EBV Microrna BART 18-5P Targets MAP3K2 to Facilitate Persistence in Vivo by Inhibiting Viral Replication in B Cells

EBV microRNA BART 18-5p targets MAP3K2 to facilitate persistence in vivo by inhibiting viral replication in B cells

Jin Qiu and David A. Thorley-Lawson1

Department of Pathology, Tufts University School of Medicine, Boston, MA 02111

Edited by Elliott Kieff, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA, and approved June 17, 2014 (received for review April 7, 2014)

EBV is an oncogenic human herpesvirus that has the ability to It is believed that, in vivo, the trigger for the virus to start infect and transform B cells latently in vitro. However, the virus replicating is the differentiation of latently infected memory cells also establishes a lifetime, benign, persistent latent infection in to become plasma cells (5). In vitro, the virus can be reactivated resting memory B cells in vivo, where the virus is quiescent (i.e., from EBV-positive cell lines by several stimuli, including phorbol expresses none of the known latent proteins). The virus encodes esters, butyrate, and cross-linking of surface Ig. However, this ∼40 micro-RNAs (miRNAs), most of which are transcribed from the process is always inefficient. Viral replication is initiated through BamH1 fragment A rightward transcript (BART) region of the virus. expression of two virally encoded immediate early (IE) transcrip- We have shown previously that a subset of these miRNAs is pres- tion factors: BamH1 fragment Z left reading frame 1 (BZLF1) ent at high copy numbers in latently infected memory B cells in and BamH1 fragment R left reading frame 1 (BRLF1) (4). In vivo, suggesting a role in maintaining latency. Here, we describe turn, these transcription factors activate a series of genes that are the role of one of these miRNAs, BART 18-5p. We show that it involved primarily in replicating the viral DNA, referred to as early targets the 3′UTR of the mRNA, encoding the important cellular (E) antigens. Subsequent to replication of the viral DNA, the viral signaling molecule MAP kinase kinase kinase 2 (MAP3K2), at ex- structural proteins are synthesized, referred to as late (L) antigens, actly the same site as the oncogenic cellular miRNA mir–26a-5p. To including the major envelope glycoprotein gp350/220 (6, 7), lead- MICROBIOLOGY our knowledge, this is the first report of a virus encoding a miRNA ing to virion assembly and release. Recently, it has become apparent that EBV encodes for that suppresses a target in the MAP kinase signaling cascade, a cen- a large number of micro-RNAs (miRNAs). miRNAs are small, tral signal transduction pathway that governs a broad spectrum of 19- to 25-nt long, evolutionarily conserved noncoding RNAs that biological processes. We further show that MAP3K2 is an interme- act posttranscriptionally to regulate gene expression. To date, diary in the signaling pathways that initiate lytic viral replication. more than 40 mature EBV miRNAs have been identified (8). Thus, 18-5p expression in latently infected B cells has the effect of These miRNAs are encoded in two major regions of the EBV blocking viral replication. We propose that the role of 18-5p is to genome: the BamH1 fragment A rightward transcript (BART) maintain latency by reducing the risk of fortuitous reactivation of gene (mir–BART1-22) and BamH1 fragment H right reading the virus in latently infected memory B cells. frame 1 (BHRF1) gene (mir-BHRF1 1-3). BHRF1 miRNAs are expressed in the growth program but not in the default or latency viral reactivation | EBV miRNA | MAPK pathway program. However, the BART miRNAs are expressed in all forms of EBV latency. The function of most EBV miRNAs is BV is a human herpesvirus that establishes a lifetime per- largely unknown. EBV lacking the BART region is able to im- Esistent latent infection in long-lived memory B cells (1). It is mortalize B cells efficiently, suggesting that the BART miRNAs a transforming virus for B cells in culture and is associated with several forms of cancer, including nasopharyngeal carcinoma, Significance gastric carcinoma, Burkitt lymphoma, and Hodgkin disease. Like many viruses that persist in the infected host, EBV replicates and EBV is a human herpesvirus that is associated with several resides in different cellular compartments with either lytic or forms of cancer. It can transform B lymphocytes into pro- latent gene expression programs. Whereas the lytic program liferating lymphoblastoid cell lines yet establishes a benign gives rise to the production of infectious viral particles, the latent lifetime latent infection in resting memory B cells in virtually all program enables the virus to express a limited number of viral human beings. EBV encodes for ∼40 micro-RNAs, small RNAs genes that maintain the viral reservoir in the face of specific that modulate the activity of cellular genes. A subset of these is immune responses. Unique for EBV is the expression of four highly expressed in latently infected memory B cells in vivo. different latency programs representing different levels of viral Here, we show that one of them, 18-5p, suppresses the ex- silencing in different stages of B-cell differentiation (2, 3). The pression of MAP kinase kinase kinase 2 (MAP3K2). We further generally accepted model is that EBV initially infects naive show that MAP3K2, a central molecule in many cellular sig- B cells and expresses the full set of latent proteins and RNAs naling pathways, mediates the signals that initiate viral repli- known collectively as the growth program. The role of this pro- cation. Thus, 18-5p favors latency in vivo by suppressing viral gram is to activate and drive proliferation of the B cells, which replication through reduction of MAP3K2. then migrate into and through the germinal center to become Author contributions: J.Q. and D.A.T.-L. designed research; J.Q. performed research; J.Q. resting memory B cells analogous to the pathway for an antigen- and D.A.T.-L. analyzed data; and J.Q. and D.A.T.-L. wrote the paper. activated B cell. In the emerging resting memory B cells, the The authors declare no conflict of interest. virus is quiescent and expresses no latent proteins. By compari- This article is a PNAS Direct Submission. son, in vitro infection of B cells also results in long-term latent Data deposition: The data reported in this paper have been deposited in the Gene Ex- infection; however, in this case, the infection process stops at the pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE58224). growth program and the activated blasts continue to proliferate 1To whom correspondence should be addressed. Email: [email protected]. in culture as lymphoblastoid cell lines expressing the growth This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. program (4). 1073/pnas.1406136111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1406136111 PNAS | July 29, 2014 | vol. 111 | no. 30 | 11157–11162 Downloaded by guest on September 30, 2021 Table 1. Representative examples of differential EBV BART miRNA expression in different tissues Germinal center B Lymphoblastoid (growth Memory B (latency (default program BART miRNA program latency III) program latency 1/0) latency II)

