Cheung, ST

Mak, CK

Tso, KK

Yip, KY

HKU Scholars Hub

Title Complete genomic sequence of Epstein-Barr virus innasopharyngeal carcinoma cell line C666-1Author(s) Tso, KK; Yip, KY; Mak, CK; Cheung, STCitation Infectious Agents and Cancer, 2013, v. 8 n. 1, article no. 29Issued Date 2013URL http://hdl.handle.net/10722/193924Rights Infectious Agents and Cancer. Copyright © BioMed Central Ltd.Tso et al. Infectious Agents and Cancer 2013, 8:29http://www.infectagentscancer.com/content/8/1/29SHORT REPORT Open AccessComplete genomic sequence of Epstein-Barr virusin nasopharyngeal carcinoma cell line C666-1Ken Kai-Yuen Tso1†, Kevin Yuk-Lap Yip1†, Cathy Ka-Yan Mak2, Grace Tin-Yun Chung2,3, Sau-Dan Lee1,Siu-Tim Cheung4, Ka-Fai To2,3 and Kwok-Wai Lo2,3*AbstractBackground: Nasopharyngeal carcinoma is a distinct type of head and neck cancer which is consistently associatedwith Epstein-Barr virus (EBV). The C666-1 cell line is the only in vitro native EBV-infected NPC cell model commonlyused for study of the viral-host interaction. Nevertheless, the complete EBV genome sequence in this in vitroEBV-infected NPC model has not been characterized.Objective: To determine the complete EBV genome sequence in C666-1 cells.Methods: The C666-1 genome was sequenced by 100-bases pair-end massive parallel sequencing. Bioinformaticsanalysis was performed to extract the EBV sequences and construct an EBV consensus sequence map. PCRamplification and Sanger DNA sequencing were used for sequence validation and gap filling. A phylogeneticanalysis of EBV strain in C666-1 cells and other reported EBV strains was performed.Results: A 171,317 bp complete EBV genome of C666-1 was successfully constructed (GenBank accession number:KC617875). Phylogenetic analysis of EBV genome in C666-1 revealed that the C666-1 EBV strain is closely related tothe reported strains in NPC primary tumors.Conclusion: C666-1 contains a representative NPC-associated EBV genome and might serve as an important modelfor studying the roles or function of viral proteins in NPC tumorigenesis.Keywords: Epstein-Barr virus, Nasopharyngeal carcinoma, Whole-genome deep sequencing, Single-nucleotidevariations, Indels, Phylogenetic analysis, BNRFFindingsNPC is a distinct type of head and neck cancer which isconsistently associated with Epstein-Barr virus (EBV).Detection of clonal EBV genome in both precancerouslesions and invasive cancers indicates that EBV latent in-fection is an early event in the tumorigenesis of NPC.Since we established the EBV-positive NPC cell lineC666-1 and reported it about fifteen years ago, it hasbeen widely used for investigating host-viral interaction,elucidating the function and transcriptional regulation ofEBV-encoded latent genes and miRNAs, and developing* Correspondence: kwlo@cuhk.edu.hk†Equal contributors2Department of Anatomical and Cellular Pathology, State Key Laboratory inOncology in South China, Prince of Wales Hospital, The Chinese University ofHong Kong, Hong Kong SAR, China3Li Ka Shing Institute of Health Science, The Chinese University of HongKong, Hong Kong SAR, ChinaFull list of author information is available at the end of the article© 2013 Tso et al. This is an Open Access articl(http://creativecommons.org/licenses/by/2.0),provided the original work is properly cited.EBV targeting therapeutic strategies [1]. The origin ofthis cell line was from an undifferentiated NPC biopsy ofa Hong Kong patient [1]. It contains normal episomalEBV genome and shows latency II EBV gene expressionpattern. A number of studies demonstrated the distinctNF-κb, STAT3, AKT and NOTCH pathways in this cellline as well as the in vivo samples including EBV-positive NPC xenografts (e.g., C15, C17, xeno-2117) andprimary tumors [2]. Recently, two novel EBV-encodedmicroRNAs, miR-BART21 and miR-BART22 have beendiscovered from this EBV-positive epithelial cell line [3].Despite C666-1 being the only in vitro native