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• W3136266807 abstract "N6-methyladenosine (m6A) is among the most abundant mRNA modifications, particularly in eukaryotes, and is found in mammals, plants, and even some viruses. Although essential for the regulation of many biological processes, the exact role of m6A modification in virus–host interaction remains largely unknown. Here, using m6A -immunoprecipitation and sequencing, we find that Epstein–Barr virus (EBV) infection decreases the m6A modification of transcriptional factor KLF4 mRNA and subsequently increases its protein level. Mechanistically, EBV immediate-early protein BZLF1 interacts with the promoter of m6A methyltransferase METTL3, inhibiting its expression. Subsequently, the decrease of METTL3 reduces the level of KLF4 mRNA m6A modification, preventing its decay by the m6A reader protein YTHDF2. As a result, KLF4 protein level is upregulated and, in turn, promotes EBV infection of nasopharyngeal epithelial cells. Thus, our results suggest the existence of a positive feedback loop formed between EBV and host molecules via cellular mRNA m6A levels, and this feedback loop acts to facilitate viral infection. This mechanism contains multiple potential targets for controlling viral infectious diseases. N6-methyladenosine (m6A) is among the most abundant mRNA modifications, particularly in eukaryotes, and is found in mammals, plants, and even some viruses. Although essential for the regulation of many biological processes, the exact role of m6A modification in virus–host interaction remains largely unknown. Here, using m6A -immunoprecipitation and sequencing, we find that Epstein–Barr virus (EBV) infection decreases the m6A modification of transcriptional factor KLF4 mRNA and subsequently increases its protein level. Mechanistically, EBV immediate-early protein BZLF1 interacts with the promoter of m6A methyltransferase METTL3, inhibiting its expression. Subsequently, the decrease of METTL3 reduces the level of KLF4 mRNA m6A modification, preventing its decay by the m6A reader protein YTHDF2. As a result, KLF4 protein level is upregulated and, in turn, promotes EBV infection of nasopharyngeal epithelial cells. Thus, our results suggest the existence of a positive feedback loop formed between EBV and host molecules via cellular mRNA m6A levels, and this feedback loop acts to facilitate viral infection. This mechanism contains multiple potential targets for controlling viral infectious diseases. Epstein–Barr virus (EBV), a large double-stranded DNA virus belonging to the γ-herpesvirus subfamily, causes latent infection of more than 95% of the adult population worldwide (1Farrell P.J. Epstein-Barr virus and cancer.Annu. Rev. Pathol. 2019; 14: 29-53Crossref PubMed Scopus (163) Google Scholar, 2Young L.S. Yap L.F. Murray P.G. Epstein-Barr virus: More than 50 years old and still providing surprises.Nat. Rev. Cancer. 2016; 16: 789-802Crossref PubMed Scopus (369) Google Scholar). As a widespread virus causing infectious mononucleosis during primary infection, EBV is also closely implicated in several malignancies in latent infection, including B-cell lymphomas, nasopharyngeal carcinoma (NPC) (3Sun X.S. Liu S.L. Liang Y.J. Chen Q.Y. Li X.Y. Tang L.Q. Mai H.Q. The role of capecitabine as maintenance therapy in de novo metastatic nasopharyngeal carcinoma: A propensity score matching study.Cancer Commun. (Lond). 2020; 40: 32-42Crossref PubMed Scopus (9) Google Scholar, 4Wu C.F. Lv J.W. Lin L. Mao Y.P. Deng B. Zheng W.H. Wen D.W. Chen Y. Kou J. Chen F.P. Yang X.L. Zheng Z.Q. Li Z.X. Xu S.S. Ma J. et al.Development and validation of a web-based calculator to predict individualized conditional risk of site-specific recurrence in nasopharyngeal carcinoma: Analysis of 10,058 endemic cases.Cancer Commun. (Lond). 