Copy number per cell 10 38 <1 <1 14* 96 <1 <1 18-5p 42 294 279

are not essential for transformation (9). Nevertheless, it is complementary oligonucleotides that hybridize with their target thought that EBV miRNAs may promote viral latency or cancer to form stable duplexes with a high melting point, essentially development by targeting both viral and cellular genes. inactivating the miRNA. As expected, cotransfection of 18-5p In a previous study, we observed that some miRNAs were with either 100 or 200 pmol of scrambled LNA [negative control more highly expressed in latently infected resting memory B cells (NC) LNA] again led to a significant decrease in MAP3K2 at the (latency/EBV nuclear antigen 1 only program) than in lympho- mRNA level, but introduction of the specific anti–18-5p LNA blastoid cell lines (growth program), and vice versa (10). BART was able to rescue this decrease (Fig. 2C). This reversal was even 18-5p is one of the miRNAs that are abundantly expressed in more striking at the protein level, with a two- to threefold in- latently infected resting memory B cells in vivo, with ∼300 copies crease in MAP3K2 protein in the presence of the 18-5p LNA per cell. Because EBV does not express any proteins in latently inhibitor compared with the control (Fig. 2D). infected memory B cells, high levels of 18-5p expression may To confirm that the activity of 18-5p was representative of the suggest a role for this miRNA in maintenance of viral latency. endogenous miRNA, the anti–18-5p LNA was introduced into In this paper, we report on the identification of a target gene for Akata 2A8.1 cells, an EBV-positive counterpart of the Akata 18-5p that implicates it in maintaining latency by suppressing 2A8 cell line, which expresses 18-5p endogenously. Anti–18-5p viral replication. LNA dramatically suppressed the levels of free endogenous Results BART 18-5p Targets Cellular Genes Functionally Related to Viral Replication. We have observed previously that 18-5p is more highly expressed in latently infected resting memory and germinal center B cells than in lymphoblastoid cell lines (Table 1). To understand the role of 18-5p better, we attempted to identify cellular genes that it regulates by expressing miRNA mimics in two different EBV-negative B-cell lines, BL2 and BJAB, followed by microarray analysis. Gene arrays have been used extensively to identify putative targets for miRNAs. A list of consensus genes down-regulated in both BL2 and BJAB cells by 18-5p was gen- erated and aligned with the same set of genes from cells expressing other miRNAs (miRNA 10 and 14*). As shown by a heat map (Fig. 1A), 18-5p exhibits a distinct pattern of down- regulated genes compared with miRNA 10 and 14*. Interestingly, many of these genes are functionally related to autophagy, viral replication, viral budding, and endocytic protein recycling, including MAP kinase kinase kinase 2 (MAP3K2). To validate down-regulation of these genes, 18-5p was expressed in a third EBV-negative B-cell line, Akata 2A8, and the mRNA levels were measured by quantitative RT-PCR (qRT-PCR) (Fig. 1B), with typical reductions in the range of 20–60%. Akata 2A8 and its EBV-positive counterpart, Akata 2A8.1, were used in subsequent studies to analyze the function of 18-5p.

BART 18-5p Specifically Down-Regulates MAP3K2. We decided to focus on MAP3K2 because MAPK family members are known to be important in herpesvirus replication (11–18). To test if 18-5p specifically targets MAP3K2, we either transiently expressed an 18-5p miRNA mimic or stably expressed an 18-5p primary miRNA (pri-miRNA; pGIZ–18-5p) in EBV-negative Akata 2A8 cells and measured the inhibition of MAP3K2 at both the mRNA and protein levels by real-time qRT-PCR and Western blot (Fig. 2 A and B). In contrast to control miRNAs [mimic control (MC) and pGIZ-MC], 18-5p expressed transiently or stably suppressed MAP3K2 gene expression by ∼40% or ∼30%, respectively. A parallel decrease in MAP3K2 at the protein level Fig. 1. BART 18-5p targets cellular genes related to viral replication. (A) (48% and 24%, respectively) was also observed. The effect was Heat map of consensus genes down-regulated in BL2 and BJAB cells by 18-5p specific, because other EBV miRNAs (BART10 and BART14*) in comparison to the same gene set for miRNA 10 and 14*. Brown and had no effect on MAP3K2 expression (Fig. S1). purple indicate up-regulation (up to 1.9-fold) and down-regulation (down to To validate the specificity of the MAP3K2 knockdown by −1.9-fold), respectively. The microarray data can be accessed in the National 18-5p further, locked nucleic acids (LNAs) were used to in- Center for Biotechnology Information Gene Expression Omnibus database activate 18-5p, followed by measurement of MAP3K2 expression (accession no. GSE58224). (B) qRT-PCR validation of selected genes down- again in the EBV-negative line, Akata 2A8. LNAs are modified, regulated in Akata 2A8 cells expressing 18-5p.

