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Efficient Epstein-Barr Virus Progeny Production Mediated by Cancer

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Efficient Epstein-Barr Virus Progeny Production Mediated by Cancer microorganisms Article Efficient Epstein-Barr Virus Progeny Production Mediated by Cancer-Derived LMP1 and Virally-Encoded microRNAs Misako Yajima 1 , Mamiko Miyata 2, Kazufumi Ikuta 1, Yasuhisa Hasegawa 3,4 , Chitose Oneyama 2 and Teru Kanda 1,2,* 1 Division of Microbiology, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, Sendai 983-8536, Japan; [email protected] (M.Y.); [email protected] (K.I.) 2 Division of Cancer Cell Regulation, Aichi Cancer Center Research Institute, Nagoya 464-8681, Japan; [email protected] (M.M.); [email protected] (C.O.) 3 Department of Head and Neck Surgery, Aichi Cancer Center Hospital, Nagoya 464-8681, Japan; [email protected] 4 Department of Head and Neck Surgery, Asahi University Hospital, Gifu 500-8523, Japan * Correspondence: [email protected]; Tel.: +81-22-290-8723 Received: 26 March 2019; Accepted: 28 April 2019; Published: 30 April 2019 Abstract: Epstein-Barr virus (EBV) genomes, particularly their latent genes, are heterogeneous among strains. The heterogeneity of EBV-encoded latent membrane protein 1 (LMP1) raises the question of whether there are functional differences between LMP1 expressed by cancer-associated EBV and that by non-cancerous strains. Here, we used bacterial artificial chromosome (BAC)-cloned EBV genomes retaining all virally encoded microRNA (miRNA) genes to investigate the functions of cancer-derived LMP1 in the context of the EBV genome. HEK293 cells were stably transfected with EBV-BAC clone DNAs encoding either nasopharyngeal carcinoma (NPC)-derived CAO-LMP1 (LMP1CAO) or LMP1 from a prototype B95-8 strain of EBV (LMP1B95-8). When an EBV-BAC clone DNA encoding LMP1CAO was stably transfected into HEK293 cells, it generated many more stable transformants than the control clone encoding LMP1B95-8. Furthermore, stably transfected HEK293 cells exhibited highly efficient production of progeny virus. Importantly, deletion of the clustered viral miRNA genes compromised the ability to produce progeny viruses. These results indicate that cancer-derived LMP1 and viral miRNAs together are necessary for efficient production of progeny virus, and that the resulting increase in efficiency contributes to EBV-mediated epithelial carcinogenesis. Keywords: Epstein-Barr virus; Nasopharyngeal carcinoma; LMP1; BART; microRNA 1. Introduction Epstein-Barr virus (EBV) is an oncogenic herpesvirus that causes various lymphoproliferative diseases, including B cell lymphomas and T/NK lymphomas [1]. EBV also plays a role in the development of epithelial cancers, such as nasopharyngeal carcinoma (NPC) and gastric cancer [2]. The mechanisms underlying EBV-mediated B cell lymphomagenesis have been studied extensively using in vitro EBV-mediated B cell immortalization as an experimental system [3]. By contrast, T/NK lymphomagenesis and epithelial carcinogenesis following EBV infection have never been reproduced in vitro. Thus, the molecular mechanisms underlying these processes remain largely unknown. EBV-encoded latent membrane protein 1 (LMP1) is essential for EBV-mediated B cell transformation [4]. LMP1, which belongs to the tumor necrosis factor receptor family, is a constitutively active mimic of cellular CD40 [5]. LMP1 activates downstream signaling pathways, such as NF-κB, c-jun N-terminal kinase, and p38/MAPK, through C-terminal activation regions 1, 2, 3 (CTAR1, CTAR2, Microorganisms 2019, 7, 119; doi:10.3390/microorganisms7050119 www.mdpi.com/journal/microorganisms Microorganisms 2019, 7, x FOR PEER REVIEW 2 of 12 Microorganisms 2019, 7, 119 2 of 12 constitutively active mimic of cellular CD40 [5]. LMP1 activates downstream signaling pathways, such as NF-κB, c-jun N-terminal kinase, and p38/MAPK, through C-terminal activation regions 1, 2, CTAR3)3 (CTAR1, [6–8], CTAR2, thereby CTAR3) triggering [6–8], cell thereby proliferation. triggering cell LMP1 proliferation. fulfills theLMP1 criteria fulfills for the acriteria classical for a viral oncoproteinclassical becauseviral oncoprotein its expression because transforms its expression rodent transforms fibroblasts rodent [9]. Numerous fibroblasts studies [9]. Numerous suggest that LMP1studies plays asuggest role in that the LMP1 pathogenesis plays a role of EBV-associated in the pathogenesis epithelial of EBV-associated cancers [2,10 epithelial]; however, cancers due to a lack of[2,10]; experimental however, systems,due to a thelack contribution of experimental of LMP1 systems, to epithelialthe contribution carcinogenesis of LMP1 is to unclear. epithelial EBV-encodedcarcinogenesis is latent unclear. gene products, such as EBV nuclear antigens (EBNAs) and LMPs, are heterogeneousEBV-encoded among latent EBVgene