Epstein–Barr virus enhances genome maintenance of Kaposi sarcoma-associated herpesvirus

Rachele Bigia, Justin T. Landisa, Hyowon Ana, Carolina Caro-Vegasa, Nancy Raab-Trauba, and Dirk P. Dittmera,1

aDepartment of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill, NC 27599

Edited by Thomas E. Shenk, Princeton University, Princeton, NJ, and approved October 18, 2018 (received for review June 13, 2018) Primary effusion lymphoma (PEL) is a B cell lymphoma that is Like all herpesviruses, KSHV has two typical life cycles, lytic always associated with Kaposi’s sarcoma-associated herpesvirus and latent (reviewed in ref. 9). During latent infection, the viral (KSHV) and in many cases also with Epstein–Barr virus (EBV); how- genome replicates as a in synchrony with the host cell ever, the requirement for EBV coinfection is not clear. Here, we cycle, and the viral DNA is replicated by the host cell DNA + demonstrate that adding exogenous EBV to KSHV single-positive polymerase (10). It is partitioned in equal copy numbers into the PEL leads to increased KSHV genome maintenance and KSHV daughter cells (11), and viral copy number per cell is maintained latency-associated nuclear antigen (LANA) expression. To show at a constant level (12). Thus far no PELs have been reported in that EBV was necessary for naturally coinfected PEL, we nucleo- + + which KSHV was integrated into the host . Neither fected KSHV /EBV PEL cell lines with an EBV-specific CRISPR/ the KSHV nor the EBV genome in PEL is defective, as both Cas9 plasmid to delete EBV and observed a dramatic decrease in viruses can be induced to replicate their genome and release cell viability, KSHV genome copy number, and LANA expression. – infectious virus. In the latent state, the viral expression is This phenotype was reversed by expressing Epstein Barr nuclear limited to a few , most notably the latency-associated nu- antigen 1 (EBNA-1) in trans, even though EBNA-1 and LANA do not clear antigen (LANA) (13–16) in the case of KSHV and the colocalize in infected cells. This work reveals that EBV EBNA- Epstein–Barr nuclear antigen 1 (EBNA-1) in the case of EBV. 1 plays an essential role in the pathogenesis of PEL by increasing – KSHV viral load and LANA expression. LANA is dispensable for lytic replication (17 19). The two es- sential functions of either protein are to support latent genome

EBV | Kaposi sarcoma | KSHV | LANA | primary effusion lymphoma replication and to partition the viral plasmid during mitosis to MICROBIOLOGY maintain the same genome copy number per cell (20–26). PELs are dependent on KSHV for survival (27, 28). About half of the ne almost never finds two different viruses in the same cell. culture-adopted PEL cell lines carry the EBV plasmid. In EBV- The odds of particles of two different viruses simultaneously O coinfected PELs, EBNA-1, the EBV miRNAs, and the EBV- hitting the same cell are astronomically small. Clinically, most encoded small RNAs are expressed, as well as low levels of infectious diseases are monocausal with regard to the infectious + LMP2A (29, 30). EBV PELs have a distinct cellular transcrip- agent; patients experience one exposure event and one acute − viral infection episode at a time. (“Acute infection” here is de- tion and protein expression profile compared with EBV PELs fined as a sufficiently high systemic viral load to hit the majority (31, 32), prompting the hypothesis that EBV provides an ad- of receptor-bearing cells under a Poisson-distributed random vantage in either cell survival or KSHV persistence in dually model.) Theoretically, only two scenarios are able to escape the infected cells. In the strongest sense this hypothesis posits that probability distribution of double-hit kinetics: (i) if the first virus dually infected PELs are dependent as much on EBV as on is absolutely dependent on the second virus, as Adeno-associated KSHV. Supporting higher KSHV plasmid numbers in infected B virus is dependent on Adenovirus, and (ii) if the first virus pre- cells would provide an advantage during asymptomatic latency exists in the majority of the target cells for the second virus, such in vivo before PEL development and may aid KSHV transmis- as during latent persistence of HIV or of two gamma-herpesviruses, sion in EBV/KSHV coendemic areas. such as Epstein–Barr Virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV). EBV/KSHV latent coinfection of primary Significance effusion lymphoma (PEL) provides a unique opportunity to study pathogen interdependence at a molecular level and in a clinically Primary effusion lymphoma (PEL) is a B cell lymphoma; the relevant disease. prognosis is poor, with a median survival around 6 months. PEL PELs represent a natural experiment in which in the majority is always associated with the presence of Kaposi’s sarcoma- of tumor cells are stably coinfected with two viruses, EBV and associated herpesvirus and in most cases is coinfected with KSHV (reviewed in ref. 1). PELs are derived from postgerminal Epstein–Barr virus (EBV); however, the role of EBV in the center B cells. PEL does not typically form a solid mass; it arises pathogenesis of the tumor is still not clear. This study dem- in serous cavities, such as the pleural and peritoneal cavities, onstrates the intricate interaction between the two herpesvi- although exceptions have been noted. PEL is a rare neoplasm, ruses, which exacerbate tumorigenesis by mutually reinforcing which occurs almost exclusively in HIV patients. Very few cases the persistence of each latent genome and by altering cell of HIV-negative PEL have been reported; one led to the es- proliferation. It establishes curing EBV and inhibiting Epstein– tablishment of the BC3 cell line (2). It is likely that these patients Barr nuclear antigen 1 as a potential treatment for PEL. nevertheless experienced immunodeficiency of some occult na- ture. The median survival for PEL patients is less than 1 y. Even Author contributions: N.R.-T. and D.P.D. designed research; R.B. and C.C.-V. performed though PELs initially respond to DNA-damaging chemotherapy, research; R.B., J.T.L., H.A., and D.P.D. analyzed data; and R.B. and D.P.D. wrote the paper. the cells rapidly develop drug resistance, which is associated with The authors declare no conflict of interest. mutations in (3). PEL can be adapted to continuous growth This article is a PNAS Direct Submission. in culture, and all PEL cell lines cause tumors as xenotransplants Published under the PNAS license. in nu/nu or SCID mice (4–6). Growth in culture or in mice is not 1To whom correspondence should be addressed. Email: [email protected]. dependent on KSHV or EBV replication, as the cells tolerate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ganciclovir at a level that inhibits the virally encoded, DNA- 1073/pnas.1810128115/-/DCSupplemental. dependent DNA polymerase (7, 8).

