Suppression of cGAS- and RIG-I-mediated innate immune signaling by Epstein-Barr virus deubiquitinase BPLF1.

Epstein-Barr virus (EBV) has developed effective strategies to evade host innate immune responses. Here we reported on mitigation of type I interferon (IFN) production by EBV deubiquitinase (DUB) BPLF1 through cGAS-STING and RIG-I-MAVS pathways. The two naturally occurring forms of BPLF1 exerted potent suppressive effect on cGAS-STING-, RIG-I- and TBK1-induced IFN production. The observed suppression was reversed when DUB domain of BPLF1 was rendered catalytically inactive. The DUB activity of BPLF1 also facilitated EBV infection by counteracting cGAS-STING- and TBK1-mediated antiviral defense. BPLF1 associated with STING to act as an effective DUB targeting its K63-, K48- and K27-linked ubiquitin moieties. BPLF1 also catalyzed removal of K63- and K48-linked ubiquitin chains on TBK1 kinase. The DUB activity of BPLF1 was required for its suppression of TBK1-induced IRF3 dimerization. Importantly, in cells stably carrying EBV genome that encodes a catalytically inactive BPLF1, the virus failed to suppress type I IFN production upon activation of cGAS and STING. This study demonstrated IFN antagonism of BPLF1 mediated through DUB-dependent deubiquitination of STING and TBK1 leading to suppression of cGAS-STING and RIG-I-MAVS signaling.

Suppression of JAK-STAT Signaling by Epstein-Barr Virus Tegument Protein BGLF2 through Recruitment of SHP1 Phosphatase and Promotion of STAT2 Degradation.

Some lytic proteins encoded by Epstein-Barr virus (EBV) suppress host interferon (IFN) signaling to facilitate viral replication. In this study, we sought to identify and characterize EBV proteins antagonizing IFN signaling. The induction of IFN-stimulated genes (ISGs) by IFN-ß was effectively suppressed by EBV. A functional screen was therefore performed to identify IFN-antagonizing proteins encoded by EBV. EBV tegument protein BGLF2 was identified as a potent suppressor of JAK-STAT signaling. This activity was found to be independent of its stimulatory effect on p38 and JNK pathways. Association of BGLF2 with STAT2 resulted in more pronounced K48-linked polyubiquitination and proteasomal degradation of the latter. Mechanistically, BGLF2 promoted the recruitment of SHP1 phosphatase to STAT1 to inhibit its tyrosine phosphorylation. In addition, BGLF2 associated with cullin 1 E3 ubiquitin ligase to facilitate its recruitment to STAT2. Consequently, BGLF2 suppressed ISG induction by IFN-ß. Furthermore, BGLF2 also suppressed type II and type III IFN signaling, although the suppressive effect on type II IFN response was milder. When pretreated with IFN-ß, host cells became less susceptible to primary infection of EBV. This phenotype was reversed when expression of BGLF2 was enforced. Finally, genetic disruption of BGLF2 in EBV led to more pronounced induction of ISGs. Our study unveils the roles of BGLF2 not only in the subversion of innate IFN response but also in lytic infection and reactivation of EBV. IMPORTANCE Epstein-Barr virus (EBV) is an oncogenic virus associated with the development of lymphoid and epithelial malignancies. EBV has to subvert interferon-mediated host antiviral response to replicate and cause diseases. It is therefore of great interest to identify and characterize interferon-antagonizing proteins produced by EBV. In this study, we perform a screen to search for EBV proteins that suppress the action of interferons. We further show that BGLF2 protein of EBV is particularly strong in this suppression. This is achieved by inhibiting two key proteins STAT1 and STAT2 that mediate the antiviral activity of interferons. BGLF2 recruits a host enzyme to remove the phosphate group from STAT1 thereby inactivating its activity. BGLF2 also redirects STAT2 for degradation. A recombinant virus in which BGLF2 gene has been disrupted can activate host interferon response more robustly. Our findings reveal a novel mechanism by which EBV BGLF2 protein suppresses interferon signaling.

Epigenetic silencing of miR-342-3p in B cell lymphoma and its impact on autophagy.

