研究者詳細 - 渡辺　崇広

Growth Transformation of B Cells by Epstein-Barr Virus Requires <i>IMPDH2</i> Induction and Nucleolar Hypertrophy

Sugimoto, A; Watanabe, T; Matsuoka, K; Okuno, Y; Yanagi, Y; Narita, Y; Mabuchi, S; Nobusue, H; Sugihara, E; Hirayama, M; Ide, T; Onouchi, T; Sato, Y; Kanda, T; Saya, H; Iwatani, Y; Kimura, H; Murata, T

MICROBIOLOGY SPECTRUM 11 巻 ( 4 ) 頁： e0044023 2023年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Microbiology Spectrum

The in vitro growth transformation of primary B cells by Epstein-Barr virus (EBV) is the initial step in the development of posttransplant lymphoproliferative disorder (PTLD). We performed electron microscopic analysis and immunostaining of primary B cells infected with wild-Type EBV. Interestingly, the nucleolar size was increased by two days after infection. A recent study found that nucleolar hypertrophy, which is caused by the induction of the IMPDH2 gene, is required for the efficient promotion of growth in cancers. In the present study, RNA-seq revealed that the IMPDH2 gene was significantly induced by EBV and that its level peaked at day 2. Even without EBV infection, the activation of primary B cells by the CD40 ligand and interleukin-4 increased IMPDH2 expression and nucleolar hypertrophy. Using EBNA2 or LMP1 knockout viruses, we found that EBNA2 and MYC, but not LMP1, induced the IMPDH2 gene during primary infections. IMPDH2 inhibition by mycophenolic acid (MPA) blocked the growth transformation of primary B cells by EBV, leading to smaller nucleoli, nuclei, and cells. Mycophenolate mofetil (MMF), which is a prodrug of MPA that is approved for use as an immunosuppressant, was tested in a mouse xenograft model. Oral MMF significantly improved the survival of mice and reduced splenomegaly. Taken together, these results indicate that EBV induces IMPDH2 expression through EBNA2-dependent and MYC-dependent mechanisms, leading to the hypertrophy of the nucleoli, nuclei, and cells as well as efficient cell proliferation. Our results provide basic evidence that IMPDH2 induction and nucleolar enlargement are crucial for B cell transformation by EBV. In addition, the use of MMF suppresses PTLD. IMPORTANCE EBV infections cause nucleolar enlargement via the induction of IMPDH2, which are essential for B cell growth transformation by EBV. Although the significance of IMPDH2 induction and nuclear hypertrophy in the tumorigenesis of glioblastoma has been reported, EBV infection brings about the change quickly by using its transcriptional cofactor, EBNA2, and MYC. Moreover, we present here, for the novel, basic evidence that an IMPDH2 inhibitor, namely, MPA or MMF, can be used for EBV-positive posttransplant lymphoproliferative disorder (PTLD).

DOI： 10.1128/spectrum.00440-23

Epstein-Barr Virus BBLF1 Mediates Secretory Vesicle Transport to Facilitate Mature Virion Release 国際誌

Uddin, MK; Watanabe, T; Arata, M; Sato, Y; Kimura, H; Murata, T

JOURNAL OF VIROLOGY 97 巻 ( 6 ) 頁： e0043723 2023年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Virology

Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function.

DOI： 10.1128/jvi.00437-23

Comprehensive Analyses of Intraviral Epstein-Barr Virus Protein-Protein Interactions Hint Central Role of BLRF2 in the Tegument Network 国際誌

Hara Yuya, Watanabe Takahiro, Yoshida Masahiro, Al Masud H. M. Abdullah, Kato Hiromichi, Kondo Tomohiro, Suzuki Reiji, Kurose Shutaro, Uddin Md Kamal, Arata Masataka, Miyagi Shouhei, Yanagi Yusuke, Sato Yoshitaka, Kimura Hiroshi, Murata Takayuki

JOURNAL OF VIROLOGY 96 巻 ( 14 ) 頁： e0051822 2022年7月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Virology