www.pnas.org/cgi/doi/10.1073/pnas.1810128115 PNAS Latest Articles | 1of9 Downloaded by guest on September 23, 2021 Previously, Mack and Sugden (33) reported that a dominant- BC3 cl6 EBV and BC3 cl10 EBV, and CRO AP6 (45), with negative EBNA-1 (dnEBNA-1) mutant was toxic to dually in- CRO-AP6 cl2 EBV and CRO-AP6 cl3 EBV. Authenticity was − fected PELs and some EBV PELs. EBNA-1 is the EBV ho- reconfirmed by HLA and short tandem repeat (STR) typing. molog to LANA in terms of structure and function (34–38). Unlike PELs that were dually infected in the patient, i.e., before Some dnEBNA-1 mutants, such as used above, are broadly toxic establishment in culture, these cell lines by definition do not + − in EBV and EBV lymphoma, presumably because EBNA- depend on EBV for survival. The EBV recombinant in these 1 also binds many sites throughout the host genome and has cells carries EGFP driven by the simian virus 40 (SV40) pro- broad transactivation activity (39–42) To directly test the hy- moter as well as the gene for G418 resistance (46). + pothesis that EBV helps maintain KSHV plasmid copy number To obtain >99% GFP populations, each cell line was sub- and thereby indirectly confers a proliferative advantage to dually jected to FACS (SI Appendix, Fig. S1). Afterwards half of the infected cells, CRISPR/Cas9 (43) was used to remove EBV. We cells were maintained with G418 selection and half without found that EBV plasmid number positively correlated with G418 selection for 5 wk. This yielded three populations: (i) the + KSHV plasmid number at the single-cell level and that in EBV- original population, (ii) the sorted and selected (G418 ) pop- − addicted dually infected PELs the removal of EBV was in- ulation, and (iii) the sorted only (G418 ) population. GFP compatible with KSHV plasmid maintenance and ultimately cell positivity correlated with EBV plasmid copy number (Fig. 1 A–C + survival. Providing EBNA-1 in trans rescued the survival of EBV- and E). All sorted and selected (G418 ) sublines showed an depleted PELs. increase of mean EBV copy number as compared with the par- ent cells. In contrast, sorted cells that were maintained in the − Results absence of selection (G418 cells) did not show such an increase. Addition of EBV Increases KSHV Copy Number and PEL Proliferation. EBV copy number was determined using a digital PCR assay Trivedi et al. (44) created dually infected PEL ex vivo by using the single-copy human ERV-3 as a normalizing gene (47). superinfecting established KSHV single-infected cell lines with a Digital PCR evaluated >20,000 individual replicates. Across all recombinant EBV in culture. This yielded independently de- biological replicates represented in Fig. 2E the coefficient of + rived, syngeneic EBV subclones for two PELs: BC3 (2), with variation was 1.48 copies (n = 26). Because digital PCR utilizes