2021; 41: 37-50Crossref PubMed Scopus (4) Google Scholar, 5Zhao Y. Hong X.H. Li K. 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Liu L.T. Li Y. Xie H.J. Tang Q.N. Liang H. et al.The diagnostic and prognostic values of plasma Epstein-Barr virus DNA for residual cervical lymphadenopathy in nasopharyngeal carcinoma patients: A retrospective study.Cancer Commun. (Lond). 2019; 39: 14Crossref PubMed Scopus (16) Google Scholar). The viral oncogenic factors, such as RNA transcripts and proteins expressing during the latent or lytic phase have been reported to drive pathogenic processes (10Li H. Liu S. Hu J. Luo X. Li N. A M.B. Cao Y. Epstein-Barr virus lytic reactivation regulation and its pathogenic role in carcinogenesis.Int. J. Biol. Sci. 2016; 12: 1309-1318Crossref PubMed Scopus (71) Google Scholar, 11Munz C. Latency and lytic replication in Epstein-Barr virus-associated oncogenesis.Nat. Rev. Microbiol. 2019; 17: 691-700Crossref PubMed Scopus (121) Google Scholar). The EBV immediate-early protein BZLF1 is highly expressed following lytic stimuli induction, and it mediates the transition from the latent to the lytic replication cycle, which is critical for viral spread (12Bristol J.A. Djavadian R. Albright E.R. Coleman C.B. Ohashi M. Hayes M. Romero-Masters J.C. Barlow E.A. Farrell P.J. Rochford R. Kalejta R.F. Johannsen E.C. Kenney S.C. A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection.PLoS Pathog. 2018; 14e1007179Crossref PubMed Scopus (48) Google Scholar, 13Packham G. Economou A. Rooney C.M. Rowe D.T. Farrell P.J. Structure and function of the Epstein-Barr virus BZLF1 protein.J. Virol. 1990; 64: 2110-2116Crossref PubMed Google Scholar, 14Schaeffner M. Mrozek-Gorska P. Buschle A. Woellmer A. Tagawa T. Cernilogar F.M. Schotta G. Krietenstein N. Lieleg C. Korber P. Hammerschmidt W. BZLF1 interacts with chromatin remodelers promoting escape from latent infections with EBV.Life Sci. Alliance. 2019; 2e201800108Crossref PubMed Scopus (22) Google Scholar, 15Wille C.K. Nawandar D.M. Henning A.N. Ma S. Oetting K.M. Lee D. Lambert P. Johannsen E.C. Kenney S.C. 5-hydroxymethylation of the EBV genome regulates the latent to lytic switch.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E7257-E7265Crossref PubMed Scopus (21) Google Scholar). As a transcription factor, BZLF1 binds to the promoters of many viral lytic genes as well as cellular genes to regulate their expression through EBV–host crosstalk (16Ramasubramanyan S. Osborn K. Al-Mohammad R. Naranjo Perez-Fernandez I.B. Zuo J. Balan N. Godfrey A. Patel H. Peters G. Rowe M. Jenner R.G. Sinclair A.J. Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression.Nucleic Acids Res. 2015; 43: 3563-3577Crossref PubMed Scopus (23) Google Scholar). It has been reported that BZLF1 indirectly inhibits the expression of IFN-γ and promotes the expression of VEGF, IL10, and IL13 via viral proteins (10Li H. Liu S. Hu J. Luo X. Li N. A M.B. Cao Y. Epstein-Barr virus lytic reactivation regulation and its pathogenic role in carcinogenesis.Int. J. Biol. Sci. 2016; 12: 1309-1318Crossref PubMed Scopus (71) Google Scholar, 17Morrison T.E. Mauser A. Wong A. Ting J.P. Kenney S.C. Inhibition of IFN-gamma signaling by an Epstein-Barr virus immediate-early protein.Immunity. 2001; 15: 787-799Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In addition, BZLF1 was also identified to directly regulate the expression of various host genes, such as FOSB and RASA3 (16Ramasubramanyan S. Osborn K. Al-Mohammad R. Naranjo Perez-Fernandez I.B. Zuo J. Balan N. Godfrey A. Patel H. Peters G. Rowe M. Jenner R.G. Sinclair A.J. Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression.Nucleic Acids Res. 2015; 43: 3563-3577Crossref PubMed Scopus (23) Google Scholar). Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor belonging to its eponymous family, which is implicated in cell growth, proliferation, and differentiation (18Rane M.J. Zhao Y. Cai L. Krupsilonppel-like factors (KLFs) in renal physiology and disease.EBioMedicine. 2019; 40: 743-750Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Recently, there is increasing evidence that KLF4 is involved in other biological processes such as viral infection. For example, KLF4 negatively regulated cellular antiviral response by inhibiting the activation of NF-κB by TNF-α and IL-1β (19Luo W.W. Lian H. Zhong B. Shu H.B. Li S. Kruppel-like factor 4 negatively regulates cellular antiviral immune response.Cell Mol. Immunol. 2016; 13: 65-72Crossref PubMed Scopus (30) Google Scholar). In addition, melatonin was found to stabilize KLF4 by preventing its m6A-dependent mRNA decay (20Yang L. Liu X. Song L. Su G. Di A. Bai C. Wei Z. Li G. Melatonin restores the pluripotency of long-term-cultured embryonic stem cells through melatonin receptor-dependent m6A RNA regulation.J. Pineal Res. 2020; 69e12669Crossref PubMed Scopus (13) Google Scholar). However, whether and how KLF4 is regulated by the N6-methyladenosine (m6A) modification via virus–host interaction need to be further elucidated. m6A is the most prevalent internal modification present in the messenger RNAs of eukaryotes viruses that replicate in the nucleus (21Meyer K.D. Saletore Y. Zumbo P. Elemento O. Mason C.E. Jaffrey S.R. Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.Cell. 2012; 149: 1635-1646Abstract Full Text Full Text PDF PubMed Scopus (2094) Google Scholar, 22Wang X. Zhao B.S. 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The modification is introduced by a multicomponent “writer” complex consisting of the enzyme methyltransferase-like 3 (METTL3) and other cofactors including METTL14 and WTAP (25Liu J. Yue Y. Han D. Wang X. Fu Y. Zhang L. Jia G. Yu M. Lu Z. Deng X. Dai Q. Chen W. He C. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation.Nat. Chem. Biol. 2014; 10: 93-95Crossref PubMed Scopus (1467) Google Scholar). The resulting m6A residues are recognized by “reader” proteins such as YTHDF2 and YTHDF3, which contain a YTH domain that directly binds to m6A (22Wang X. Zhao B.S. Roundtree I.A. Lu Z. Han D. Ma H. Weng X. Chen K. Shi H. He C. N(6)-methyladenosine modulates messenger RNA translation efficiency.Cell. 2015; 161: 1388-1399Abstract Full Text Full Text PDF PubMed Scopus (1503) Google Scholar, 26Li A. Chen Y.S. Ping X.L. Yang X. Xiao W. Yang Y. Sun H.Y. Zhu Q. Baidya P. Wang X. Bhattarai D.P. Zhao Y.L. Sun B.F. Yang Y.G. Cytoplasmic m(6)A reader YTHDF3 promotes mRNA translation.Cell Res. 2017; 27: 444-447Crossref PubMed Scopus (393) Google Scholar, 27Wang X. Lu Z. Gomez A. Hon G.C. Yue Y. Han D. Fu Y. Parisien M. Dai Q. Jia G. Ren B. Pan T. He C. N6-methyladenosine-dependent regulation of messenger RNA stability.Nature. 2014; 505: 117-120Crossref PubMed Scopus (1964) Google Scholar, 28Shi H. Wang X. Lu Z. Zhao B.S. Ma H. Hsu P.J. Liu C. He C. YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA.Cell Res. 2017; 27: 315-328Crossref PubMed Scopus (718) Google Scholar). There are multiple lines of evidence that m6A plays an important role in regulating viral infection. Previous studies characterized the m6A RNA methylomes of many RNA viruses, including human immunodeficiency virus, Zika virus, hepatitis C virus, enterovirus 71, murine leukemia virus, and the plant virus alfalfa mosaic virus (29Courtney D.G. Kennedy E.M. Dumm R.E. Bogerd H.P. Tsai K. Heaton N.S. Cullen B.R. 