was held for 7 minutes to re-equilibrate the column.Buffer A was comprised of 20 mM ammonium hydroxide/20 mM ammonium acetate (pH = 9.0) in 95:5 water:acetonitrile.Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v3.0.2 software (AB/SCIEX).Metabolites with p-values < 0.05, log2(fold change) > 1 or <-1 were used for pathway analysis using MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/MetaboAnalyst/ModuleView.xhtml).

U-13C-Glutamine tracing
EBV infected primary B-cells were collected at 4DPI, treated with DMSO or 2µM AD for 16h, then U-13C glutamine was applied to the cells.Ten million cells were cultured in glutamine-free media containing 10% dialyzed FBS-and 2 mM 13 C5-L-Glutamine (184161-19-1, Cambridge Isotope Laboratories) for 8 h.Cell samples were collected and processed as mentioned in intracellular metabolite pro ling.Metabolic ux analysis was performed as described 63 .

Seahorse mitochondrial stress test
The Agilent Seahorse XF Assay was conducted as described preiously 12 .cally, the sensor cartridge was rst hydrated with water overnight and incubated with XF Calibrant for 1h.Add  Flow cytometry analysis mitochondrial mass was determined by the MitoTracker Green FM (M7514, Thermo Fisher Scienti c) and the mitochondrial membrane potential was determined by the uorescence intensity of TMRM (Tetramethyl-rhodamine methyl ester perchlorate, T668, ThermoFisher Scienti c) following the manual.1×10 6 of Cells were collected and resuspended in 500 µL cell culture media with 1.5 µL 100 µM of MitoTracker Green or TMRM.Cells were then incubated in 37°C incubator for 30 min.Then cells were washed once with 1×PBS and resuspended in PBS buffer with 2% FBS for FACS.For cell viability analysis, cells were washed and resuspended with PBS buffer with 2% FBS.Then cells were incubated with 1 µM of 17-Aminoactinomycin D (7AAD, A1310, Invitrogen) for 5 min before analyzing.For CFSE (C345544, Invitrogen) cell proliferation staining, 10 millions of primary B cells were resuspended in PBS with 0.1% BSA, then the cells were mixed with the same volume of 1µM CFSE for 10 min at 37°C.Cells were then neutralized by prechilled 10% FBS RMPI-1640 for 5 min.
After washing the cell with culture media, cells were resuspended and infected with virus.1h after infection, cells were treated with 100 µM Aminooxyacetic acid (AOA), CFSE were analyzed at 5DPI.For cell cycle analysis, cells were xed with ice-cold 70% ethanol for at least 24h.At the day for analysis, cells were centrifuged at 3000 rpm for 10 min to remove ethanol, and resuspended in PBS buffer.Cells were incubated in 1 mL of staining buffer (propidium iodide, 5 µg/ml; RNase A, 40 µg/ml; 0.1% Triton X-100 in PBS) at room temperature for 30 min in dark.For MitoSOX analysis, 1 million cells were collected and washed with HBSS (Hank's Balanced Salt Solution, Gibco) buffer, then incubated with 1uM MitoSOX™ Green (M36006, Thermo Fisher Scienti c) for 30 min.cells gently washed with warm HBSS buffer.Flow cytometry was performed on a BD FACS Calibur instrument.Data were analyzed with FlowJo V10.

Quantitative real-time PCR (qRT PCR)
Extraction of total RNA from cells was conducted by the RNeasy Mini Kit (Qiagen).RNase-Free DNase Set (Qiagen) were used to remove genomic DNA and SYBR Green RNA-to-CT 1-Step Kit (Applied Biosystems) were applied to assemble the qRT PCR reaction.Primer sequences are listed in the Supplementary Table .Samples were run in technical triplicates.The