Fig. 1. EBV and KSHV copy numbers in PELs and their EBV-GFP sublines by digital qPCR. (A–D) Scatter plots from digital PCR analysis show signal from VIC reporter dye on the x axis against signal from fluorescein amidite (FAM) reporter dye on the y axis. Data points represent individual PCR reactions and are color-coded for reporter dye signals: blue for FAM, red for VIC (ERV-3), green for FAM and VIC, yellow for no amplification. (A) Namalwa cells were used as positive control for EBV (FAM: green and blue). (B and C) Signal from EBV (FAM: green and blue) is completely absent in BC3 cells serving as negative control; only the single-copy human gene (ERV-3: red) is visible (B). This is in contrast to the BC3 EBV-GFP sorted and selected subline (C). (D) Signal from the KSHV FAM-labeled probe. (E and F) EBV (E) and KSHV (F) copy numbers in BC3 (red dots) and CRO-AP6 (green dots) cells and their EBV-GFP sublines (blue dots for CRO-AP6 EBV clone 3). Total DNA was analyzed by digital qPCR; values are from two biological replicates per cell line. Gray boxes represent the 25th to 75th percentiles of the data. Data were analyzed by ANOVA adjusted for multiple comparisons by Tukey method. ***P < 0.01.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1810128115 Bigi et al. Downloaded by guest on September 23, 2021 the Poisson distribution as the basis of measurement, we were KSHV , akin to the set point for fixed-copy bacterial able to obtain absolute copy numbers. This technique provided plasmids, and that EBV increased this set point. the most accurate viral copy numbers to date, and it allowed us to determine twofold differences in viral copy number with Addition of EBV Increases KSHV Plasmid Copy Number per Cell. The 95% confidence. number of “LANA dots” in an interphase nucleus correlates with To test the hypothesis that EBV conferred a growth advantage the number of KSHV genomes (48, 49). We used 3D immuno- to dually infected cells, growth curves were recorded. The EBV- fluorescence coupled to image reconstruction to count the + + sorted and selected (G418 ) cells showed an increased pro- number of distinct LANA foci (Fig. 2 and Movie S1). Three liferation in comparison with either the parent cells or cells not slides were prepared for each cell line, and from each slide we − maintained under selection (G418 cells) (SI Appendix, Fig. obtained three 3D images (50 z stacks per field) to analyze >100 S2A). This experiment suggests that EBV increased the pro- individual nuclei per sample. LANA was detected using a mono- liferative potential, i.e., decreased the doubling time. The effect clonal antibody against LANA followed by Alexa-Fluor 350- was small but significant (P ≤ 0.001 by linear regression across all conjugated secondary antibody. Actin was stained by Acti-Stain SI Appendix cell lines after adjustment for multiple comparisons by Dunnett’s 488 (phalloidin) to delineate the cytoplasm ( , Fig. S3 method). To confirm these results, seeding efficiency was de- and Movie S2), which in PEL is minimal. The number of LANA SI Appendix B dots in each nucleus was recorded. First, the naturally dually termined by limiting dilution ( , Fig. S2 ). We ob- + + infected (KSHV /EBV ) PELs BC1 and JSC1 (4, 50) were served a small but significant (P ≤ 0.001 after adjustment for + − multiple comparisons) increase in the fraction of positive wells of compared with the single-infected (KSHV /EBV ) BC3 and A the sorted and selected cultures at the limit of dilution, consis- CRO-AP6 PELs (Fig. 3 ). The dually infected cells exhibited a significantly higher number of LANA dots per nucleus com- tent with increased proliferative capacity. P ≤ To test the hypothesis that EBV genome copy number cor- pared with the singly infected cells ( 0.05 based on two-way related with KSHV genome copy number, both were measured ANOVA of Anscombe-transformed counts). Variance was sim- by digital PCR (Fig. 1 D and F). KSHV plasmid copy number ilar across all cell lines. Of note, in naturally infected PELs both was consistently higher (P ≤ 0.001 after adjustment for multiple EBV and KSHV plasmids are maintained ad infinitum in the comparison) in sorted cells maintained under selection for EBV absence of G418 selection. To confirm the count data, LANA protein levels were evaluated by Western blot. Sorted and then (>95% of cells carry EBV) than in the parental populations. Cell + selected (G418 ) sublines exhibited higher levels [Fig. 3 B and D, lines that showed a higher EBV plasmid copy number also had a −

+ MICROBIOLOGY higher KSHV plasmid copy number. In the absence for selection positive ( ) lanes] than the not-selected (G418 ) sublines [Fig. 3 − B and D, negative (−) lanes] and unsorted cells [Fig. 3 B and D, of EBV (G418 cells) the KSHV plasmid copy number reverted unsorted (u) lanes]. The amount of actin protein was comparable back to the set point established in the parental cell line. This across all samples. suggested that in cells there is a fixed set point for the number of Next, the number of LANA dots per cell was determined for experimentally ex vivo EBV-infected BC3 and CRO-AP6 cells. In addition to LANA and actin, EBNA-1 was stained to establish the presence of EBV in the same nucleus (SI Appendix, Fig. S4). EBNA-1 and LANA dots did not overlap. Again, the selected + (G418 ) cells exhibited a significantly higher number of LANA − dots per nucleus than the unselected (G418 ) cells (P ≤ 0.05 based on ANOVA of Anscombe-transformed counts) (Fig. 3 C and E). These experiments demonstrate that the presence of EBV results in a higher set point of LANA dots per cell, which correlates with higher KSHV genome copy number as previously determined by digital PCR.

Depletion of EBV Decreases KSHV Copy Number and Survival. To test the hypothesis that EBV regulates KSHV plasmid copy number, we removed EBV from in-patient coinfected PELs. We nucleo- + + fected the KSHV /EBV JSC1 and BC1 cells with (i) three single-guide (sg) RNAs (sgEBV1, -2, and -6) directed against the BamHI-W region and the PstI repeats near the lytic origin of replication or (ii) two sgRNAs (sgEBV4 and -5) against both ends of the EBNA-1 coding region (SI Appendix, Fig. S5A)or (iii) an sgRNA against GFP (sgGFP) (43). sgEBV1, -2, and -6 induce large deletions and lead to rapid loss of the EBV plasmid. By contrast, sgEBV4 and-5 induce a precise deletion of only EBNA-1. This leads to gradual loss, as the EBV genome remains Fig. 2. Representative example of a 3D-IFA image created by Imaris soft- intact and is diluted out during cell division. As expected, EBV ware. (A–D) Multipage TIFF format, which encloses 50 individual optical copy number was decreased at 24 h in cells that were nucleo- sections from the same sample in a single file, of a BC3 sorted G418+ clone. fected with sgEBV1, -2, and -6, and EBV copy number was A–C show monochrome, conventionally captured images of single-channel unaltered or was only slightly affected in cells that were nucle- signals from LANA, β-actin, and EBNA-1, respectively, in which all the signals ofected with sgEBV4 and -5 at this early time point (Fig. 4A). overlap and bleed into each other. (D) In the merged image LANA (labeled The specificity of CRISPR/Cas9 was confirmed by PCR against in blue) localizes within the nucleus of the cells in foci in characteristic dots; the target region (SI Appendix, Fig. S5 B–E). The differential EBNA-1 (in magenta) is labeled to confirm the presence of EBV-GFP after β efficacy in PELs paralleled the differential efficacy as originally infection. -Actin is labeled in green. (E and F) 3D images created from the + ’ multipage file after signal processing. The signal from LANA was maintained reported for EBV Burkitt s lymphoma (BL) (51). It was con- in dots; the signal from EBNA-1 has also been represented as dots to facili- sistent with target choice (single copy vs. repeat) and purported tate counting of LANA dots and to provide a clearer view of possible mechanism of action (rapid destruction vs. gradual dilution colocalization between the two proteins. (Scale bars: 5 μm.) during cell division). To confirm that host genome integrity was