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Addition of m6A to SV40 late mRNAs enhances viral structural gene expression and replication.PLoS Pathog. 2018; 14e1006919Crossref PubMed Scopus (82) Google Scholar, 40Tan B. Liu H. Zhang S. da Silva S.R. Zhang L. Meng J. Cui X. Yuan H. Sorel O. Zhang S.W. Huang Y. Gao S.J. Viral and cellular N(6)-methyladenosine and N(6),2'-O-dimethyladenosine epitranscriptomes in the KSHV life cycle.Nat. Microbiol. 2018; 3: 108-120Crossref PubMed Scopus (88) Google Scholar, 41Ye F. Chen E.R. Nilsen T.W. Kaposi's sarcoma-associated herpesvirus utilizes and manipulates RNA N6-adenosine methylation to promote lytic replication.J. Virol. 2017; 91e00466-17Crossref PubMed Scopus (73) Google Scholar, 42Hesser C.R. Karijolich J. Dominissini D. He C. 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Glaunsinger B.A. N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection.PLoS Pathog. 2018; 14e1006995Crossref PubMed Scopus (104) Google Scholar, 47Tirumuru N. Wu L. HIV-1 envelope proteins up-regulate N (6)-methyladenosine levels of cellular RNA independently of viral replication.J. Biol. Chem. 2019; 294: 3249-3260Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Recently, the role of m6A in EBV-associated tumorigenesis has been investigated by Lang et al. (44Lang F. Singh R.K. Pei Y. Zhang S. Sun K. Robertson E.S. EBV epitranscriptome reprogramming by METTL14 is critical for viral-associated tumorigenesis.PLoS Pathog. 2019; 15e1007796Crossref PubMed Scopus (61) Google Scholar), who examined the viral epitranscriptome of EBV-transformed cell lines. Here, we focused on the modification of cellular genes upon EBV infection in nasopharyngeal cells. We found that EBV infection decreased the METTL3-mediated m6A modification of the host gene KLF4, thereby increasing its expression to promote EBV infection. Mechanistically, the expression of METTL3 was inhibited by BZLF1 during viral infection, which increased the mRNA stability of KLF4. Overall, our results demonstrated a positive feedback loop between BZLF1 and KLF4 that facilitated EBV infection, providing new insights into the crosstalk between virus and host cell. Previous studies found that viral infection alters the m6A modification of host genes (32Lichinchi G. Gao S. Saletore Y. Gonzalez G.M. Bansal V. Wang Y. Mason C.E. Rana T.M. Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells.Nat. Microbiol. 2016; 1: 16011Crossref PubMed Scopus (272) Google Scholar, 40Tan B. Liu H. Zhang S. da Silva S.R. Zhang L. Meng J. Cui X. Yuan H. Sorel O. Zhang S.W. Huang Y. Gao S.J. Viral and cellular N(6)-methyladenosine and N(6),2'-O-dimethyladenosine epitranscriptomes in the KSHV life cycle.Nat. Microbiol. 2018; 3: 108-120Crossref PubMed Scopus (88) Google Scholar, 42Hesser C.R. Karijolich J. Dominissini D. He C. Glaunsinger B.A. N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection.PLoS Pathog. 2018; 14e1006995Crossref PubMed Scopus (104) Google Scholar, 48Gokhale N.S. McIntyre A.B.R. Mattocks M.D. Holley C.L. Lazear H.M. Mason C.E. Horner S.M. Altered m(6)A modification of specific cellular transcripts affects flaviviridae infection.Mol. Cell. 2020; 77: 542-555.e8Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To identify the m6A modification changes in cellular mRNAs following EBV infection, we performed immunoprecipitation and sequencing of methylated RNA (MeRIP-seq) in infected and uninfected nasopharyngeal epithelial cells (Fig. 1A). After extracting the mRNA from NP460 and HK1 cells at 24 h postinfection with EBV, we used an anti-m6A antibody to enrich m6A-modified RNA fragments. This was followed by RNA sequencing