BACKGROUND: miR-342-3p, localized to 14q32, is a tumor suppressor miRNA implicated in carcinogenesis. Given the presence of a promotor-associated CpG island for its host gene, EVL, we hypothesized that intronic miR-342-3p is a tumor suppressor co-regulated with host gene by promoter DNA methylation in B cell lymphoma. RESULTS: By bisulfite pyrosequencing-verified methylation-specific PCR (MSP), EVL/MIR342 methylation was detected in five (50%) lymphoma cell lines but not normal peripheral blood and tonsils. EVL/MIR342 methylation correlated with repression of both miR-342-3p and EVL in cell lines. In completely methylated SU-DHL-16 cells, 5-AzadC treatment resulted in promoter demethylation and re-expression of miR-342-3p and EVL. In 132 primary lymphoma samples, EVL/MIR342 was preferentially methylated in B cell lymphomas (N = 68; 68.7%) than T cell lymphoma (N = 8; 24.2%) by MSP (P < 0.0001). Moreover, EVL/MIR342 methylation was associated with lower miR-342-3p expression in 79 primary NHL (P = 0.0443). In SU-DHL-16 cells, the tumor suppressor function of miR-342-3p was demonstrated by the inhibition of cellular proliferation and increase of cell death upon over-expression of miR-342-3p. Mechanistically, overexpression of miR-342-3p resulted in a decrease of LC3-II, a biomarker of autophagy, which was pro-survival for SU-DHL-16. Pre-treatment with 3-methyladenine, an autophagy inhibitor, abrogated tumor suppression associated with miR-342-3p overexpression. By luciferase assay, MAP1LC3B, a precursor of LC3-II, was confirmed as a direct target of miR-342-3p. Finally, in SU-DHL-16 cells, overexpression of miR-342-3p downregulated the known target DNMT1, with promoter demethylation and re-expression of tumor suppressor E-cadherin. CONCLUSIONS: Intronic miR-342-3p is co-regulated with its host gene EVL by tumor-specific promoter DNA methylation in B cell lymphoma. The tumor suppressor function of miR-342-3p was mediated via inhibition of pro-survival autophagy by targeting MAP1LC3B and downregulation of DNMT1 with demethylation and re-expression of tumor suppressor genes.

Epstein-Barr Virus and Innate Immunity: Friends or Foes?

Epstein-Barr virus (EBV) successfully persists in the vast majority of adults but causes lymphoid and epithelial malignancies in a small fraction of latently infected individuals. Innate immunity is the first-line antiviral defense, which EBV has to evade in favor of its own replication and infection. EBV uses multiple strategies to perturb innate immune signaling pathways activated by Toll-like, RIG-I-like, NOD-like, and AIM2-like receptors as well as cyclic GMP-AMP synthase. EBV also counteracts interferon production and signaling, including TBK1-IRF3 and JAK-STAT pathways. However, activation of innate immunity also triggers pro-inflammatory response and proteolytic cleavage of caspases, both of which exhibit proviral activity under some circumstances. Pathogenic inflammation also contributes to EBV oncogenesis. EBV activates NFκB signaling and induces pro-inflammatory cytokines. Through differential modulation of the proviral and antiviral roles of caspases and other host factors at different stages of infection, EBV usurps cellular programs for death and inflammation to its own benefits. The outcome of EBV infection is governed by a delicate interplay between innate immunity and EBV. A better understanding of this interplay will instruct prevention and intervention of EBV-associated cancers.

Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is capable of inducing a storm of proinflammatory cytokines. In this study, we show that the SARS-CoV open reading frame 3a (ORF3a) accessory protein activates the NLRP3 inflammasome by promoting TNF receptor-associated factor 3 (TRAF3)-mediated ubiquitination of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). SARS-CoV and its ORF3a protein were found to be potent activators of pro-IL-1ß gene transcription and protein maturation, the 2 signals required for activation of the NLRP3 inflammasome. ORF3a induced pro-IL-1ß transcription through activation of NF-κB, which was mediated by TRAF3-dependent ubiquitination and processing of p105. ORF3a-induced elevation of IL-1ß secretion was independent of its ion channel activity or absent in melanoma 2 but required NLRP3, ASC, and TRAF3. ORF3a interacted with TRAF3 and ASC, colocalized with them in discrete punctate structures in the cytoplasm, and facilitated ASC speck formation. TRAF3-dependent K63-linked ubiquitination of ASC was more pronounced in SARS-CoV-infected cells or when ORF3a was expressed. Taken together, our findings reveal a new mechanism by which SARS-CoV ORF3a protein activates NF-κB and the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of p105 and ASC.-Siu, K.-L., Yuen, K.-S., Castaño-Rodriguez, C., Ye, Z.-W., Yeung, M.-L., Fung, S.-Y., Yuan, S., Chan, C.-P., Yuen, K.-Y., Enjuanes, L., Jin, D.-Y. Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC.