Bigi et al. PNAS Latest Articles | 3of9 Downloaded by guest on September 23, 2021 + Fig. 3. LANA expression is significantly increased in naturally KSHV and EBV coinfected PEL lines and in the EBV-GFP–sorted G418 cells. (A) The number of LANA dots per cell is shown on the vertical axis. They are significantly increased in BC1 and JSC1 cells, which are naturally EBV and KSHV coinfected, in comparison with BC3 and CRO-AP6 cells, which do not carry EBV. Data were analyzed by ANOVA. ***P < 0.001. (C and E) The number of LANA dots per cell is + − shown on the vertical axis for BC3 (C) and CRO-AP6 (E) parental cells and after sorting and maintenance in either G418 or G418 medium. Data were analyzed by ANOVA. ***P < 0.001. (B and D) LANA protein levels were ascertained by Western blotting using anti-LANA and anti-actin antibody. +, sorted G418+ cells; −, sorted G418− cells; u, unsorted cells. An EBV+, KSHV− LCL cell line was used as negative control. LANA dot analysis was performed by counting the dots of more than 100 cells for each cell line using 3D IFA images; for EBV-GFP sublines, only one clone was used for each cell line (clone 6 for BC3 and clone 2 for CRO-AP6).

not affected, cells were stained with phosphorylated histone EBV from natively dual-infected cells decreases KSHV genome family member X (p-H2AX) (SI Appendix, Fig. S6). p-H2AX is a copy number. marker for DNA damage. It was rapidly induced by etoposide The loss of EBV from in-patient dually infected PELs resulted in reduced proliferation (Fig. 4 F and G). By comparison, treatment but not by CRISPR/Cas9 treatment. In sum, the − CRISPR/Cas9 reagents performed as expected based on prior nucleofection of EBV PEL (BC3) with Cas9/sgEBV1, -2, and -6 work applying this technology to herpesviruses (43, 52). Off- or Cas9/sgEBV4 and -5 had no effect on cell proliferation (Fig. D SI Appendix target activity was negligible, and CRISPR/Cas9 did not yield 4 ) or on LANA expression ( , Fig. S7). This dem- detrimental genomic instability, as is consistent with experi- onstrates that the proliferation loss is dependent on the pres- ence with multigene-targeted approaches in other studies ence of EBV and is unlikely to be an off-target effect of these EBV-directed guide RNAs on genes in the human genome. (53, 54). + The levels of the KSHV plasmid in the same samples were Nucleofection of EBV Namalwa cells, in which EBV is stably measured at the same time. Loss of the EBV resulted in a re- integrated rather than maintained as a plasmid, with Cas9/ sgEBV1, -2, and -6 or Cas9/sgEBV4 and -5 had no effect on cell duction of KSHV copy number (Fig. 4B). LANA protein levels proliferation (Fig. 4E). These observations suggest the EBV were reduced in PELs nucleofected with sgRNAs directed provides a selective advantage to dually infected PELs analogous against EBV, but not in PELs that were nucleofected with + B C to the selective advantage it provides to EBV BL. In sum, sgRNAs directed against an irrelevant target (Fig. 4 and ). forced introduction of EBV into single-infected PELs increases The degree to which LANA protein expression was reduced KSHV plasmid copy number per cell, and deletion of EBV from correlated with the degree to which KSHV plasmid copy number natively dually infected cells decreases KSHV plasmid number to was reduced. Nucleofection with sgEBV1, -2, and -6, which in- the point that KSHV is no longer able to support PEL growth. duced a rapid loss of EBV, completely abrogated LANA ex- pression; nucleofection with sgEBV4 and -5, which induced a EBNA-1 Restores KSHV Copy Number and LANA Expression in EBV- gradual loss of EBV, resulted in a gradual reduction of LANA Depleted PELs. To test the hypothesis that EBNA-1 alone was expression. These experiments demonstrate that deletion of necessary and sufficient to account for the EBV effect on KSHV

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1810128115 Bigi et al. Downloaded by guest on September 23, 2021 MICROBIOLOGY