Protein-protein interactions (PPIs) are crucial for various biological processes. Epstein-Barr virus (EBV) proteins typically form complexes, regulating the replication and persistence of the viral genome in human cells. However, the role of EBV protein complexes under physiological conditions remains unclear. In this study, we performed comprehensive analyses of EBV PPIs in living cells using the NanoBiT system. We identified 195 PPIs, many of which have not previously been reported. Computational analyses of these PPIs revealed that BLRF2, which is only found in gammaherpesviruses, is a central protein in the structural network of EBV tegument proteins. To characterize the role of BLRF2, we generated two BLRF2 knockout EBV clones using CRISPR/Cas9. BLRF2 knockout significantly decreased the production of infectious virus particles, which was partially restored by exogenous BLRF2 expression. In addition, self-association of BLRF2 protein was found, and mutation of the residues crucial for the self-association affected stability of the protein. Our data imply that BLRF2 is a tegument network hub that plays important roles in progeny virion maturation.

DOI： 10.1128/jvi.00518-22

Epstein-Barr virus tegument protein BGLF2 in exosomes released from virus-producing cells facilitates de novo infection 国際誌

Sato Yoshitaka, Yaguchi Masahiro, Okuno Yusuke, Ishimaru Hanako, Sagou Ken, Ozaki Somi, Suzuki Takeshi, Inagaki Tomoki, Umeda Miki, Watanabe Takahiro, Fujimuro Masahiro, Murata Takayuki, Kimura Hiroshi

CELL COMMUNICATION AND SIGNALING 20 巻 ( 1 ) 頁： 95 - 95 2022年6月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell Communication and Signaling

Background: Viruses must adapt to the environment of their host cells to establish infection and persist. Diverse mammalian cells, including virus-infected cells, release extracellular vesicles such as exosomes containing proteins and miRNAs, and use these vesicles to mediate intercellular communication. However, the roles of exosomes in viral infection remain unclear. Results: We screened viral proteins to identify those responsible for the exosome-mediated enhancement of Epstein–Barr virus (EBV) infection. We identified BGLF2 protein encapsulated in exosomes, which were released by EBV-infected cells. BGLF2 protein is a tegument protein that exists in the space between the envelope and nucleocapsid, and it is released into the cytoplasm shortly after infection. BGLF2 protein-containing exosomes enhanced viral gene expression and repressed innate immunity, thereby supporting the EBV infection. Conclusions: The EBV tegument protein BGLF2 is encapsulated in exosomes and released by infected cells to facilitate the establishment of EBV infection. These findings suggest that tegument proteins support viral infection not only between the envelope and nucleocapsid, as well as in extraviral particles such as exosomes. Graphical abstract: [Figure not available: see fulltext.] [MediaObject not available: see fulltext.].

DOI： 10.1186/s12964-022-00902-7

EBV Exploits RNA m(6)A Modification to Promote Cell Survival and Progeny Virus Production During Lytic Cycle 国際誌

Yanagi Yusuke, Watanabe Takahiro, Hara Yuya, Sato Yoshitaka, Kimura Hiroshi, Murata Takayuki

FRONTIERS IN MICROBIOLOGY 13 巻頁： 870816 - 870816 2022年6月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers in Microbiology

N6-methyladenosine (m6A) mediates various biological processes by affecting RNA stability, splicing, and translational efficiency. The roles of m6A modification in Epstein-Barr virus (EBV) infection in the lytic phase are unclear. Here, knockout of the m6A methyltransferase, N6-methyladenosine methyltransferase-like 3 (METTL3), or inhibition of methylation by DAA or UZH1a decreased the expression of viral lytic proteins and reduced progeny virion production. Interestingly, cell growth and viability were decreased by induction of the lytic cycle in METTL3-knockout or inhibitor-treated cells. Apoptosis was induced in those conditions possibly because of a decreased level of the anti-apoptotic viral protein, BHRF1. Therefore, m6A shows potential as a target of lytic induction therapy for EBV-associated cancers, including Burkitt lymphoma.

DOI： 10.3389/fmicb.2022.870816

PD-L1 upregulation by lytic induction of Epstein-Barr Virus.