EBV-infected NPC model worldwide, the EBV genome in thiscell line has not been fully characterized until now. To fa-cilitate the EBV-related studies using this unique cell line,we constructed the EBV genome map through bioin-formatic analysis and experimental validation of our re-cent whole-genome deep sequencing results (Additionale distributed under the terms of the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium,Tso et al. Infectious Agents and Cancer 2013, 8:29 Page 2 of 4http://www.infectagentscancer.com/content/8/1/29file 1 Supplementary methodology). By 100-base pair-endgenomic sequencing on Illumina HiSeq 2000 genome se-quencer, the C666-1 genome was sequenced with average >75-fold coverage as described [4]. A total of 2,511,210,660reads (251 Gb) were collected from the sample. By using anapproach that combines the results of two alignment strat-egies, namely aligning the reads to both human and EBVreference genomes (EBV-WT; GeneBank accession numberAJ507799) at the same time, and aligning them first to theFigure 1 Characterization of the EBV genome sequence derived fromCircos plot demonstrates the genome-wide comparison of SNVs and indelsstrains (HKNPC1, red bars; GD2, orange bars; GD1, blue bars; AG876, grey bSummary of SNVs and indels identified in C666-1 strain. (c) Phylogenetic anGD1, GD2, AG876 and EBV-WT. (d) A nonsense mutation in codon 333 (Q tsequence from the NPC xenograft xeno-2117 is also shown.human genome and then the remaining reads to the EBVreference genome, we extracted a total of 857,595 kb EBVsequences from the collected C666-1 data. A high coveragevalue of 504 folds to EBV genome was yielded. All uniquelymapped EBV sequences were assembled into a 143,734 bpconsensus sequence with a read depth of at least 10 reads.We validated the poorly aligned and questionable regionsand filled up the gaps by PCR amplification and con-ventional Sanger DNA sequencing. The regions failed to bewhole-genome deep sequencing of NPC cell line C666-1. (a)in EBV genome of C666-1 (green bars) and those of other reportedars). The WT-EBV genome sequence was used as reference. (b)alysis of the genome sequences in five EBV strains, C666-1, HKNPC1,o stop) of BNRF1 identified in the C666-1 strain. The wild typeTso et al. Infectious Agents and Cancer 2013, 8:29 Page 3 of 4http://www.infectagentscancer.com/content/8/1/29assembled (e.g. with highly repetitive sequences) are repre-sented by tracts of Ns as described previously [5]. A171,317 bp complete EBV genome of C666-1 was cons-tructed (Figure 1a). This newly assembled C666-1 EBV se-quence was submitted to GenBank with accession numberKC617875. The study was approved by the University Ani-mal Experimentation Ethics Committee (AEEC) (13-036-MIS) of the Chinese University of Hong Kong.In this study, we have assembled the EBV genome inC666-1 using high-coverage genome sequencing data.Since no PCR amplification was involved, both homo-genous and heterogeneous genome variations are accur-ately determined. Comparing with the EBV-WT referencegenomic sequence (AJ507799), we have revealed a total of1,268 homogenous and 87 heterogeneous sequence varia-tions. These changes include 127 indels and 1,228 SNVs.Among the SNVs, 907 are located within the coding re-gions and 41.3% (386/907) of them are nonsynonymous(Figure 1b). The sequence variations in selected SNVs wereconfirmed by Sanger DNA sequencing. Phylogeneticanalysis of whole EBV genomes in C666-1 and the reportedstrains (EBV-WT, AG876, GD1, GD2, and HKNPC1)showed that C666-1 is closely related to the GD2 andHKNPC1 strains (Figure 1c) [5,6]. It has great divergenceEBNA1LMP2ABZLF1Figure 2 Phylogenetic analysis of EBNA1, LMP1, LMP2A, LMP2B, BZLFEBV strains (HKNPC1, GD1, GD2, AG876 and WT-EBV).with the AG876 and reference EBV-WT genome. Similarresults were observed when we compared the protein se-quences of various EBV lytic (BZLF1, BLLF1) and latent(EBNA1, LMP1, LMP2) genes (Figure 2). A number ofstudies have also shown that BZLF1 and LMP1 sequencesof the isolates from Hong Kong NPC patients are distinctfrom that of the EBV-infected lymphoid cells derived in Af-rica or Western countries [7–9]. The findings imply thatC666-1 might serve as an important model for studying theroles or functions of viral proteins in NPC tumorigenesis.Among the four EBV strains from South China, the isolatefrom NPC patient’s saliva (GD1) shows the greatestdivergence with those from the tumors (C666-1, GD2,HKNPC1). This finding suggests the presence of tumor-associated EBV strain(s) in NPC patients. Nevertheless, acomprehensive sequencing of EBV isolates from saliva,peripheral blood and tumor specimens in a panel of NPCpatients may prove this hypothesis. A summary of non-synonymous SNVs in the majority of EBV-encoded lyticand latent genes of C666-1 strain versus those of GD2 andHKNPC1 is shown in Additional file 2: Table S1. In thelatent genes including EBNA1, EBNA3B/3C, LMP1 andLMP2B genes, high frequencies of C666-1 specific non-synonymous SNVs were observed. The prevalence andLMP1BLLF1LMP2B1 and BLLF1 protein sequences of C666-1 and other reportedTso et al. Infectious Agents and Cancer 2013, 8:29 Page 4 of 4http://www.infectagentscancer.com/content/8/1/29function of these SNVs in NPC need further elucidations.Previously, we have demonstrated that multiple EBV-encoded BART miRNAs (miR-BART1-5p, miR-BART16and miR-BART17-5p) target the 3′UTR of the LMP1 gene[10]. The predicted target sequences of these 3 EBV-encoded BART miRNAs in the 3′UTR of the LMP1 geneare highly conserved in the NPC-derived EBV strains. Inthis study, we also found no polymorphism in the predictedtarget sequences of the miR-BART1-5p, 16, and 17-5p inthe C666-1 EBV strain.Apart from the missense mutations, a homogenousnonsense mutation in the lytic gene, BNRF1, which en-codes an EBV major tegument protein was found. Weconfirmed the mutation in C666-1 by PCR amplificationand Sanger Sequencing (Figure 1d). This finding indi-cates the deficiency of BNRF1 protein expression in thisin vitro EBV-positive NPC models. Notably, it wasreported that EBV with BNRF1 deletion also showed ef-ficient lytic replication and production of mature viralparticles. There are no major structural alterations inthe BNRF1-deleted virus [11]. Further elucidation of thevirus production and lytic cycle of this BNRF1-deficientC666-1 strain is needed. On the other hand, a recentstudy has reported that BNRF1 activates viral early geneBZLF1 transcription via disrupting cellular DAXX-ATRX in 293 cells. Thus, BNRF1 deficiency may help tomaintain the latent EBV genome in NPC cells [12]. Onthe other hand, loss of BNRF1 in the C666-1 strain mayimpact the escape from the host immune responses inthe NPC patients since BNRF1 is a defined target of theEBV-specific T-helper-cell response.In summary, we delineated the whole EBV genome se-quence in C666-1, which might serve as an importantresource for NPC studies. The phylogenetic analysis in-dicates the C666-1 strain as a representative strain forEBV-associated NPC.Additional files:Additional file 1: Supplementary methodology.Additional file 2: Table S1. Non-synonymous mutations and aminoacid changes commonly found in NPC tumor samples (C666-1, HKNPC1and GD2).AbbreviationsEBV: Epstein-Barr virus; NPC: Nasopharyngeal carcinoma; Indels: Insertions/deletions; SNVs: Single-nucleotide variations; CDS: Coding sequence.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsKWL and KYLY designed the study; KWL, KYLY, and KFT drafted themanuscript; KKYT, KYLY and SDL participated in the bioinformatics analysisand sequence alignment; CKYM, GTYC, STC carried out the molecular geneticstudies. All authors read and approved the final manuscript.Authors’ informationKen Kai-Yuen Tso and Kevin Yuk-Lap Yip are co-first authors.AcknowledgementsThe research was supported by Focused Investments Scheme-A from theChinese University of Hong Kong, and Hong Kong Research Grant Council –GRF (471610, 471211), CRF (CUHK8/CRF/11R), Theme-Based Research Scheme(T12-403/11 and T12-401/13-R) and AoE NPC (AoE/M-06/08).Author details1Department of Computer Science and Engineering, The Chinese Universityof Hong Kong, Hong Kong SAR, China. 