Fig. 4. EBV-specific CRISPR/Cas9 nucleofection leads to a decrease in KSHV copy number, LANA expression, and cell viability in naturally coinfected PEL cells. (A) KSHV and EBV viral load as determined by digital PCR is shown on the vertical axis for BC1, JSC1, BC3 (KSHV only), and Namalwa (EBV only) cells at 24 h after nucleofection with the indicated plasmids. Individual values are shown for three independent experiments (a, b, c). Gray boxes represent the 25th to 75th percentiles of the data. (B and C) Western blotting for LANA protein in JSC1 (B) and BC1 (C) cells harvested 48 h after nucleofection with indicated plasmids. β-Actin was loaded for normalization. The BJAB cell line was used as a negative control. Also indicated are molecular weight markers. (D–G) Relative cell viability at 24, 48, and 72 h after nucleofection for EBV− BC3 (D), KSHV− Namalwa (E), EBV+KSHV+ JSC1 (F) and BC1 (G) cells. Values are measured in log10 RLU of CTG reagent. Individual data for three independent biological experiments (indicated by symbol shape), each with multiple technical replicates, are shown with boxplot of the 25th to 75th percentiles of the data.

plasmid copy number, the CRISPR/Cas-9 experiments were re- EBV; however, the rescue of cells exposed to sgEBV1, -2, and -6 peated in the presence of pCEP4, a plasmid vector carrying was only partial. Providing EBNA-1 in trans to EBV-depleted EBNA-1. As before, cells were harvested and DNA was analyzed PELs also restored cell survival (Fig. 5C). This demonstrates at 24 h after nucleofection (Fig. 5A). EBNA-1 complemented the that EBNA-1 is necessary and sufficient to maintain KSHV in copy number defect associated with the loss of EBV. Even dual-infected PELs addicted to EBV. though EBV copy number was reduced in response to targeting There are multiple mechanisms that could explain these ob- by sgEBV1, -2, and -6 or sgEBV4 and -5 in JSC1 and BC1 cells, servations. The particular CRISPR/Cas9 design used here (43) is KSHV copy number was not reduced. EBNA-1 restored LANA not trivial, as EBV-targeting sgRNAs were expressed from a expression: Cells subjected to CRISPR with sgEBV1, -2, and -6 plasmid that itself required EBNA-1 expression from EBV for or sgEBV4 and -5 in the presence of pCEP4 maintained LANA extended persistence and that did not carry a selection marker. protein levels; cells subjected to CRISPR with sgEBV1, -2, and -6 Several confirmatory studies were performed to place these ob- or sgEBV4 and -5 without pCEP4 did not (Fig. 5B). As servations in context. To confirm that targeting either the expected, providing EBNA-1 in trans from pCEP4 completely EBNA-1 ORF (sgEBV4 and -5) or cis-regulatory regions in EBV restored the phenotype of sgEBV4 and -5 targeting EBNA-1 on (sgEBV1, -3, and -6) reduced EBNA-1 expression as well as

Bigi et al. PNAS Latest Articles | 5of9 Downloaded by guest on September 23, 2021 Fig. 5. (A) KSHV and EBV genome copy number is shown on the vertical axis for sgEBV1, -2, and -6 or sgEBV4 and -5 or GFP- or mock-nucleofected cells either alone or with pCEP4. Individual values for two experiments are shown. (B) Western blots for LANA protein expression. β-Actin was loaded for normali- zation. The BJAB cell line was used as a negative control. (C) Cell viability assay performed by CTG at 24 (yellow), 48 (orange), or 72 (red) h after pCEP4 or mock nucleofection. Values for three independent ex- periments are shown. The y axis represents log10 RLU.

LANA expression and EBV viral load, an immunofluorescence canonical latent KSHV pattern in PEL (15, 16, 56). assay (IFA) for EBNA-1 was conducted at 72 h after treatment. In cases of two isogenic PEL clones, in which EBV was introduced (SI Appendix, Fig. S8A). EBNA-1 expression in BC1 cells was ex vivo, the results were more differentiated. Again, the majority abolished upon treatment. At 72 h after pCEP4 transfection into of signal and all high-confidence peaks were identical, irrespective + the CRISPR-treated cells EBNA-1 expression was restored as of the presence of EBV; however, the KSHV genomes in EBV expected. To exclude the possibility that the treatment with PEL had more low-confidence peaks, indicative of open EBV-specific CRISPR-Cas9 was activating the KSHV lytic cycle, regions, randomly distributed throughout the genome. a Western blot analysis for the KSHV lytic marker ORF45 was Human mRNA profiling using Affymetrix arrays was performed + conducted (SI Appendix, Fig. S8 B and C). Now induction of across multiple clones of BC3 cells compared with EBV BC3 cells, + ORF45 was visible under any condition. as well as multiple clones of CRO-AP6 cells compared with pa- One means by which EBV could aid KSHV plasmid persis- rental CRO-AP6 cells. The addition of EBV followed by extensive tence is through epigenetic modification of the KSHV genome, passage under selection did not induce significant changes in human specifically by fostering the expression of progrowth lytic genes. mRNA levels compared with parental PEL lines as ascertained Multiple reports showed that KSHV DNA entered a tightly la- by hierarchical clustering and principal component analyses (SI tent conformation in PEL. Formaldehyde-assisted isolation of Appendix, SI Materials and Methods and Fig. S10). The fluctua- + regulatory elements sequencing (FAIRE-seq) is one method to tion in mRNA levels between EBV subclones was no greater − interrogate the chromatin state of KSHV (55). Using this tech- than the fluctuation between EBV subclones. The majority of dif- nique (SI Appendix, SI Materials and Methods and Fig. S9), KSHV ferential transcription was due to patient origin. genome accessibility was probed in naturally coinfected vs. in-culture Thus far all CRISPR experiments had been conducted short- EBV-infected PEL. The KSHV FAIRE profiles in naturally and term, as EBNA-1 and LANA protein expression was already dually infected PEL were indistinguishable, as is consistent with reduced at 72 h and as the great majority of cells die over a prior mRNA profiling studies by us and others who identified the period of 7 d congruent with a gradual reduction in viral DNA at