Yanagi Y, Hara Y, Mabuchi S, Watanabe T, Sato Y, Kimura H, Murata T

Virology 568 巻頁： 31 - 40 2022年3月

記述言語：英語出版者・発行元：Virology

Epstein-Barr virus (EBV) is an etiologic agent of infectious mononucleosis and several malignancies. Here, we found that reactivation of EBV resulted in increased programmed cell death-ligand 1 (PD-L1) expression in a cell type-dependent manner. Lytic induction in EBV-positive Akata, AGS, MutuI, and Jijoye cell lines increased PD-L1 levels, but cells such as EBV-negative Akata, MutuIII, and P3HR1 did not have increased PD-L1. EBV in the P3HR1 cell line has a deletion in the EBNA2 gene, while EBV in its parental cell line, Jijoye, has the complete EBNA2 gene. PD-L1 expression by lytic induction was reduced when EBNA2 was knocked down. In addition, pharmacological inhibition indicated involvement of nuclear factor kappa B, mitogen-activated protein kinase, and AKT signaling. These results suggest that EBV likely evades immunity by inducing PD-L1 upon reactivation, through the increased expression of EBNA2 and activation of signaling pathways.

DOI： 10.1016/j.virol.2022.01.006

Molecular Basis of Epstein-Barr Virus Latency Establishment and Lytic Reactivation 国際誌

Murata Takayuki, Sugimoto Atsuko, Inagaki Tomoki, Yanagi Yusuke, Watanabe Takahiro, Sato Yoshitaka, Kimura Hiroshi

VIRUSES-BASEL 13 巻 ( 12 ) 2021年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Viruses

Epstein–Barr virus (EBV) is a causative agent of infectious mononucleosis and several types of cancer. Like other herpesviruses, it establishes an asymptomatic, life-long latent infection, with occasional reactivation and shedding of progeny viruses. During latency, EBV expresses a small number of viral genes, and exists as an episome in the host–cell nucleus. Expression patterns of latency genes are dependent on the cell type, time after infection, and milieu of the cell (e.g., germinal center or peripheral blood). Upon lytic induction, expression of the viral immediate-early genes, BZLF1 and BRLF1, are induced, followed by early gene expression, viral DNA replication, late gene expression, and maturation and egress of progeny virions. Furthermore, EBV reactivation involves more than just progeny production. The EBV life cycle is regulated by signal transduction, transcription factors, promoter sequences, epigenetics, and the 3D structure of the genome. In this article, the molecular basis of EBV latency establishment and reactivation is summarized.

DOI： 10.3390/v13122344

Deletion of Viral microRNAs in the Oncogenesis of Epstein-Barr Virus-Associated Lymphoma

A STING inhibitor suppresses EBV-induced B cell transformation and lymphomagenesis 国際誌

Miyagi Shouhei, Watanabe Takahiro, Hara Yuya, Arata Masataka, Uddin Md. Kamal, Mantoku Keisuke, Sago Ken, Yanagi Yusuke, Suzuki Takeshi, Masud H. M. Abdullah Al, Kawada Jun-ichi, Nakamura Shigeo, Miyake Yasuyuki, Sato Yoshitaka, Murata Takayuki, Kimura Hiroshi

CANCER SCIENCE 2021年10月

Kimura Hiroshi, Okuno Yusuke, Sato Yoshitaka, Watanabe Takahiro, Murata Takayuki

FRONTIERS IN MICROBIOLOGY 12 巻頁： 667968 2021年7月

記述言語：日本語出版者・発行元：Frontiers in Microbiology

Epstein–Barr virus (EBV), which encodes >80 genes and nearly 50 non-coding RNAs, is a double-stranded DNA virus. EBV is associated with various types of lymphomas and lymphoproliferative disorders not only of B-cell but also T/NK-cell origin. However, the oncogenic mechanism remains poorly understood, including the EBV receptors expressed on T/NK cells, relationship of EBV with host genes, and epigenetic regulation of EBV and host genes. The roles of host and viral non-coding RNAs during tumorigenesis have been elucidated. EBV encodes at least 49 mature microRNAs (miRNAs), of which 44 are located in BamHI-A rightward transcripts (BARTs) region, and the remaining five are located in BamHI-H rightward fragment 1. BART miRNAs modulate cell differentiation, proliferation, apoptosis, and the cell cycle, and they are considered positive regulators of oncogenesis. We and others have recently reported that EBV-positive lymphomas frequently possess large deletions in BART miRNA clusters, suggesting that some viral miRNAs have suppressive effects on oncogenesis, and that deletion of these miRNAs may aid lymphoma formation.