A novel transcript isoform of STING that sequesters cGAMP and dominantly inhibits innate nucleic acid sensing.

STING is a core adaptor in innate nucleic acid sensing in mammalian cells, on which different sensing pathways converge to induce type I interferon (IFN) production. Particularly, STING is activated by 2'3'-cGAMP, a cyclic dinucleotide containing mixed phosphodiester linkages and produced by cytoplasmic DNA sensor cGAS. Here, we reported on a novel transcript isoform of STING designated STING-ß that dominantly inhibits innate nucleic acid sensing. STING-ß without transmembrane domains was widely expressed at low levels in various human tissues and viral induction of STING-ß correlated inversely with IFN-ß production. The expression of STING-ß declined in patients with lupus, in which type I IFNs are commonly overproduced. STING-ß suppressed the induction of IFNs, IFN-stimulated genes and other cytokines by various immunostimulatory agents including cyclic dinucleotides, DNA, RNA and viruses, whereas depletion of STING-ß showed the opposite effect. STING-ß interacted with STING-α and antagonized its antiviral function. STING-ß also interacted with TBK1 and prevented it from binding with STING-α, TRIF or other transducers. In addition, STING-ß bound to 2'3'-cGAMP and impeded its binding with and activation of STING-α, leading to suppression of IFN-ß production. Taken together, STING-ß sequesters 2'3'-cGAMP second messenger and other transducer molecules to inhibit innate nucleic acid sensing dominantly.

Suppression of Epstein-Barr virus DNA load in latently infected nasopharyngeal carcinoma cells by CRISPR/Cas9.

Epstein-Barr virus (EBV) infects more than 90% of the world's adult population. Once established, latent infection of nasopharyngeal epithelial cells with EBV is difficult to eradicate and might lead to the development of nasopharyngeal carcinoma (NPC) in a small subset of individuals. In this study we explored the anti-EBV potential of CRISPR/Cas9 targeting of EBV genome in infected NPC cells. We designed gRNAs to target different regions of the EBV genome and transfected them into C666-1 cells. The levels of EBV DNA in transfected cells were decreased by about 50%. The suppressive effect on EBV DNA load lasted for weeks but could not be further enhanced by re-transfection of gRNA. Suppression of EBV by CRISPR/Cas9 did not affect survival of C666-1 cells but sensitized them to chemotherapeutic killing by cisplatin and 5-fluorouracil. Our work provides the proof-of-principle for suppressing EBV DNA load with CRISPR/Cas9 and a potential new strategy to sensitize EBV-infected NPC cells to chemotherapy.

Mutagenesis and Genome Engineering of Epstein-Barr Virus in Cultured Human Cells by CRISPR/Cas9.

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 nuclease (Cas9) system is a powerful genome-editing tool for both chromosomal and extrachromosomal DNA. DNA viruses such as Epstein-Barr virus (EBV), which undergoes episomal replication in human cells, can be effectively edited by CRISPR/Cas9. We have demonstrated targeted editing of the EBV genome by CRISPR/Cas9 in several lines of EBV-infected cells. CRISPR/Cas9-based mutagenesis and genome engineering of EBV provides a new method for genetic analysis, which has some advantages over bacterial artificial chromosome-based recombineering. This approach might also prove useful in the cure of EBV infection. In this chapter, we use the knockout of the BART promoter as an example to detail the experimental procedures for construction of recombinant EBV in human cells.