Supplementary Files
dCas9 fused with KRAB.Single guide RNAs against targeting upstream CRLS1 enhancers were designed using GPP sgRNA Designer at the Broad Institute.sgRNA oligos were obtained from Integrated DNA Technologies and cloned into the pLentiGuide-Puro vector (Addgene plasmid #52963, a gift from Feng Zhang).Lentiviruses were produced in HEK-293 cells by co-transfection of pLentiGuid-puro with psPAX2 and VSV-G packaging.At 24 hours post transfection, cell culture media was changed to RPMI-1640 + 10% FBS.Two rounds of lentiviral transduction were performed at 48 and 72 hours post plasmids transfection.Cells were selected by puromycin (3 µg/ml), added 48 hours post-transduction.Depletion of target gene encoded protein expression was con rmed by immunoblot.cDNArescueThe PTPMT1 cDNA with silent PAM site mutations was purchased from IDT and was inserted into pLX-TRC313 (a gift from John Doench) by Hi Assembly (New England Bioloabs).GM12878 -Cas9 with stable C-terminal V5 epitope-tagged PTPMT1 cDNA expression was established by lentiviral transduction and hygromycin selection as described above.Ten days post hygromycin selection, PTPMT1-V5 expression was con rmed by immunoblot.The sequence of PTPMT1 rescue cDNA is listed below.sg PTPMT1 targeting sequences are highlighted in underlined bold.PAM sequences are underlined.Mutation sites are indicated in italics bold.Overlapping sequences for Hi Assembly reaction are highlighted in underlined.

Figures
Figures

Figure 6 Inhibiting
Figure 6 To Fix primary B cells, 1 million cells were collected and washed with Dulbecco's Phosphate Buffered Saline (DPBS, 14190, Gibco) one time, remove the residue buffer, then gently add the diluted xative to the primary B cells.The cells were xed at RT for 1h.The cells were then post-xed for 30 min in 1% Osmium tetroxide (OsO4)/1.5% potassium ferrocyanide (KFeCN6), washed in water 3x and incubated in 1% aqueous uranyl acetate for 30 minutes.Samples were then washed twice in water and dehydrated in grades of alcohol (5min each; 50%, 70%, 95%, 2x 100%).Cells were removed from the dish in propyleneoxide, pelleted at 3000 rpm for 3 minutes and in ltrated for 2 hours in a 1:1 mixture of propyleneoxide and TAAB Epon (Marivac Canada Inc.St. Laurent, Canada).Samples were subsequently embedded in TAAB Epon and polymerized at 60 degrees C for 48 hrs.Ultrathin sections (about 60nm) were cut on a Reichert Ultracut-S microtome, picked up on to copper grids stained with lead citrate and examined in a JEOL 1200EX transmission electron microscope or a TecnaiG² Spirit BioTWIN.Images were recorded with an AMT 2k CCD camera.
CRISPR/Cas9 editing CRISPR/Cas9 knock-out was performed in cells with stably Cas9 expression, using Broad Institute Brunello or Avana library sgRNA sequences as listed in TableS1.CRISPR/dCas9 interference (CRISPRi) was performed in GM12878 LCL expressing 12 µL Cell-Tak solution (1.3 mL of 0.1M sodium bicarbonate, 11.2 µL of 0.1M NaOH, 22.4 µL of Cell-Tak solution) to each well of the V7-PS 96-well cell culture plate.The Cell-Tak solution was washed with sterile water twice and 0.25 million primary B cells (resuspension in 180 µL of RPMI-1640 with 10% FBS and 5 mM pyruvate) were seed on a Seahorse plate.Then the cells were placed in a non-CO2 37°C for 30 minutes.The Oxygen consumption rates (OCR) were simultaneously recorded by a Seahorse XFe96 Analyzer (Agilent).The cells were sequentially probed by 20 µL of 3.5 µM oligomycin, 20 µL of 2 µM CCCP, and 20 µL of 100 nM piericidin A. For Seahorse in GM12878 cells, cells were seeded as 0.1 million/well in a 96-well cell culture plate.Data were analyzed by Seahorse Wave Desktop Software (Agilent).