11158 | www.pnas.org/cgi/doi/10.1073/pnas.1406136111 Qiu and Thorley-Lawson Downloaded by guest on September 30, 2021 18-5p Physically Interacts with the MAP3K2 Transcript at the 3′UTR. We performed bioinformatics analysis on the MAP3K2 gene using the miRNA target prediction software MiRanda in an at- tempt to identify potential binding site(s) for 18-5p. This analysis revealed a single complementary site that was located in the 3′UTRs of the MAP3K2 mRNA (Fig. 3B). This region had eight nucleotides of perfect complementarity with the seed region of mature 18-5p (positions 2–8 at the 5′ end), the region believed to be the key for posttranscriptional repression of mRNA targets by miRNAs. Interestingly, this is the identical seed sequence targeted by the oncogenic human mir–26a-5p, which also down- regulates MAP3K2 (19). To validate the functionality of the putative 18-5p binding site, the 3′UTR (3,018 bp), 5′UTR (457 bp), and coding regions (1,860 bp) of the MAP3K2 mRNA were cloned downstream of the Renilla luciferase coding region in the psiCheck2 vector. HEK 293 cells, highly transfectable cells rou- tinely used for luciferase assays, were cotransfected with this vector and either the control (MC) or 18-5p miRNA, together with either the NC LNA or anti–18-5p LNA. Luciferase activity was measured at 24 h posttransfection. As shown in Fig. 3C,18-5p significantly reduced the activity of the luciferase vector contain- Fig. 2. BART 18-5p specifically targets MAP3K2 in B cells. (A) mRNA level of ing the full-length 3′UTR (40% down-regulation; P < 0.01), MAP3K2 in EBV-negative Akata 2A8 cells transfected with miRNA mimics whereas the MC had no effect. In contrast, 18-5p mimics had no (MC and 18-5p) or cells infected with lentiviruses that express primary miRNAs effect on the luciferase expression of cells expressing the empty (pGIZ-MC and pGIZ–18-5p). (B) Protein level of MAP3K2 from the same experi- reporter vector, 5′UTR, or the coding region of MAP3K2. The ments as in A shown by Western blot. (C) Knockdown of Akata 2A8 cells reduction of luciferase activity in the 3′UTR group was directly transfected with an 18-5p miRNA mimic using 100 or 200 pmol of anti–18-5p proportional to 18-5p expression, because treatment with a LNA reversed the reduction of MAP3K2 by 18-5p at the RNA level. (D) Protein specific anti–18-5p inhibitor, but not NC LNA, fully restored levels of MAP3K2 in the same experiment as C.(E) 18-5p miRNA expression in P <