DOI： 10.3389/fmicb.2021.667968

RNAseq analysis identifies involvement of EBNA2 in PD-L1 induction during Epstein-Barr virus infection of primary B cells 査読有り国際誌

Yanagi Yusuke, Okuno Yusuke, Narita Yohei, Al Masud H. M. Abdullah, Watanabe Takahiro, Sato Yoshitaka, Kanda Teru, Kimura Hiroshi, Murata Takayuki

VIROLOGY 557 巻頁： 44 - 54 2021年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Virology

Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and several types of malignancy. RNAseq of peripheral blood primary B cell samples infected with wild-type EBV revealed that expression of programmed cell death ligand-1 (PD-L1) is markedly induced by infection. This induction of PD-L1 was alleviated by knockout of the EBNA2 gene, but knockout of LMP1 had little effect. ChIPseq, ChIA-PET, and reporter assays further confirmed that EBNA2-binding sites in the promoter region and at 130 kb downstream of the PD-L1 gene played important roles in PD-L1 induction. Our results indicate that EBV mainly utilizes the EBNA2 gene for induction of PD-L1 and to evade host immunity on infection of primary B cells. Furthermore, pathway analysis revealed that genes involved in the cell cycle, metabolic processes, membrane morphogenesis, and vesicle regulation were induced by EBNA2, and that EBNA2 suppressed genes related to immune signaling.

DOI： 10.1016/j.virol.2021.02.004

Anti-tumor effect of IL2-expressing HSV amplicon vector and oncolytic virus HF10 査読有り国際誌

Esaki Shinichi, Goshima Fumi, Takano Gaku, Watanabe Takahiro, Sato Yoshitaka, Murata Takayuki, Kimura Hiroshi

CANCER SCIENCE 112 巻頁： 394 - 394 2021年2月

Role of Epstein-Barr Virus C Promoter Deletion in Diffuse Large B Cell Lymphoma 査読有り国際共著国際誌

Mabuchi Seiyo, Hijioka Fumiya, Watanabe Takahiro, Yanagi Yusuke, Okuno Yusuke, Al Masud H. M. Abdullah, Sato Yoshitaka, Murata Takayuki, Kimura Hiroshi

CANCERS 13 巻 ( 3 ) 頁： 561 - 561 2021年2月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancers

The Epstein–Barr virus (EBV) is the cause of several malignancies, including diffuse large B cell lymphoma (DLBCL). We recently found that EBV genomes in EBV‐positive cancer specimens have various deletions (Okuno et al. Nat Microbiol. 2019). Here, we focus on the deletion of C promoter (Cp), which transcribes EBV nuclear antigen (EBNA) genes in type III latency. The Cp deletion found in a DLBCL patient (332 bp) was introduced into EBV‐BAC of the B95‐8 strain. Interestingly, the dCp virus transformed B cells more efficiently than WT and revertant strains. Deletion of Cp also promoted tumor formation and severe pathogenicity in a mouse xenograft model. RNA sequencing and qRT–PCR analyses revealed that Cp transcription was undetectable in the dCp cells. Instead, transcription from the W promoter (Wp), an alternative promoter for EBNA, was activated in the dCp mutant. We also found that the expression of latent membrane protein 2A (LMP2A) was somehow induced in the dCp mutant. Double knockout of Cp and LMP2A indicated that LMP2A is crucial for B cell transformation, but the increased transformation induced by Cp deletion cannot be explained by LMP2A alone. We also tested the effect of an anti‐apoptotic viral BCL2 homolog, BHRF1, because its expression was reportedly induced more efficiently by that of Wp. However, increased growth transformation via Cp deletion was not due to the BHRF1 gene. Taken together, the results indicated that deletion of a specific region in Cp increased in vitro transformation and the rate of progression of EBV‐positive lymphoproliferative disorders in vivo. Our data suggest that genomic alteration not only of the host but also the virus promotes EBVpositive tumor generation and expansion, although the molecular mechanism underlying this phenomenon is still unclear. However, LMP2A and BHRF1 are not involved.

DOI： 10.3390/cancers13030561