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1810128115 Bigi et al. Downloaded by guest on September 23, 2021 each cell division. To expand on these observations, we analyzed molecules that disrupt EBV plasmid maintenance in EBV lym- cells that were rescued by pCEP4:EBNA-1:hygromycin and were phomas (66) may have efficacy in dually infected PELs. resistant to hygromycin at 3 wk (SI Appendix, Fig. S11A). To Thus, far, no one has been able to cure naturally EBV- verify that the sgEBV4 and -5 CRISPR/Cas-9 targeted the viral coinfected PELs of EBV. Akata is the only BL cell line that EBNA-1 locus, we used a PCR with primers against regions could be cured of EBV. After extensive selection an Akata outside the EBNA-1 ORF. Those regions are present in EBV subclone maintained its proliferative capacity in culture; how- but not in the pCEP4 plasmid. These assays confirmed the de- ever, the cells were impaired for growth in the more demanding letion of EBNA-1 in clones exposed to sgRNA4 and -5/CRISPR environment of nude mice (51). Other BLs, particularly from + but not in clones exposed to sgEBV1,-2, and -6/CRISPR (SI HIV patients, do not carry EBV but nevertheless grow readily Appendix, Fig. S11B). KSHV and EBV viral copy number anal- in culture or form tumors in nude mice. Some BLs, such as ysis confirmed the presence of both viral genomes (SI Appendix, BJAB, can be infected with either EBV or KSHV post facto (67, Fig. S11C). At this point, however, we have not been able to + − 68), and some EBV LCL cell lines can be infected with KSHV produce a single-cell EBV BC1 clone after CRISPR, perhaps (69); under either scenario the addition of the virus does not since the CRISPR/Cas-9 vectors did not have a selection marker. change the proliferative potential of these cells. This suggests the presence of two evolutionary pathways toward the development Discussion of PEL, one that is absolutely dependent on the presence of The majority of the people are latently infected with multiple EBV coinfection and another that is not. A given PEL isolate herpesviruses, although each herpesvirus has a different tissue belongs to one or the other but cannot switch. tropism. There are eight human herpesviruses (HHV1–8). Im- How can we explain these observations with regard to plasmid munocompetent individuals tend to experience episodes of in- maintenance and tumor survival? The stochastic model repre- fection or reactivation by one herpesvirus at a time. In HIV- sents the null hypothesis: There is no interaction between KSHV associated immune deficiency, herpesvirus-associated disease and EBV. Dually infected PELs represent the historical outcome and viremia tend to occur at different levels of immune sup- of two viruses that happen to encounter the same B cell. Then pression. For instance, EBV/HHV4-associated non-Hodgkin the dually infected cells accumulate additional transforming lymphoma of the central nervous system is prevalent in HIV mutations in the human genome. Certainly, infection with KSHV < 3 patients with the lowest CD4 counts ( 50 cells/mm ) and highest does not prevent superinfection with EBV, or vice versa, as the immune deficiency, whereas EBV-associated Hodgkin disease is 3 two viruses use different receptors; however, this model is in-