luciferase expression (1.89-fold increase; 0.01). Finally, to MICROBIOLOGY – EBV-positive Akata 2A8.1 cells treated with the anti 18-5p inhibitor or NC LNA. confirm that 18-5p exerted its effect specifically through the (F and G) LNA knockdown of endogenous 18-5p expression leads to elevated putative binding site identified in the 3′UTR, all seven bases that levels of MAP3K2 in the EBV-positive BL cell lines Akata 2A8.1 and BL36. matched the seed region of 18-5p were mutated (Fig. 3B). This completely abolished the inhibitory effects of 18-5p, because no 18-5p miRNA (Fig. 2E), leading to a significantly higher level difference in luciferase expression was observed between MC and 18-5p–expressing cells. We conclude therefore that 18-5p (40% increase) of MAP3K2 mRNA (Fig. 2F) To confirm this directly binds to this sequence in the 3′UTR to target the observation in a second, independent cell line, we tested the MAP3K2 mRNA. EBV-positive Burkitt lymphoma line BL36. In this case, we saw an even more striking effect, with a 70% increase in MAP3K2 18-5p Impairs EBV Lytic replication upon Reactivation by B-Cell mRNA upon suppression of the endogenous 18-5p (Fig. 2G). Receptor Cross-Linking. MAP3K2 has been implicated previously We conclude that MAP3K2 is a specific target for 18-5p. as a crucial mediator of replication by the mouse gammaherpesvirus Murid herpesvirus 68 (MHV 68) (11). Thus, the 18-5p Specifically Interacts with the MAP3K2 Transcript in the RNA- ability of 18-5p to repress MAP3K2 raises the possibility that Induced Silencing Complex. Mature miRNAs stably associate with the function of 18-5p could be to block EBV lytic replication. the Argonaute (Ago) proteins in the RNA-induced silencing No fully permissive culture system for EBV replication is complex (RISC) before targeting the mRNA. Overexpression available. Current experimental models are usually based on of exogenous miRNAs, followed by immunoprecipitation of the reactivation of latently infected cell lines with inducing Ago proteins, permits the rapid isolation and identification of chemicals or B-cell receptor (BCR) stimulation. To determine miRNA-bound mRNAs. To test if MAP3K2 is a physiological if 18-5p disrupts the BCR signaling pathway, we transiently target for 18-5p, endogenous Ago2 was immunoprecipitated – overexpressed 18-5p mimics in Akata 2A8.1, followed by from Akata 2A8 cells stably expressing either pGIZ 18-5p or anti-Ig treatment. Overexpression of 18-5p down-regulated pGIZ-MC and examined for enrichment of MAK3K2 mRNA by MAP3K2 expression in Akata 2A8.1 (Fig. S3A). In parallel, qRT-PCR. Specific precipitation of Ago2 resulted in a 400-fold the transcription of lytic genes, including the IE BZLF1 and A B enrichment of 18-5p (Fig. S2 and ). The enrichment was the L glycoprotein gp350, acting as indicators for viral repli- specific, because a positive control cellular miRNA, mir-200c, cation, was examined by qRT-PCR. We observed a substantial was also enriched by nearly 130-fold in the Ago2 complexes, repression of viral replication by 18-5p. BZLF1 was reduced whereas the small, noncoding NC RNA U1 was not associated fourfold, and gp350 was reduced twofold (P < 0.05 and P < 0.01, with Ago2 (Fig. S2 C and D). When the level of mRNAs was respectively) (Fig. 4A). In parallel, cell-free virions (DNase- measured, it was possible to demonstrate a 3.5-fold enrichment resistant) in the culture supernatant, representative of freshly of the MAP3K2 mRNA in the pGIZ–18-5p/Ago2 precipitates encapsidated virus, were measured by gp350 DNA PCR. The level (Fig. 3A). This association was specific, because no enrichment of extracellular virus was reduced sixfold by 18-5p (1.8 × 105 was seen in the pGIZ–18-5p/Ago2 precipitates for the mRNA copies per milliliter vs. 3.3 × 104 copies per milliliter; P < 0.05) of a control gene, beta-actin. The mRNA for AB12, another (Fig. 4B). putative 18-5p target from the microarray analysis, was not sig- Because 18-5p is expressed endogenously in Akata 2A8.1 cells, nificantly enriched in the Ago2 complex, suggesting that it might to demonstrate the direct action of endogenous 18-5p on the in- be an indirect target for 18-5p. As a further control, we analyzed duction of viral replication by endogenous MAP3K2, we knocked ZEB1, a known target of mir-200c. No enrichment of ZEB1 down endogenous 18-5p with the anti–18-5p LNA. In this case, mRNA was observed in the pGIZ–18-5p/Ago2 precipitates (Fig. lytic gene transcription was enhanced fivefold for BZLF1 and 3A). This confirms the specificity of the MAP3K2 mRNA asso- threefold for gp350 (Fig. 4C), and virus production was increased ciation with the 18-5p/Ago2 complex. We conclude that endog- twofold (Fig. 4D). Taken together, we observed a 20-fold change enous MAP3K2 interacts with 18-5p in the RISC. in BZLF1, a sixfold change in gp350, and a 12-fold change in

Qiu and Thorley-Lawson PNAS | July 29, 2014 | vol. 111 | no. 30 | 11159 Downloaded by guest on September 30, 2021 virion production (Fig. 5B). To confirm the relationship between 18-5p, MAP3K2, and viral replication, we performed add-back experiments with MAP3K2 in B95.8-LCL cells ectopically expressing 18-5p and observed the effect on viral replication. We observed that overexpression of MAP3K2 (Fig. S5B) did indeed significantly restore the expression of BZLF1 (increased from 2.1-fold to 12.8-fold after TPA/BA induction; P < 0.01) and gp350 (from 3.6-fold to 10-fold; P < 0.01) (Fig. 5C). However, we failed to detect an increase in virions (Fig. S6). The failure to recover virus production suggests that the reduction of EBV lytic replication by 18-5p is only partially mediated through MAP3K2 and that 18-5p interferes with other genes associated with viral assembly and budding (Fig. 1). Discussion In this study, we have demonstrated that the EBV-encoded BART 18-5p miRNA targets the mRNA for the MAP kinase MAP3K2. We further demonstrate the relevance of this finding by showing that this kinase plays a central role in the signaling cascade that drives reactivation and replication of the virus. Consequently, the effect of 18-5p is to reduce the level of viral Fig. 3. BART 18-5p physically interacts with the 3′UTR of MAP3K2. (A) BART replication. The 18-5p miRNA had an impact on viral replication 18-5p interacts with MAP3K2 in the RISC complex. Fold enrichment of MAP3K2, induced by two different in vitro signals, TPA and BA, which pri- beta-actin (ACTB), ZEB1, and abl-interactor 2 (ABI2) mRNAs in the immuno- marily act through the PKC signaling pathway, and by BCR cross- precipitated 18-5p/Ago2 complex from Akata 2A8 cells stably expressing pGIZ– linking, which involves four signaling pathways: PI3K, Rac1, Ras, 18-5p. (B) Potential 18-5p binding site in the 3′UTR of the MAP3K2 mRNA. The and phospholipase C. As can be seen in Fig. 6, both pathways 18-5p miRNA has identical nucleotides (blue) in the seed region as the human miRNA 26a-5p. The seed region of 18-5p has a complementary binding site in the 3′UTR (red) but not the 5′UTR or coding region. For the mutated 3′UTR (mut), the potential binding nucleotides were replaced to disrupt the complementarity (red). (C) Luciferase assays in HEK 293 cells cotransfected by luciferase reporter vectors encoding 3′UTR WT (wt), 3′UTR mut, 5′UTR, and the coding region with or without LNA inhibitors. Open bars represent vector alone, dotted bars represent vector + MC miRNA, black bars represent vector + 18–5p, gray bars represent vector + 18-5p + anti–18-5p LNA, and striped bars represent vector + 18–5p + anti-MC LNA. *not significant (N.S); **P < 0.01.