of both the input and immunoprecipitated fractions. A significant number of cellular genes with altered m6A modification in response to EBV infection were screened out. As indicated, 25,902 m6A peaks spanning on 11,601 cellular genes in HK1 cells, as well as 27,007 m6A peaks spanning on 12,110 cellular genes in NP460 cells, were identified (Tables S1 and S2). In total, 6758 peaks were downregulated and 1157 peaks were upregulated in HK1 cells (Fig. 1B). In total, 1935 peaks were downregulated and 947 peaks were upregulated in NP460 cells (Fig. 1C). Among these, 553 differential peaks were present in both cell lines (fold change ≧ 1.2), 68 of which showed increased m6A levels, while 485 showed decreased m6A levels, termed as hyper- and hypomethylated peaks, respectively (Fig. 1D). Almost 87% of the altered m6A peaks were hypomethylated, indicating that the m6A levels of total mRNAs of the host were impaired upon viral infection. To verify this hypothesis, we used a cellular RNA m6A level quantification kit to assess the m6A level of EBV-infected HK1, NP460, and NPEC1-Bmil cells. As expected, the m6A levels of cellular genes were lower in the EBV-infected cells (Fig. S1). Next, the hypomethylated genes were screened out for further analysis combined with the differential gene expression. As for the differential gene expression, a total of 607 genes were downregulated and 504 were upregulated following EBV infection in HK1 and NP460 cells (Fig. 1E). KEGG pathway analysis indicated that these genes were mainly enriched in the NF-κB signaling pathway (Fig. 1F), which was consistent with a previous report that viral infection primarily triggered the activation of the transcription factors NF-κB and IRF3 to elicit antiviral response (19Luo W.W. Lian H. Zhong B. Shu H.B. Li S. Kruppel-like factor 4 negatively regulates cellular antiviral immune response.Cell Mol. Immunol. 2016; 13: 65-72Crossref PubMed Scopus (30) Google Scholar). Combined analysis of the m6A levels and differential gene expression revealed 3231 and 985 differential peaks in the EBV-infected HK1 cells and NP460 cells, respectively (Fig. 1, G and H and Tables S1 and S2). Taken together, the results indicated that EBV infection not only decreased cellular m6A modification, but also regulated the mRNA levels of cellular genes which was mainly related to the NF-κB pathway. We therefore set out to further elucidate the crucial genes participating in the regulation. After overlap analysis of the differential expression genes with m6A hypomethylated in both EBV infected cells, the MeRIP-seq data revealed that 33 hypomethylated genes were differentially expressed following EBV infection (Fig. 2A). Among these genes, KLF4, a transcriptional regulator of the NF-κB signaling pathway, has been reported to induce lytic EBV reactivation in Burkitt lymphoma cells (49Nawandar D.M. Wang A. Makielski K. Lee D. Ma S. Barlow E. Reusch J. Jiang R. Wille C.K. Greenspan D. Greenspan J.S. Mertz J.E. Hutt-Fletcher L. Johannsen E.C. Lambert P.F. et al.Differentiation-dependent KLF4 expression promotes lytic Epstein-Barr virus infection in epithelial cells.PLoS Pathog. 2015; 11e1005195Crossref PubMed Scopus (60) Google Scholar, 50Shaverdashvili K. Padlo J. Weinblatt D. Jia Y. Jiang W. Rao D. Laczko D. Whelan K.A. Lynch J.P. Muir A.B. Katz J.P. KLF4 activates NFkappaB signaling and esophageal epithelial inflammation via the Rho-related GTP-binding protein RHOF.PLoS One. 2019; 14e0215746Crossref PubMed Scopus (9) Google Scholar). As indicated in our MeRIP-seq data, KLF4 exhibited lower m6A modification and higher expression levels in the context of EBV infection. Furthermore, EBV infection decreased the m6A levels in the