Middle East respiratory syndrome coronavirus M protein suppresses type I interferon expression through the inhibition of TBK1-dependent phosphorylation of IRF3.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection has claimed hundreds of lives and has become a global threat since its emergence in Saudi Arabia in 2012. The ability of MERS-CoV to evade the host innate antiviral response may contribute to its severe pathogenesis. Many MERS-CoV-encoded proteins were identified to have interferon (IFN)-antagonizing properties, which correlates well with the reduced IFN levels observed in infected patients and ex vivo models. In this study, we fully characterized the IFN-antagonizing property of the MERS-CoV M protein. Expression of MERS-CoV M protein suppressed type I IFN expression in response to Sendai virus infection or poly(I:C) induction. This suppressive effect was found to be specific for the activation of IFN regulatory factor 3 (IRF3) but not nuclear factor-κB. MERS-CoV M protein interacted with TRAF3 and disrupted TRAF3-TBK1 association leading to reduced IRF3 activation. M proteins from MERS-CoV and SARS-CoV have three highly similar conserved N-terminal transmembrane domains and a C-terminal region. Using chimeric and truncation mutants, the N-terminal transmembrane domains of the MERS-CoV M protein were found to be sufficient for its inhibitory effect on IFN expression, whereas the C-terminal domain was unable to induce this suppression. Collectively, our findings suggest a common and conserved mechanism through which highly pathogenic MERS-CoV and SARS-CoV harness their M proteins to suppress type I IFN expression at the level of TBK1-dependent phosphorylation and activation of IRF3 resulting in evasion of the host innate antiviral response.

Suppression of Type I Interferon Production by Human T-Cell Leukemia Virus Type 1 Oncoprotein Tax through Inhibition of IRF3 Phosphorylation.

UNLABELLED: Infection with human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and tropical spastic paraparesis. Type I interferons (IFNs) are key effectors of the innate antiviral response, and IFN-α combined with the nucleoside reverse transcriptase inhibitor zidovudine is considered the standard first-line therapy for ATL. HTLV-1 oncoprotein Tax is known to suppress innate IFN production and response but the underlying mechanisms remain to be fully established. In this study, we report on the suppression of type I IFN production by HTLV-1 Tax through interaction with and inhibition of TBK1 kinase that phosphorylates IRF3. Induced transcription of IFN-ß was severely impaired in HTLV-1-transformed ATL cells and freshly infected T lymphocytes. The ability to suppress IRF3 activation was ascribed to Tax. The expression of Tax alone sufficiently repressed the induction of IFN production by RIG-I plus PACT, cGAMP synthase plus STING, TBK1, IKKε, IRF3, and IRF7, but not by IRF3-5D, a dominant-active phosphomimetic mutant. This suggests that Tax perturbs IFN production at the step of IRF3 phosphorylation. Tax mutants deficient for CREB or NF-κB activation were fully competent in the suppression of IFN production. Coimmunoprecipitation experiments confirmed the association of Tax with TBK1, IKKε, STING, and IRF3.In vitrokinase assay indicated an inhibitory effect of Tax on TBK1-mediated phosphorylation of IRF3. Taken together, our findings suggested a new mechanism by which HTLV-1 oncoprotein Tax circumvents the production of type I IFNs in infected cells. Our findings have implications in therapeutic intervention of ATL. IMPORTANCE: Human T-cell leukemia virus type 1 (HTLV-1) is the cause of adult T-cell leukemia (ATL), an aggressive and fatal blood cancer, as well as another chronic disabling disease of the spinal cord. Treatments are unsatisfactory, and options are limited. A combination of antiviral cellular protein alpha interferon and zidovudine, which is an inhibitor of a viral enzyme called reverse transcriptase, has been recommended as the standard first-line therapy for ATL. Exactly how HTLV-1 interacts with the cellular machinery for interferon production and action is not well understood. Our work sheds light on the mechanism of action for the inhibition of interferon production by an HTLV-1 oncogenic protein called Tax. Our findings might help to improve interferon-based anti-HTLV-1 and anti-ATL therapy.

SIRT1 Suppresses Human T-Cell Leukemia Virus Type 1 Transcription.