2Department of Anatomical andCellular Pathology, State Key Laboratory in Oncology in South China, Princeof Wales Hospital, The Chinese University of Hong Kong, Hong Kong SAR,China. 3Li Ka Shing Institute of Health Science, The Chinese University ofHong Kong, Hong Kong SAR, China. 4Department of Surgery, University ofHong Kong, Hong Kong SAR, China.Received: 20 April 2013 Accepted: 4 July 2013Published: 2 August 2013References1. Cheung ST, Huang DP, Hui AB, Lo KW, Ko CW, Tsang YS, Wong N, WhitneyBM, Lee JC: Nasopharyngeal carcinoma cell line (C666-1) consistentlyharbouring Epstein-Barr virus. Int J Cancer 1999, 83(1):121–126.2. Lo KW, Chung GT, To KF: Deciphering the molecular genetic basis of NPCthrough molecular, cytogenetic, and epigenetic approaches.Semin Cancer Biol 2012, 22(2):79–86.3. Lung RW, Tong JH, Sung YM, Leung PS, Ng DC, Chau SL, Chan AW, Ng EK,Lo KW, To KF: Modulation of LMP2A expression by a newly identifiedEpstein-Barr virus-encoded microRNA miR-BART22. Neoplasia 2009,11(11):1174–1184.4. Ju YS, Lee WC, Shin JY, Lee S, Bleazard T, Won JK, Kim YT, Kim JI, Kang JH,Seo JS: A transforming KIF5B and RET gene fusion in lungadenocarcinoma revealed from whole-genome and transcriptomesequencing. Genome Res 2012, 22(3):436–445.5. Liu P, Fang X, Feng Z, Guo YM, Peng RJ, Liu T, Huang Z, Feng Y, Sun X,Xiong Z, Guo X, Pang SS, Wang B, Lv X, Feng FT, Li DJ, Chen LZ, Feng QS,Huang WL, Zeng MS, Bei JX, Zhang Y, Zeng YX: Direct sequencing andcharacterization of a clinical isolate of Epstein-Barr virus fromnasopharyngeal carcinoma tissue by using next-generation sequencingtechnology. J Virol 2011, 85(21):11291–11299.6. Kwok H, Tong AH, Lin CH, Lok S, Farrell PJ, Kwong DL, Chiang AK: Genomicsequencing and comparative analysis of Epstein-Barr virus genomeisolated from primary nasopharyngeal carcinoma biopsy. PLoS One 2012,7(5):e36939.7. Cheung ST, Leung SF, Lo KW, Chiu KW, Johnson PJ, Lee JCK, Huang DP:Specific latent membrane protein 1 gene sequences in type 1 and type2 Epstein-Barr virus from nasopharyngeal carcinoma in Hong Kong.Int J Cancer 1998, 76(3):399–406.8. Tong JH, Lo KW, Au FW, Huang DP, To KF: Re: Discrete alterations in theBZLF1 promoter in tumor and non-tumor-associated Epstein-Barr virus.J Natl Cancer Inst 2003, 95(13):1008–1009.9. Edwards RH, Sitki-Green D, Moore DT, Raab-Traub N: Potential selection ofLMP1 variants in nasopharyngeal carcinoma. J Virol 2004, 78(2):868–881.10. Lo AK, To KF, Lo KW, Lung RW, Hui JW, Liao G, Hayward SD: Modulation ofLMP1 protein expression by EBV-encoded microRNAs. Proc Natl Acad SciU S A 2007, 104(41):16164–16169.11. Feederle R, Neuhierl B, Baldwin G, Bannert H, Hub B, Mautner J, Behrends U,Delecluse HJ: Epstein-Barr virus BNRF1 protein allows efficient transferfrom the endosomal compartment to the nucleus of primary Blymphocytes. J Virol 2006, 80(19):9435–9443.12. Tsai K, Thikmyanova N, Wojcechowskyj JA, Delecluse HJ, Lieberman PM: EBVtegument protein BNRF1 disrupts DAXX-ATRX to activate viral earlygene transcription. PLoS Pathog 2011, 7(11):e1002376.doi:10.1186/1750-9378-8-29Cite this article as: Tso et al.: Complete genomic sequence of Epstein-Barr virus in nasopharyngeal carcinoma cell line C666-1. Infectious Agentsand Cancer 2013 8:29.

Infectious Agents and Cancer

Complete genomic sequence of Epstein-Barr virus in nasopharyngeal carcinoma cell line C666-1

http://hub.hku.hk/bitstream/10722/193924/1/Content.pdf

Complete genomic sequence of Epstein-Barr virus in nasopharyngeal carcinoma cell line C666-1

Abstract

Similar works

Full text

Available Versions

HKU Scholars Hub