KLF4 mRNA (Fig. 2B), indicating that it may be modified and regulated by EBV. Next, RIP assays were performed using an anti-m6A antibody or IgG control. The results indicated that KLF4 was highly m6A-modified in HK1, NPEC1-Bmi1, and NP460 cells (Fig. 2C). To determine the role of KLF4, we performed MeRIP and qRT-PCR assays to measure its m6A modification and expression levels in EBV infected HK1 and NPEC1-Bmi1 cells. As indicated, the m6A modification level of KLF4 was downregulated in response to viral infection, accompanied by increased expression of KLF4 (Fig. 2, D and E). To further validate the role of KLF4 in EBV infection, we transfected HK1 and NPEC1-Bmi1 cells with a KLF4 plasmid, followed by EBV-GFP infection. The fluorescence microscopy and flow cytometry results revealed that nasopharyngeal epithelial cells with higher expression of KLF4 were more susceptible to acute EBV infection, as revealed by their higher fluorescence ratio (Fig. 2, F and G). Overall, EBV infection reduced the m6A modification of KLF4 and increased its expression, which promoted the EBV infection of nasopharyngeal epithelial cells. We next investigated the mechanism underlying the EBV-induced decrease of KLF4 m6A modification. Since METTL3 is the key enzyme in the m6A writer complex, we next sought to determine whether EBV infection affected the expression level of METTL3. As predicted, the mRNA level of METTL3 was significantly downregulated in HK1 and NP460 cells after EBV infection (Fig. 3A). 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Persisting oncogenic herpesvirus induced by the tumour promotor TPA.Nature. 1978; 272: 373-375Crossref PubMed Scopus (475) Google Scholar). Consistently, the expression of METTL3 was reduced after induction of lytic virus replication in CNE2EBV cells by TPA/NaB treatment, while expression of BZLF1 was increased (Fig. 3B). Mounting evidence supports the idea that BZLF1 is the major transcription factor regulating numerous viral and cellular genes in response to EBV infection. BZLF1 is not only a transcriptional activator, but also as a repressor (13Packham G. Economou A. Rooney C.M. Rowe D.T. Farrell P.J. Structure and function of the Epstein-Barr virus BZLF1 protein.J. Virol. 1990; 64: 2110-2116Crossref PubMed Google Scholar, 55Morrison T.E. Mauser A. Klingelhutz A. Kenney S.C. Epstein-Barr virus immediate-early protein BZLF1 inhibits tumor necrosis factor alpha-induced signaling and apoptosis by downregulating tumor necrosis factor receptor 1.J. Virol. 2004; 78: 544-549Crossref PubMed Scopus (65) Google Scholar, 56Hahn A.M. Huye L.E. Ning S. Webster-Cyriaque J. Pagano J.S. Interferon regulatory factor 7 is negatively regulated by the Epstein-Barr virus immediate-early gene, BZLF-1.J. Virol. 2005; 79: 10040-10052Crossref PubMed Scopus (83) Google Scholar, 57Balan N. Osborn K. Sinclair A.J. Repression of CIITA by the Epstein-Barr virus transcription factor Zta is independent of its dimerization and DNA binding.J. Gen. Virol. 2016; 97: 725-732Crossref PubMed Scopus (8) Google Scholar). We therefore hypothesized that BZLF1 might regulate the promoter activity of METTL3. By analyzing the BZLF1 ChIP-seq data in the NPC cell line HONE1 (58Liu X. Hong T. Parameswaran S. Ernst K. Marazzi I. Weirauch M.T. Fuxman Bass J.I. Human virus transcriptional regulators.Cell. 2020; 182: 24-37Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) from the GEO datasets, we found that" @default.
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• W3136266807 title "Identification of an N6-methyladenosine-mediated positive feedback loop that promotes Epstein–Barr virus infection" @default.
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