UNLABELLED: Human T-cell leukemia virus type 1 (HTLV-1)-associated diseases are poorly treatable, and HTLV-1 vaccines are not available. High proviral load is one major risk factor for disease development. HTLV-1 encodes Tax oncoprotein, which activates transcription from viral long terminal repeats (LTR) and various types of cellular promoters. Counteracting Tax function might have prophylactic and therapeutic benefits. In this work, we report on the suppression of Tax activation of HTLV-1 LTR by SIRT1 deacetylase. The transcriptional activity of Tax on the LTR was largely ablated when SIRT1 was overexpressed, but Tax activation of NF-κB was unaffected. On the contrary, the activation of the LTR by Tax was boosted when SIRT1 was depleted. Treatment of cells with resveratrol shunted Tax activity in a SIRT1-dependent manner. The activation of SIRT1 in HTLV-1-transformed T cells by resveratrol potently inhibited HTLV-1 proviral transcription and Tax expression, whereas compromising SIRT1 by specific inhibitors augmented HTLV-1 mRNA expression. The administration of resveratrol also decreased the production of cell-free HTLV-1 virions from MT2 cells and the transmission of HTLV-1 from MT2 cells to uninfected Jurkat cells in coculture. SIRT1 associated with Tax in HTLV-1-transformed T cells. Treatment with resveratrol prevented the interaction of Tax with CREB and the recruitment of CREB, CRTC1, and p300 to Tax-responsive elements in the LTR. Our work demonstrates the negative regulatory function of SIRT1 in Tax activation of HTLV-1 transcription. Small-molecule activators of SIRT1 such as resveratrol might be considered new prophylactic and therapeutic agents in HTLV-1-associated diseases. IMPORTANCE: Human T-cell leukemia virus type 1 (HTLV-1) causes a highly lethal blood cancer or a chronic debilitating disease of the spinal cord. Treatments are unsatisfactory, and vaccines are not available. Disease progression is associated with robust expression of HTLV-1 genes. Suppressing HTLV-1 gene expression might have preventive and therapeutic benefits. It is therefore critical that host factors controlling HTLV-1 gene expression be identified and characterized. This work reveals a new host factor that suppresses HTLV-1 gene expression and a natural compound that activates this suppression. Our findings not only provide new knowledge of the host control of HTLV-1 gene expression but also suggest a new strategy of using natural compounds for prevention and treatment of HTLV-1-associated diseases.

CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

Requirement of CRTC1 coactivator for hepatitis B virus transcription.

Transcription of hepatitis B virus (HBV) from the covalently closed circular DNA (cccDNA) template is essential for its replication. Suppressing the level and transcriptional activity of cccDNA might have anti-HBV effect. Although cellular transcription factors, such as CREB, which mediate HBV transcription, have been well described, transcriptional coactivators that facilitate this process are incompletely understood. In this study we showed that CREB-regulated transcriptional coactivator 1 (CRTC1) is required for HBV transcription and replication. The steady-state levels of CRTC1 protein were elevated in HBV-positive hepatoma cells and liver tissues. Ectopic expression of CRTC1 or its homolog CRTC2 or CRTC3 in hepatoma cells stimulated the activity of the preS2/S promoter of HBV, whereas overexpression of a dominant inactive form of CRTC1 inhibited HBV transcription. CRTC1 interacts with CREB and they are mutually required for the recruitment to the preS2/S promoter on cccDNA and for the activation of HBV transcription. Accumulation of pregenomic RNA (pgRNA) and cccDNA was observed when CRTC1 or its homologs were overexpressed, whereas the levels of pgRNA, cccDNA and secreted HBsAg were diminished when CRTC1 was compromised. In addition, HBV transactivator protein HBx stabilized CRTC1 and promoted its activity on HBV transcription. Our work reveals an essential role of CRTC1 coactivator in facilitating and supporting HBV transcription and replication.

Middle east respiratory syndrome coronavirus 4a protein is a double-stranded RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in the innate antiviral response.