> MICROBIOLOGY more prevalent in HIV patients with CD4 counts 200 cells/mm . consistent with the dependence on EBV in the dually infected In transplant-associated immunodeficiency, herpesvirus reac- PEL. The temporal model assumes that EBV infection of naive + tivation tends to cluster at different times posttransplant. The risk B cell precedes KSHV infection and that EBV naive B cells for herpes simplex virus (HHV1) reactivation is greatest imme- support higher levels of KSHV plasmids. After coinfection the diately after transplantation, necessitating acyclovir prophylaxis; dually infected cells accumulate additional transforming muta- episodes of human cytomegalovirus (HHV5) reactivation tend to tions in the human genome. As 90% of the population carries − occur later; and posttransplant disease caused by EBV is typically EBV, cases of EBV PEL are rare. Finally, the dependency a late complication (years out). Kaposi sarcoma caused by KSHV/ model would ascribe a selective, cell-autonomous advantage to HHV8 also is a late complication. EBV and KSHV persist si- EBV coinfection and a continued need for the prosurvival multaneously for extended periods of time in the B cells of function provided by EBV miRNAs or proteins. This is the case coexposed patients. for naturally coinfected PELs as demonstrated here. The majority of PELs are coinfected with EBV and KSHV, but some PELs carry only KSHV. This demonstrates that EBV is Materials and Methods not required for PEL lymphomagenesis; however, EBV is nec- Cell Culture. BC3 (2) and CRO-AP6 (45) cells and lymphoblastoid cell lines LCLs essary for the survival of the PEL cell lines that are derived from were cultured in RPMI 1640 medium supplemented with 20% FBS, 1% L- in vivo KSHV/EBV-coinfected effusions but not for the survival glutamine, 100 IU penicillin, and 100 μg/mL streptomycin. BC1, JSC1, BJAB, of PEL cell lines into which EBV was artificially introduced ex Raji, and Namalwa cells were cultured in RPMI 1640 medium supplemented vivo. This suggests the existence of two subtypes of PEL: those with 10% FBS, 1% L-glutamine, 100 IU penicillin, and 100 μg/mL strepto- that have grown addicted to prosurvival signals provided by mycin. EBV-superinfected sublines from BC3 and CRO-AP6 cells were cul- EBV, and others that have not. Indeed, genomic and tran- tured in a selection medium containing G418 (44). Cells were grown at 37 °C + − scriptomic difference between EBV and EBV PELs have been in a humidified environment supplemented with 5% CO2. Cells were tested < reported (31, 57), and KSHV is absolutely required for PEL for mycoplasma routinely (Lonza) and were used within 2 mo and 20 pas- survival (27). Using single-cell measurements, this study dem- sages after initial thawing. onstrated (i) that there exists a set point or fixed carrying ca- Cell authenticity was verified by STR and HLA typing at the University of North Carolina (UNC) Vironomics Core using Ion Torrent (Precision ID pacity for KSHV plasmids in PEL, (ii) that EBV increases this iii GlobalFiler NGS STR Panel; Thermo Fisher Scientific) sequencing, and cell set point, and ( ) that EBV provides a growth advantage to lines were compared with each other and with STR cell line profiles gen- dually infected cells that have become addicted to EBV. erated from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, The experiments reported here complement prior work, that ATCC, Japanese Collection of Research Bioresources Cell Bank, and RIKEN showed that a dnEBNA-1 mutant was toxic to EBV/KSHV du- databases. − − ally infected PELs, some EBV PELs, and some KSHV BLs but not others and to some lymphoblastoid cell lines (LCLs) but FACS. EBV-infected clones were sorted for the presence of GFP at the UNC – Flow Cytometry Core Facility using a BD FACSAria II flow cytometer (BD not others (33, 58 60). Overall, BLs were heterogeneous in their + response to a dnEBNA-1. In PEL, EBNA-1 and LANA did not Biosciences) to obtain a 100% EBV population. Details are shown in SI colocalize, and the EBV and KSHV plasmids did not commingle, Appendix, Fig. S1. as demonstrated by the (now well-validated) assumption that LANA-dots represent plasmid-chromosome attachment sites Cell Viability Assay. The CellTiter-Glo (CTG) Luminescence Cell Viability Assay (Promega) was used to determine cell proliferation. Cells (1 × 105) were (12, 13, 23, 48, 61, 62). Recent genome structure data are not plated in 100 μL of medium per well in a 96-well plate and at the indicated directly comparable. Some support a very compact organization times and conditions were incubated with an additional 10 μL of CTG for of KSHV plasmids (63, 64); others find evidence for a clustering 10 min at room temperature. Luminescence readings were recorded in rel- of multiple KSHV plasmids and for higher-order assemblies (37, ative light units (RLU) using a FLUORstar OPTIMA microplate reader (BMG 65). These findings raise the interesting possibility that small Labtech). All assays were conducted in technical triplicates.

Bigi et al. PNAS Latest Articles | 7of9 Downloaded by guest on September 23, 2021 Table 1. Digital PCR probe and primers sequences Gene Probe Primers

KSHV (LANA) 5′-FAM-gtgggtcatcgtctggtgta Forward: 5′-gcctcatacgaactccaggt Reverse: 5′-gagcccataatcttgcacgg EBV (EBNA-3C) 5′-FAM-ccaaaaaggcctcgagtaga Forward: 5′-aaggtgcatttaccccatg Reverse: 5′-ggtgttcccaagctactgct ERV-3 5′-VIC-tcttccctcgaacctgcaccatcaagtca Forward: 5′-catgggaagcaagggaactaatg Reverse: 5′-cccagcgagcaatacagaattt