virion production associated with the difference between suppression of endogenous 18-5p and ectopic expression of 18-5p.

18-5p Impairs EBV Lytic Replication upon Reactivation by 12-o- Tetradecanoylphorbol-13-Acetate/Sodium Butyrate. An alternate pathway for reactivating EBV is through induction with 12-o- tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate (BA). To test the effect of 18-5p on chemical induction of the virus, we induced a lymphoblastoid cell line (LCL; previously selected for maximal inducibility) transformed with the B95-8 strain of virus (known to lack 18-5p). Overexpression of 18-5p specifically down-regulated MAP3K2 expression in the LCL (Fig. S3B). After 48 h of TPA/BA stimulation, transcription of the lytic genes BZLF1 and gp350 was induced 12-fold and sev- enfold, respectively, compared with the uninduced cells (Fig. 4E). However, the induction was significantly disrupted by the presence of 18-5p, as evidenced by the reduced levels of gene transcription of both lytic genes (two- to 2.5-fold) in the presence of 18-5p and its reversal with the 18-5p LNA inhibitor (Fig. 4E). In parallel, we observed a 60% reduction in released virus by 18-5p (15 × 103 copies per milliliter with MC vs. 5.1 × 103 copies per milliliter with 18-5p; P < 0.05) (Fig. 4F), which, again, was reversed with the specific inhibitory LNA. Similar results were obtained with the parental B95.8 cell line (Fig. S4). We conclude therefore that 18-5p significantly inhibits EBV lytic replication in response to both TPA/BA and surface Ig activation. Fig. 4. BART 18-5p impairs viral lytic replication upon reactivation. (A) Lytic genes (IE BZLF1 and L gp350) were quantified by qRT-PCR in Akata 2A8.1 cells transfected with MC or 18-5p mimics upon reactivation by BCR cross-linking for MAP3K2 Mediates Reactivation Signaling and Rescues 18-5p– 72 h. (B) Copy numbers of EBV virions in the supernatants from A.(C) BZLF1 and Mediated Repression. To test if MAP3K2 indeed mediates viral gp350 expression in Akata 2A8.1 cells transfected with NC LNA or anti–18-5p LNA reactivation, we have overexpressed MAP3K2 in the B95-8 upon reactivation by BCR cross-linking for 72 h. (D) Copy numbers of EBV virions LCL (Fig. S5A) and observed the effect upon reactivation by in the supernatant collected from C.(E) BZLF1 and gp350 expression in a B95.8- TPA/BA. Overexpression of MAP3K2 markedly enhanced viral infected LCL transfected with miRNA mimics and LNAs upon TPA/BA treatment replication as judged by both lytic gene expression (Fig. 5A) and for 48 h. (F) Copy numbers of EBV virions in the supernatant collected from E.

11160 | www.pnas.org/cgi/doi/10.1073/pnas.1406136111 Qiu and Thorley-Lawson Downloaded by guest on September 30, 2021 G3BP2 plays a pivotal role in Sindbis virus replication via inter- acting with virus-encoded nonstructural proteins (32). HNRPUL1 is recruited to the viral replication center, and thus modulates adenovirus replication (33). Therefore, 18–5p may have global effects that suppress multiple functions related to viral replication. Down-regulation of these genes by 18-5p only ranged from 20 to 40%, consistent with the current belief that miRNAs typically exert subtle inhibitory effects on many mRNAs embedded within intricate regulatory networks, thereby augmenting the effect and producing a stronger response. Limiting lytic replication would confer a selective survival advantage to the EBV-infected cells, especially in the long-lived Fig. 5. Overexpression of MAP3K2 enhances viral replication. (A) BZLF1 and memory B-cell population. The production of virus triggers gp350 expression in B95.8-LCL cells transfected with empty vector or MAP3K2 alone, followed by TPA/BA induction. (B) Copy numbers of EBV virions in the a potent cytotoxic T-cell and neutralizing antibody response, supernatant collected from A.(C) BZLF1 and gp350 expression in B95.8-LCL which controls virus spread (34). This provides a strong selective cells expressing 18-5p with or without MAP3K2 upon TPA/BA induction. pressure for EBV to develop molecular strategies to evade host attack and only reactivate at a specific place and time. These strategies include limited expression of potentially immuno- converge on MAP3K2, the now-identified target of 18-5p, genic viral proteins (e.g., shutoff of all latent proteins in EBV- explaining why the miRNA has an impact on both signaling infected memory B cells) (35), expression of genes with the mechanisms. ability to block presentation of viral antigens (36), and Interestingly, we have also shown that 18-5p shares a high avoidance of unnecessary viral replication. In this study, the degree of sequence homology at the 5′ end with the human identification of 18-5p as having a role in restricting viral oncogenic miRNA mir–26a-5p, especially in the seed sequence. replication in the memory compartment falls into the last cate- The mir–26a-5p also down-regulates MAP3K2 by targeting ex- gory and is in accord with the observation that viral reactivation actly the same region in its 3′UTR. The coincidence is satisfying, is restricted to the oropharyngeal lymphoid tissues and is not because it further strengthens our conclusion that MAP3K2 is seen in the memory compartment (5, 37). a bona fide target for 18-5p and supports the idea that EBV In conclusion, we have shown that a previously reported but

