EBV-Encoded MicroRNA-BART17-3p Targets DDX3X and Promotes EBV Infection in EBV-Associated T/Natural Killer-Cell Lymphoproliferative Diseases

Abstract

Background: Epstein-Barr virus (EBV) persistently infects T/natural killer (NK) cells causing an array of refractory EBV-associated T/NK-cell lymphoproliferative disorders. EBV-encoded microRNAs are important regulators for EBV latent infection and tumorigenesis. However, the roles of most EBV microRNAs in EBV-infected T/NK cells remain poorly understood.

Methods: On the basis of a search of the doRiNA database and the BiBiServ2-RNAhybrid website, we predicted that EBV-miR-BART17-3p targeted DDX3X, and we verified the hypothesis by dual-luciferase reporter assay and cell function experiments. In addition, we collected 50 EBV-positive T-, B-, and NK-cell samples from the peripheral blood of EBV-positive cases to examine the role of EBV-miR-BART17-3p in the disease.

Results: We found that EBV-miR-BART17-3p directly targeted DDX3X and downregulated DDX3X expression. By analyzing EBV-positive cell samples from cell lines and patients, we found that EBV-miR-BART17-3p was highly expressed only in EBV-positive NK cells and that the overexpression was significantly related to high EBV loads in EBV-infected NK cells. Furthermore, we found that EBV-miR-BART17-3p downregulated the RIG-I-like receptor antiviral pathway and promoted the expression of EBV-encoded proteins in EBV-infected NK cells by targeting DDX3X.

Conclusions: Our study showed that EBV-miR-BART17-3p was abundantly expressed in EBV-infected NK cells and inhibited the important antivirus immune responses of hosts by targeting DDX3X of the RIG-I-like receptor pathway. These findings could help us gain insights into the pathogenic mechanisms underlying EBV-associated T/NK-cell lymphoproliferative disorders and find the potential therapeutic target.

Keywords: DDX3X; EBV-associated T/NK-cell lymphoproliferative disorders; EBV-miR-BART17-3p; RIG-I–like receptor pathway.

Figure 1.

Epstein-Barr virus miRNAs might regulate DDX3X expression. A, Relative expression of DDX3X was measured in YT cells overexpressing DDX3X induced 2, 3, and 5 weeks after viral transfection. B, The protein expression levels of DDX3X and flag tag were detected in YT cells. C, The mRNA expression of DDX3X was measured in HEK293T cells. D, The protein expression levels of DDX3X and flag tag were detected in HEK293T cells. Statistical analysis was performed with an unpaired t test or analysis of variance. **P < .01. ***P < .001. ****P < .0001. Data are presented as mean ± SD.

Figure 2.

EBV-miR-BART17-3p directly targeted DDX3X. A, Matching results between the DDX3X 3′-UTR region and the EBV-miR-BART17-3p sequence, including the wild type and mutant type of the matching sequence. B, Negative control (DDX3X-NCs), wild type (DDX3X-WT), or mutated (DDX3X-MUT) plasmids were cotransfected with EBV-miR-BART17-3p mimics or miR-NCs into HEK293T cells. The luciferase activities were measured for 24 hours. Statistical analysis was performed with analysis of variance. NS, not significant (P > .05). **P < .01. C, The relative expression levels of EBV-miR-BART17-3p were measured in B-cell lines (NCI-BL2009 and Raji) and NK-cell lines (KAI3, SNK-6, IMC-1, NKYS, YT, and NK92). D, The protein expression level of DDX3X was detected in B-cell lines (NCI-BL2009 and Raji) and NK-cell lines (KAI3, SNK-6, IMC-1, NKYS, YT, and NK92). E, Comparison of DDX3X expression levels among NK-cell lines (NKYS, IMC-1, KAI3) transfected with EBV-miR-BART17-3p negative controls, inhibitors, or mimics. Data are presented as mean ± SD. EBV, Epstein-Barr virus; MUT, mutated; NC, negative control; NK, natural killer; WT, wild type.

Figure 3.

EBV-miR-BART17-3p promoted EBV infection in EBV + NK cells by modulating the expression of DDX3X. A, Comparison of EBV loads between NK-cell samples and T/B-cell samples from patients. B, Comparison of the relative expression levels of EBV-miR-BART17-3p in B-, NK-, and T-cell samples from patients with EBV loads between 10⁶ and 10⁷ copies/2 × 10⁵ cells. C, Analysis of the correlation between EBV-miR-BART17-3p expression and DDX3X levels in NK-cell samples (r = −0.6961, P = .0005). D, Analysis of the correlation between EBV-miR-BART17-3p expression and EBV loads in NK-cell samples (r = 0.5162, P = .0083). Statistical analysis was performed with the Mann-Whitney test. Correlation was evaluated in terms of the Spearman correlation coefficient. NS, not significant (P > .05). *P < .05. ****P < .0001. Data are presented as median (line), IQR (box), and range (error bars). EBV, Epstein-Barr virus; NK, natural killer.

Figure 4.

EBV-miR-BART17-3p downregulated the RLR pathway and promoted EBV gene expression. A and B, The expression levels of EBV-miR-BART17-3p and genes in the RLR pathway, including IRF3, IRF7, IFNB, RIG-1, and TRAF3, in 18 NK-cell samples from patients. C, The protein expression levels of DDX3X and representative EBV lytic/latent proteins (BZLF1, EBNA1, LMP1) were determined in NKYS cells transfected with EBV-miR-BART17-3p negative controls and mimics by Western blotting. D, The transcriptional levels of EBV latent genes (LMP1, LMP2A, EBNA1), EBV immediate early lytic genes (BZLF1, BRLF1), EBV early lytic genes (BMRF, BGLF5), and late lytic genes (BILF1, GP350) were determined in NKYS cells transfected with EBV-miR-BART17-3p negative controls and mimics by real-time polymerase chain reaction. Statistical analysis was performed with analysis of variance. Correlation was evaluated in terms of the Spearman correlation coefficient. NS, not significant (P > .05). ****P < .0001. Data are presented as mean ± SD. EBV, Epstein-Barr virus; NC, negative control; NK, natural killer; RLR, RIG-I–like receptor.

Figure 5.

The proposed mechanism of persistent EBV infection in NK cells. Normally, RLR family members RIG-1 and MDA5, supported by LGP2, are able to sense the viral RNAs once NK cells are infected by EBV. After activation, RLRs interact with MAVS to recruit downstream signaling molecules (IKKε, TBK1, and IKKα/β) and activate IRF-3/7, which leads to IFN secretion, thereby enhancing the antiviral immune response. Defects in the host DDX3X gene negatively affect multiple molecules of RLR signaling (blue background), including its interactions with MAVS and TBK1, thus downregulating the secretion of IFNs. In addition, EBV can further inhibit the RLR antiviral pathway by expressing EBV-miR-BART17-3p targeting DDX3X (pink background). In conclusion, the genetic defects and viral factors in the RLR pathway might synergistically result in impaired immune function and persistent EBV infection in NK cells. EBV, Epstein-Barr virus; IFN, interferon; IKK, inhibitor of NF-κB kinase; IRF3/7, IFN regulatory factor 3/7; LGP2, laboratory of genetics and physiology 2; MAVS, mitochondrial antiviral signaling; MDA-5 (also IFIH1), melanoma differentiation–associated gene 5; NK, natural killer; RIG-1, retinoic acid–inducible gene 1; RLR, RIG-I–like receptors; TBK, TANK-binding kinase; TRAF3/6, TNF receptor–associated factor 3/6.