3D IFA. Cells (1 × 105) were deposited on a clean microscope cover glass, fixed digital PCR, and cell viability was measured 24, 48, and 72 h after nucleo- with 3% paraformaldehyde (Electron Microscopy Science) at room temper- fection via CTG. The same experimental setting was used to nucleofect the ature for 30 min, and washed three times for 5 min each with TBS-T [0.02 M EBV-CRISPRed cells with pCEP4 plasmid (Thermo Fisher Scientific) carrying Tris·HCl (pH 7.6), 0.137 M NaCl, Tween 0.1%]. Afterwards cells were in- the gene for EBNA-1. DNA extracted from mock- and sgEBV-nucleofected cubated with PBS/10% goat serum (Vector Laboratories)/0.2% Triton X-100. cells was also PCR amplified using sgEBV primers (43) with Phusion DNA Then cells were incubated overnight at 4 °C with blocking buffer containing polymerase to check the proper operation of the CRISPR/Cas9 system (SI the primary antibodies monoclonal mouse HHV-8-LANA clone 13B10 (1:100; Appendix, Fig. S5 B–E) and run on a LabChip GX Touch HT (PerkinElmer). Leica Biosystems) and biotinylated polyclonal goat EBNA-1 (1:100; Abcam). The following day, cells were incubated with blocking buffer containing the sec- Digital qPCR. DNA was extracted from 1 × 106 cells using a MagNA Pure ondary antibodies Alexa Fluor 350 goat anti-mouse (1:250; Life Technologies), Compact instrument and the MagNA Pure Compact Nucleic Acid Isolation Kit Texas Red streptavidin (1:250; Vector Laboratories), and 100 nM Acti-Stain 488 I (Roche) according to the manufacturer’s protocols. Five nanograms of DNA (Cytoskeleton) to stain the cytoplasm. The impetus for this procedure was to was used to determine KSHV and EBV plasmid copy number using a reduce background and improve image reconstruction of cells with no clear QuantStudio 3D Digital PCR system (Thermo Fisher Scientific). Chips were cytoplasmic/nuclear features. PEL cells are suspension cells and as such have minimal cytoplasm. A nuclear dye such as DAPI tends to bleed extensively and loaded with a sample mix of DNA, QuantStudio 3D Digital PCR Master Mix allow less precise 3D caging of the nucleus. For each sample, a negative control (Thermo Fisher Scientific), labeled probes, and primers (Life Technologies). slide was prepared in which the cells were treated only with secondary anti- The single-copy human gene ERV-3 (47) was used to normalize to cell bodies. Images were obtained using a LEICA DM4000B epi-fluorescence mi- number. Absolute quantification was analyzed with QuantStudio 3D Ana- croscope equipped with a Leica apochromatic 63×/1.40 numerical aperture, a lysisSuite Cloud Software (Thermo Fisher Scientific). The following PCR PL Apo oil objective, and a QImaging Retiga 2000RV monochrome camera. To conditions were used: 96 °C for 10 min; 40 cycles of 56 °C for 2 min and 98 °C obtain 3D images, the microscope was set up by identifying the lowest and for 30 s; and 60 °C for 2 min. Probe and primer sequences are reported in highest plane of the z axis on which the protein of interest was still visible; Table 1. The lower limit of detection was set to 0.01 copy virus per ERF- then, 50 sequential images of the individual optical sections between these 3 copy, i.e., per cell. reference planes were taken (z-stack). To eliminate background fluorescence, each image was subjected to deconvolution using Simple PCI_5 version Statistical Analysis. Statistical analysis was performed using R software version 6.2 microscope software. The individual optical sections were exported to 3.3.3 (March 6, 2017). To analyze LANA dots, negative binomial distributions multipage TIFF format and were used to create a tridimensional image via were fitted, and their means were compared. The function glht in the R IMARIS software version 7.1 (Bitplane). LANA and EBNA-1 dots were gener- package multicomp was used to obtain maxT-type adjusted P values for ated by utilizing the Imaris Spots surfacing. LANA dots were counted in more multiple comparisons. To analyze CRISPR/Cas9 experiments, a linear mixed than 100 cells for each cell line. For each examined sample, three independent effects model was fitted to each group, and the plate effect was treated as a immunofluorescence assays were carried out. Regular IFAs were performed for random effect. Then the analysis of covariance method was used to obtain P p-H2AX (1:100; Cell Signaling) using anti-rabbit IgG fluorescein (1:250; Vector values of the exact F-test adjusted by the Bonferroni method for multiple Laboratories) as the secondary antibody and DAPI (1:100) to stain nuclei. comparisons.

Western Blotting. Protein samples were prepared by lysing with radio- FAIRE-Seq Assay. The results of the FAIRE-seq assay are reported in SI Ap- immunoprecipitation assay (RIPA) buffer [150 mM NaCl, 1%Nonidet P-40, pendix, SI Materials and Methods. 50 mM Tris·HCl (pH 8), 0.5% deoxycholic acid, 0.1%SDS] and were sepa- rated on 6% SDS/PAGE. Proteins were detected with monoclonal mouse Affymetrix Analysis. Affymetrix analysis is reported in SI Appendix, SI Ma- antibody HHV-8-LANA clone 13B10 (Leica Biosystems), monoclonal mouse ORF45 (Sigma), and monoclonal mouse β-actin (Sigma). The anti-mouse HRP- terials and Methods. coupled secondary antibody (Vector Laboratories) and Pierce ECL Western Blotting Substrate (Thermo Scientific) were used to visualize the bands. Each ACKNOWLEDGMENTS. We thank Dr. Alberto Faggioni and Dr. Pankaj – Western blotting experiment was performed in triplicate. Trivedi (University La Sapienza of Rome) for PEL EBV-GFP infected cells and Dr. Stephen R. Quake (Stanford University) for providing EBV-directed CRISPR/Cas9 reagents. CRISPR/Cas-9 constructs were obtained under a mate- Plasmids Nucleofection. EBV-specific CRISPR/Cas9 plasmids were obtained rial transfer agreement from Stanford University. The Affymetrix assay was × 6 from S. Quake (Stanford University, Stanford, CA). Cells (3 10 ) were performed by Quintiles Genomic Service. The UNC Flow Cytometry Core nucleofected with a Lonza Nucleofector II instrument using the T-001 Facility and the UNC High-Throughput Sequencing Facility are supported program. The CRISPR/Cas9 constructs were introduced transiently with no in part by Cancer Center Core Support Grant P30 CA016086 to the UNC selection for expression of Cas9. Of note, these constructs contain the B95.8 Lineberger Comprehensive Cancer Center. This work was supported by Pub- origin of replication of EBV and thus are retained only as long as EBNA-1 is lic Health Service Grants DE018304 (to D.P.D.) and CA019014 (to N.R.-T.) and present. Plasmid copy number was measured 24 h after nucleofection via the UNC Vironomics Core (https://www.med.unc.edu/vironomics).

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