miRNAs, like some other virus-encoded miRNAs, exploit the uncharacterized EBV miRNA, BART 18-5p, can inhibit viral MICROBIOLOGY host by using homologous seed sequences (20, 21). This seems to replication in EBV latently infected B cells upon in vitro be a common strategy, because it was estimated that 26% of reactivation through repression of an important cellular sig- annotated virus-derived miRNAs have seed regions that are naling molecule, MAP3K2. To our knowledge, this is the first identical to host miRNAs (22). It has been shown previously that report of a virus encoding a miRNA that suppresses a target in mir–26a-5p inhibits JNK-dependent apoptosis in human glio- the MAPK signaling cascade, a central signal transduction blastoma cells, thereby promoting tumor growth (19). Although pathway that governs a broad spectrum of biological processes. there is no known association of EBV with glioblastoma, it is The recognition of cross-talk between viral miRNAs and interesting to note that 18-5p is highly expressed in all of the EBV-associated tumors and that through analogy with mir–26a-5p, it may play a role in oncogenesis. Activation of the MAPK signaling pathway is required for efficient viral replication, especially the initiation of the lytic cascade, for several gammaherpesvirinae, including EBV, Kaposi sarcoma-associated herpesvirus, and MHV68 (11, 13, 15). The full process of EBV lytic replication has three stages: initial induction of the lytic gene cascade, viral assembly and transport, and egress of infectious progeny. The lytic gene cascade, in turn, has three stages: induction of the IE transcription factors that, in turn, activate the E genes responsible for initiating viral DNA replication and, finally, expression of the L genes, including the virion structural proteins. Transcription factors, such as CREB, ATF1-2, and c-JUN, downstream of the MAPK pathway bind to the promoter regions of the IE gene BZLF1 (8), which encodes a transcriptional activator that is sufficient to drive the entire lytic replication program in both B cells and epithelial cells (23). Thus, 18-5p–mediated down-regulation of MAP3K2 should have an impact on the IE stage of lytic replication, as verified by the observed down-regulation of BZLF1 with a consequent decrease in L antigen and virion production. However, the inhibitory effect of 18-5p on viral replication is only partially mediated by MAP3K2, because reconstitution of MAP3K2 reversed the de- creased expression of the IE and L genes tested but did not increase the production of virions. This implies that 18-5p also targets other cellular genes involved in the production of virions. Indeed, in our initial microarray screen for 18-5p targets, we Fig. 6. Proposed model for 18-5p–mediated inhibition of viral reactivation noticed that many of the genes are functionally related to through the MAPK signal transduction pathway. Briefly, TPA triggers the autophagy, viral replication, viral budding, and endocytic protein PKC signaling pathway, and BCR stimulation activates the PI3K, PLC, Ras, and recycling (Fig. 1). For instance, RAB11FIP1, KIF3B, and KIAA Rac1 pathways. Both pathways are mediated through MAP3K2 and activate are key players in regulating endocytic protein transport and downstream effector transcription factors that can activate the BZLF1 pro- autophagy, with potential involvement in virion packaging moter (Zp), followed by BZLF1 protein expression and induction of the lytic (24–27). ABI2 is associated with virus assembly and viral release cycle gene cascade. BART 18-5p represses MAP3K2 via binding to its 3′UTR, for Arenavirus, hepatitis B virus, Sendai virus, and HIV (28–31). and thereby inhibits viral reactivation.