UNLABELLED: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes severe disease in human. MERS-CoV is closely related to bat coronaviruses HKU4 and HKU5. Evasion of the innate antiviral response might contribute significantly to MERS-CoV pathogenesis, but the mechanism is poorly understood. In this study, we characterized MERS-CoV 4a protein as a novel immunosuppressive factor that antagonizes type I interferon production. MERS-CoV 4a protein contains a double-stranded RNA-binding domain capable of interacting with poly(I · C). Expression of MERS-CoV 4a protein suppressed the interferon production induced by poly(I · C) or Sendai virus. RNA binding of MERS-CoV 4a protein was required for IFN antagonism, a property shared by 4a protein of bat coronavirus HKU5 but not by the counterpart in bat coronavirus HKU4. MERS-CoV 4a protein interacted with PACT in an RNA-dependent manner but not with RIG-I or MDA5. It inhibited PACT-induced activation of RIG-I and MDA5 but did not affect the activity of downstream effectors such as RIG-I, MDA5, MAVS, TBK1, and IRF3. Taken together, our findings suggest a new mechanism through which MERS-CoV employs a viral double-stranded RNA-binding protein to circumvent the innate antiviral response by perturbing the function of cellular double-stranded RNA-binding protein PACT. PACT targeting might be a common strategy used by different viruses, including Ebola virus and herpes simplex virus 1, to counteract innate immunity. IMPORTANCE: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging and highly lethal human pathogen. Why MERS-CoV causes severe disease in human is unclear, and one possibility is that MERS-CoV is particularly efficient in counteracting host immunity, including the sensing of virus invasion. It will therefore be critical to clarify how MERS-CoV cripples the host proteins that sense viruses and to compare MERS-CoV with its ancestral viruses in bats in the counteraction of virus sensing. This work not only provides a new understanding of the abilities of MERS-CoV and closely related bat viruses to subvert virus sensing but also might prove useful in revealing new strategies for the development of vaccines and antivirals.

Perturbation of biogenesis and targeting of Epstein-Barr virus-encoded miR-BART3 microRNA by adenosine-to-inosine editing.

Epstein-Barr virus (EBV) encodes at least 44 mature microRNAs (miRNAs), some of which are abundantly expressed in nasopharyngeal carcinoma cells. EBV-encoded miR-BART6 miRNA is known to undergo adenosine-to-inosine (A-to-I) RNA editing, which impacts on processing and function. Whether additional EBV miRNAs might be A-to-I edited remains to be determined. In this study, we have reported on A-to-I editing of EBV miR-BART3. The A-to-I editing enzyme was expressed abundantly in EBV-infected epithelial carcinoma cells. pri-miR-BART3 was found to be edited at four sites in these cells and in nasopharyngeal carcinoma samples. Whereas editing of the second site located within the seed region prevented the targeting of DICE1 mRNA, editing of the third site effectively crippled the biogenesis of mature miR-BART3. Thus, A-to-I editing perturbs biogenesis and targeting of miR-BART3 and may contribute to its differential expression and function in EBV-infected epithelial cells.

Targeting of DICE1 tumor suppressor by Epstein-Barr virus-encoded miR-BART3* microRNA in nasopharyngeal carcinoma.

Latent infection with Epstein-Barr virus (EBV) is associated with several types of malignancies including nasopharyngeal carcinoma (NPC), which is particularly more prevalent in Southern China. EBV expresses at least 44 mature microRNAs (miRNAs) to modulate the activity of viral and cellular RNAs, but the targets of these EBV-encoded miRNAs in NPC are not well understood. In this report, we characterized DICE1 tumor suppressor to be a cellular target of EBV miR-BART3* miRNA. miR-BART3* was abundantly expressed in NPC cells. The target site of miR-BART3* located in the 3'-untranslated region of DICE1 transcript was identified and characterized. Enforced expression of miR-BART3* or its precursor pre-miR-BART3 led to down-regulation of endogenous DICE1 expression. Inhibition of endogenous miR-BART3* in NPC cells with anti-miR-BART3* oligonucleotide inhibitor resulted in increased expression of DICE1 protein. On the contrary, expression of miR-BART3* overcame the growth suppressive activity of DICE1 and stimulated cell proliferation. Consistent with its tumor suppressive function, DICE1 was underexpressed in EBV-expressing NPC tumor tissues. Taken together, our findings suggest that EBV encoded miR-BART3* miRNA targets DICE1 tumor suppressor to promote cellular growth and transformation in NPC.