strainsproducts, [11 such,12], as and EBV LMP1 nuclear is theantigens most (EBNAs) heterogeneous. and LMPs, LMP1 are is an integralheterogeneous membrane among protein EBV strains comprising [11,12], a and short LMP1 N-terminal is the most cytoplasmic heterogeneous. domain, LMP1 sixis an hydrophobic integral membrane protein comprising a short N-terminal cytoplasmic domain, six hydrophobic transmembrane domains, and a C-terminal cytoplasmic tail [3]. Heterogeneities reside within the six transmembrane domains, and a C-terminal cytoplasmic tail [3]. Heterogeneities reside within the six transmembrane domains as well as within the N- and C-terminal cytoplasmic domains. Heterogeneity transmembrane domains as well as within the N- and C-terminal cytoplasmic domains. withinHeterogeneity the C-terminal within cytoplasmic the C-terminal domain cytoplasmic includes domain variations includes in variations the copy in number the copy of number repetitive of 11 aminorepetitive acid (a.a.) 11 amino repeat acid motifs (a.a.) and repeat a 10 motifs a.a. deletion and a 10 commonly a.a. deletion found commonly in EBV found in Asian in EBV countries in Asian [13]. Acountries Chinese [13]. NPC cell CAO, propagated in nude mice, encodes LMP1 [14–16], designated hereafter as LMP1CAOA Chinese, which isNPC distinct cell CAO, from LMP1propagated expressed in nu byde prototypemice, encodes B95-8 LMP1 strain [14–16], EBV (LMP1 designatedB95-8)[ 17]. LMP1hereafterCAO contains as LMP1 multipleCAO, which a.a. is substitutions distinct from withinLMP1 expr its transmembraneessed by prototype and B95-8 N- andstrain C-terminal EBV cytoplasmic(LMP1B95-8 domains) [17]. LMP1 (FigureCAO contains1). It also multiple harbors a.a. a substitutions 10 a.a. deletion within in theits transmembrane C-terminal domain, and N- which and is C-terminal cytoplasmic domains (Figure 1). It also harbors a 10 a.a. deletion in the C-terminal common to a specific subtype of LMP1 known as China 1 type [13]. LMP1CAO is functionally different domain, which is common to a specific subtype of LMP1 known as China 1 type [13]. LMP1CAO is from LMP1B95-8 in that the former is less cytostatic than the latter. This functional difference is due functionally different from LMP1B95-8 in that the former is less cytostatic than the latter. This to the a.a. heterogeneity of the transmembrane domains rather than to the 10 a.a. deletion at the C functional difference is due to the a.a. heterogeneity of the transmembrane domains rather than to terminus [15,16,18,19]. In addition to LMP1 , numerous NPC-associated LMP1 variants have been the 10 a.a. deletion at the C terminusCAO [15,16,18,19]. In addition to LMP1CAO, numerous identifiedNPC-associated using recently LMP1 developed variants have deep been sequencing identified technologies, using recently and developed an elucidation deep ofsequencing their roles in NPCtechnologies, pathogenesis and is underwayan elucidation [20 of]. their roles in NPC pathogenesis is underway [20]. FigureFigure 1. Comparison 1. Comparison of of the the amino amino acid acid sequences sequences ofof B95-8 LMP1 LMP1 (LMP1 (LMP1B95-8B95-8) and) and CAO-LMP1 CAO-LMP1 (LMP1CAO). Sequence alignment was performed by CLUSTALW (left); schematic illustrations of the (LMP1CAO). Sequence alignment was performed by CLUSTALW (left); schematic illustrations of the LMP1 domains (right). The amino acids in LMP1CAO that differ from those in LMP1B95-8 are shown. LMP1 domains (right). The amino acids in LMP1CAO that differ from those in LMP1B95-8 are shown. Note that LMP1CAO contains three additional copies of 11 amino acid repeats (indicated by brown Note that LMP1CAO contains three additional copies of 11 amino acid repeats (indicated by brown squares) and a 10 amino acid deletion in the C-terminal cytoplasmic domain. squares) and a 10 amino acid deletion in the C-terminal cytoplasmic domain. A previous study used a recombinant EBV (so-called ‘Maxi-EBV’ [21]) to investigate functional A previous study used a recombinant EBV (so-called ‘Maxi-EBV’ [21]) to investigate functional differences between NPC-derived LMP1 and LMP1B95-8 [22]. The results were somewhat unexpected: differences between NPC-derived LMP1 and LMP1B95-8 [22]. The results were somewhat unexpected: Replacing LMP1B95-8 with NPC-derived LMP1 decreased rather than increased the EBV copy number Replacingin latently LMP1 infectedB95-8 with cells NPC-derived [22]. Maxi-EBV, LMP1 an EBV-BAC decreased clone rather of thanB95-8 increased strain EBV, the harbors EBV copy a 12 numberkb in latentlydefect infected in the BamHI cells [ 22rightward]. Maxi-EBV, transcripts an EBV-BAC (BARTs) clonelocus of[23], B95-8 which strain encodes EBV, a harbors number aof 12 BART kb defect in themicroRNAs BamHI rightward (miRNAs). transcripts Since BART (BARTs) miRNAs locus [ 23likely], which contribute encodes to
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