Qiu and Thorley-Lawson PNAS | July 29, 2014 | vol. 111 | no. 30 | 11161 Downloaded by guest on September 30, 2021 cellular genes is of particular importance because it provides using the Affymetrix Human HGU133 Plus 2.0 Array (Broad Institute) (details insight into the function of viral components in establishing and are provided in SI Materials and Methods). maintaining latency. qRT-PCR and Virion DNA PCR. Analysis of mature miRNA expression was Materials and Methods performed using miRNA-specific qRT-PCR as described previously (10). Gene expression was assessed by qRT-PCR. The housekeeping gene GAPDH was Cell Lines. BL2 and Akata 2A8 are EBV-negative BL lines. Akata 2A8.1 is an used as an internal control for normalization. For the EBV virion PCR assay, EBV-positive variant of Akata 2A8. BJAB is an EBV-negative B-cell line, B95-8 is real-time DNA PCR for the gp350 gene was used (details are provided in SI an EBV-positive marmoset B-cell line, and B95.8-LCL cells are human pe- Materials and Methods). Absolute virion copy numbers were calculated ripheral B cells transformed with B95.8 virus. based on a standard curve of serial 10-fold dilutions of EBV (B95-8 strain)- quantitated viral DNA (Advanced Biotechnologies). MiRNA Mimics, Inhibitors, and Cell Transfection. Synthetic miRNA mimics (Thermo Scientific) and LNAs (Exqion) were transfected into 1.5–2.5 × 106 Western Blot. Western blot analysis was carried out by standard procedures cells using an Amaxa nucleofector, the Kit V solution, and the program C-009 (details are provided in SI Materials and Methods). (all from Lonza). Luciferase plasmids and pGIZ plasmids were transiently transfected into HEK 392 T cells using Lipofectamine 2000 reagent (Life Ago2 Immunoprecipitation. Ago2 immunoprecipitation was performed using Technologies) according to the manufacturer’s manual. In total, 2 × 106 the Magna RIP Kit (Millipore) according to the manufacturer’s instructions. B95.8-LCL cells were electroporated with 3 μg of pCMV6-MAP3K2 (human origin) and pCMV6-XL5 control plasmids (a gift from Ren Sun, University of Luciferase Reporter Assay. Assays were carried out in triplicate (details are California, Los Angeles, Los Angeles) using the nucleofector. provided in SI Materials and Methods). Luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) according to the Cloning. The lentivirus plasmid pGIZ vector (Thermo Scientific) was used to manufacturer’s instructions. express miRNAs stably (details are provided in SI Materials and Methods). Region 145,921–146,050 on the EBV genome (European Molecular Biology Viral Reactivation and EBV Virion Isolation. B95.8 or B95.8-LCL cells were Laboratory AJ507799.2) encompassing the pri–mir–18-5p sequence was treated in replicate with TPA and BA, and Akata 2A8.1 cells were treated with used. For the NC, the Caenorhabditis elegans miR-67 stem-loop sequence anti-human IgG/IgM antibodies (Jackson ImmunoResearch) (details are was used (mirBase accession ID MI0000038). For luciferase studies, the provided in SI Materials and Methods). MAP3K2 cDNA was isolated from the BL36 cell line and 3′UTR (19), coding region, 5′UTR, and mutated 3′UTR were cloned into the psiCHECK2 dual- ACKNOWLEDGMENTS. We thank Francisco Navarro and Judy Lieberman luciferase vector (Promega) (details are provided in SI Materials and Methods). for assistance with the initial microarray analysis and Dr. Ren Sun for the pCMV6-MAP3K2 and pCMV6-XL5 control plasmids. This work was sup- Microarray Analysis. EBV miRNAs and MC were transiently expressed in the ported by Public Health Service Grants R01 CA65883 and R01 AI18757 EBV-negative B cells BL2 and BJAB, followed by microarray analysis 24 h later (to D.A.T.-L.).

1. Babcock GJ, Decker LL, Volk M, Thorley-Lawson DA (1998) EBV persistence in memory 19. Kim H, et al. (2010) Integrative genome analysis reveals an oncomir/oncogene cluster B cells in vivo. Immunity 9(3):395–404. regulating glioblastoma survivorship. Proc Natl Acad Sci USA 107(5):2183–2188. 2. Thorley-Lawson DA (2001) Epstein-Barr virus: Exploiting the immune system. Nat Rev 20. Ghosh Z, Mallick B, Chakrabarti J (2009) Cellular versus viral microRNAs in host-virus Immunol 1(1):75–82. interaction. Nucleic Acids Res 37(4):1035–1048. 3. Babcock GJ, Decker LL, Freeman RB, Thorley-Lawson DA (1999) Epstein-barr virus- 21. Gottwein E, et al. (2007) A viral microRNA functions as an orthologue of cellular miR- infected resting memory B cells, not proliferating lymphoblasts, accumulate in the 155. Nature 450(7172):1096–1099. peripheral blood of immunosuppressed patients. J Exp Med 190(4):567–576. 22. Kincaid RP, Sullivan CS (2012) Virus-encoded microRNAs: An overview and a look to 4. Kieff E, Rickinson AB (2007) Epstein-Barr virus and its replication. Fields Virology, eds the future. PLoS Pathog 8(12):e1003018. Knipe DM, Howley PM (Lippincott Williams & Wilkins, Philadelphia), 5th Ed, Vol 2, pp 23. Countryman J, Miller G (1985) Activation of expression of latent Epstein-Barr her- 2603–2654. pesvirus after gene transfer with a small cloned subfragment of heterogeneous viral 5. Laichalk LL, Thorley-Lawson DA (2005) Terminal differentiation into plasma cells in- DNA. Proc Natl Acad Sci USA 82(12):4085–4089. itiates the replicative cycle of Epstein-Barr virus in vivo. J Virol 79(2):1296–1307. 24. Schonteich E, et al. (2008) The Rip11/Rab11-FIP5 and kinesin II complex regulates 6. Thorley-Lawson DA, Edson CM (1979) Polypeptides of the Epstein-Barr virus mem- endocytic protein recycling. J Cell Sci 121(Pt 22):3824–3833. brane antigen complex. J Virol 32(2):458–467. 25. Matsunaga K, et al. (2009) Two Beclin 1-binding proteins, Atg14L and Rubicon, re- 7. Thorley-Lawson DA, Geilinger K (1980) Monoclonal antibodies against the major ciprocally regulate autophagy at different stages. Nat Cell Biol 11(4):385–396. glycoprotein (gp350/220) of Epstein-Barr virus neutralize infectivity. Proc Natl Acad Sci 26. Sir D, Ou JH (2010) Autophagy in viral replication and pathogenesis. Mol Cells 29(1):1–7. USA 77(9):5307–5311. 27. Cavignac Y, Esclatine A (2010) Herpesviruses and autophagy: Catch me if you can! 8. Klinke O, Feederle R, Delecluse HJ (2014) Genetics of Epstein-Barr virus microRNAs. Viruses 2(1):314–333. Semin Cancer Biol 26C:52–59. 28. Irie T, Nagata N, Yoshida T, Sakaguchi T (2008) Recruitment of Alix/AIP1 to the plasma 9. Kempkes B, Pich D, Zeidler R, Sugden B, Hammerschmidt W (1995) Immortalization of membrane by Sendai virus C protein facilitates budding of virus-like particles. Virol- human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein-Barr virus ogy 371(1):108–120. DNA. J Virol 69(1):231–238. 29. Strack B, Calistri A, Craig S, Popova E, Göttlinger HG (2003) AIP1/ALIX is a binding 10. Qiu J, et al. (2011) A novel persistence associated EBV miRNA expression profile is partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114(6):689–699. disrupted in neoplasia. PLoS Pathog 7(8):e1002193. 30. Shtanko O, Watanabe S, Jasenosky LD, Watanabe T, Kawaoka Y (2011) ALIX/AIP1 is 11. Li X, et al. (2010) Tpl2/AP-1 enhances murine gammaherpesvirus 68 lytic replication. required for NP incorporation into Mopeia virus Z-induced virus-like particles. J Virol J Virol 84(4):1881–1890. 85(7):3631–3641. 12. Pan H, Xie J, Ye F, Gao SJ (2006) Modulation of Kaposi’s sarcoma-associated herpes- 31. Watanabe T, et al. (2007) Involvement of host cellular multivesicular body functions virus infection and replication by MEK/ERK, JNK, and p38 multiple mitogen-activated in hepatitis B virus budding. Proc Natl Acad Sci USA 104(24):10205–10210. protein kinase pathways during primary infection. J Virol 80(11):5371–5382. 32. Cristea IM, et al. (2010) Host factors associated with the Sindbis virus RNA-dependent 13. Adamson AL, et al. (2000) Epstein-Barr virus immediate-early proteins BZLF1 and RNA polymerase: Role for G3BP1 and G3BP2 in virus replication. JVirol84(13):6720–6732. BRLF1 activate the ATF2 transcription factor by increasing the levels of phosphory- 33. Blackford AN, et al. (2008) A role for E1B-AP5 in ATR signaling pathways during lated p38 and c-Jun N-terminal kinases. J Virol 74(3):1224–1233. adenovirus infection. J Virol 82(15):7640–7652. 14. SatohT,HoshikawaY,SatohY,KurataT,Sairenji T (1999) The interaction of mitogen-activated 34. Rickinson AB, Kieff E (2007) Epstein-Barr virus. Virology, eds Knipe DM, Howley PM protein kinases to Epstein-Barr virus activation in Akata cells. Virus Genes 18(1):57–64. (Lippincott Williams & Wilkins, New York), 5th Ed, Vol 2, pp 2655–2700. 15. Xie J, Ajibade AO, Ye F, Kuhne K, Gao SJ (2008) Reactivation of Kaposi’s sarcoma- 35. Hochberg D, et al. (2004) Demonstration of the Burkitt’s lymphoma Epstein-Barr virus associated herpesvirus from latency requires MEK/ERK, JNK and p38 multiple mitogen- phenotype in dividing latently infected memory cells in vivo. Proc Natl Acad Sci USA activated protein kinase pathways. Virology 371(1):139–154. 101(1):239–244. 16. Stahl JA, et al. (2013) Phosphoproteomic analyses reveal signaling pathways that 36. Zuo J, et al. (2011) The Epstein-Barr virus-encoded BILF1 protein modulates immune facilitate lytic gammaherpesvirus replication. PLoS Pathog 9(9):e1003583. recognition of endogenously processed antigen by targeting major histocompatibility 17. Yuan J, Cahir-McFarland E, Zhao B, Kieff E (2006) Virus and cell RNAs expressed during complex class I molecules trafficking on both the exocytic and endocytic pathways. Epstein-Barr virus replication. J Virol 80(5):2548–2565. J Virol 85(4):1604–1614. 18. Gao X, Ikuta K, Tajima M, Sairenji T (2001) 12-O-tetradecanoylphorbol-13-acetate 37. Decker LL, Klaman LD, Thorley-Lawson DA (1996) Detection of the latent form of induces Epstein-Barr virus reactivation via NF-kappaB and AP-1 as regulated by pro- Epstein-Barr virus DNA in the peripheral blood of healthy individuals. J Virol 70(5): tein kinase C and mitogen-activated protein kinase. Virology 286(1):91–99. 3286–3289.

11162 | www.pnas.org/cgi/doi/10.1073/pnas.1406136111 Qiu and Thorley-Lawson Downloaded by guest on September 30, 2021