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RNA Sequencing Analyses of Gene Expression during Epstein-Barr Virus Infection of Primary B Lymphocytes.
J Virol. 2019 07 01; 93(13)JV

Abstract

Epstein-Barr virus (EBV) infection of human primary resting B lymphocytes (RBLs) leads to the establishment of lymphoblastoid cell lines (LCLs) that can grow indefinitely in vitro EBV transforms RBLs through the expression of viral latency genes, and these genes alter host transcription programs. To globally measure the transcriptome changes during EBV transformation, primary human resting B lymphocytes (RBLs) were infected with B95.8 EBV for 0, 2, 4, 7, 14, 21, and 28 days, and poly(A) plus RNAs were analyzed by transcriptome sequencing (RNA-seq). Analyses of variance (ANOVAs) found 3,669 protein-coding genes that were differentially expressed (false-discovery rate [FDR] < 0.01). Ninety-four percent of LCL genes that are essential for LCL growth and survival were differentially expressed. Pathway analyses identified a significant enrichment of pathways involved in cell proliferation, DNA repair, metabolism, and antiviral responses. RNA-seq also identified long noncoding RNAs (lncRNAs) differentially expressed during EBV infection. Clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) and CRISPR activation (CRISPRa) found that CYTOR and NORAD lncRNAs were important for LCL growth. During EBV infection, type III EBV latency genes were expressed rapidly after infection. Immediately after LCL establishment, EBV lytic genes were also expressed in LCLs, and ∼4% of the LCLs express gp350. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) and POLR2A chromatin interaction analysis followed by paired-end tag sequencing (ChIA-PET) data linked EBV enhancers to 90% of EBV-regulated genes. Many genes were linked to enhancers occupied by multiple EBNAs or NF-κB subunits. Incorporating these assays, we generated a comprehensive EBV regulome in LCLs.IMPORTANCE Epstein-Barr virus (EBV) immortalization of resting B lymphocytes (RBLs) is a useful model system to study EBV oncogenesis. By incorporating transcriptome sequencing (RNA-seq), chromatin immune precipitation followed by deep sequencing (ChIP-seq), chromatin interaction analysis followed by paired-end tag sequencing (ChIA-PET), and genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen, we identified key pathways that EBV usurps to enable B cell growth and transformation. Multiple layers of regulation could be achieved by cooperations between multiple EBV transcription factors binding to the same enhancers. EBV manipulated the expression of most cell genes essential for lymphoblastoid cell line (LCL) growth and survival. In addition to proteins, long noncoding RNAs (lncRNAs) regulated by EBV also contributed to LCL growth and survival. The data presented in this paper not only allowed us to further define the molecular pathogenesis of EBV but also serve as a useful resource to the EBV research community.

Authors+Show Affiliations

Division of Infectious Disease, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.Division of Infectious Disease, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.Division of Infectious Disease, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. Department of Medicine, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.Division of Infectious Disease, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.Division of Infectious Disease, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.Division of Infectious Disease, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.Division of Infectious Disease, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.Division of Infectious Disease, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.Department of Medicine, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.Division of Infectious Disease, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.Department of Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA Mingxiang.teng@moffitt.org bzhao@bwh.harvard.edu.Division of Infectious Disease, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA Mingxiang.teng@moffitt.org bzhao@bwh.harvard.edu.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

31019051

Citation

Wang, Chong, et al. "RNA Sequencing Analyses of Gene Expression During Epstein-Barr Virus Infection of Primary B Lymphocytes." Journal of Virology, vol. 93, no. 13, 2019.
Wang C, Li D, Zhang L, et al. RNA Sequencing Analyses of Gene Expression during Epstein-Barr Virus Infection of Primary B Lymphocytes. J Virol. 2019;93(13).
Wang, C., Li, D., Zhang, L., Jiang, S., Liang, J., Narita, Y., Hou, I., Zhong, Q., Zheng, Z., Xiao, H., Gewurz, B. E., Teng, M., & Zhao, B. (2019). RNA Sequencing Analyses of Gene Expression during Epstein-Barr Virus Infection of Primary B Lymphocytes. Journal of Virology, 93(13). https://doi.org/10.1128/JVI.00226-19
Wang C, et al. RNA Sequencing Analyses of Gene Expression During Epstein-Barr Virus Infection of Primary B Lymphocytes. J Virol. 2019 07 1;93(13) PubMed PMID: 31019051.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - RNA Sequencing Analyses of Gene Expression during Epstein-Barr Virus Infection of Primary B Lymphocytes. AU - Wang,Chong, AU - Li,Difei, AU - Zhang,Luyao, AU - Jiang,Sizun, AU - Liang,Jun, AU - Narita,Yohei, AU - Hou,Isabella, AU - Zhong,Qian, AU - Zheng,Zeguang, AU - Xiao,Haipeng, AU - Gewurz,Benjamin E, AU - Teng,Mingxiang, AU - Zhao,Bo, Y1 - 2019/06/14/ PY - 2019/02/08/received PY - 2019/04/05/accepted PY - 2019/4/26/pubmed PY - 2020/5/29/medline PY - 2019/4/26/entrez KW - ChIA-PET KW - ChIP-seq KW - EBNA KW - EBV KW - RNA-seq KW - lncRNA JF - Journal of virology JO - J Virol VL - 93 IS - 13 N2 - Epstein-Barr virus (EBV) infection of human primary resting B lymphocytes (RBLs) leads to the establishment of lymphoblastoid cell lines (LCLs) that can grow indefinitely in vitro EBV transforms RBLs through the expression of viral latency genes, and these genes alter host transcription programs. To globally measure the transcriptome changes during EBV transformation, primary human resting B lymphocytes (RBLs) were infected with B95.8 EBV for 0, 2, 4, 7, 14, 21, and 28 days, and poly(A) plus RNAs were analyzed by transcriptome sequencing (RNA-seq). Analyses of variance (ANOVAs) found 3,669 protein-coding genes that were differentially expressed (false-discovery rate [FDR] < 0.01). Ninety-four percent of LCL genes that are essential for LCL growth and survival were differentially expressed. Pathway analyses identified a significant enrichment of pathways involved in cell proliferation, DNA repair, metabolism, and antiviral responses. RNA-seq also identified long noncoding RNAs (lncRNAs) differentially expressed during EBV infection. Clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) and CRISPR activation (CRISPRa) found that CYTOR and NORAD lncRNAs were important for LCL growth. During EBV infection, type III EBV latency genes were expressed rapidly after infection. Immediately after LCL establishment, EBV lytic genes were also expressed in LCLs, and ∼4% of the LCLs express gp350. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) and POLR2A chromatin interaction analysis followed by paired-end tag sequencing (ChIA-PET) data linked EBV enhancers to 90% of EBV-regulated genes. Many genes were linked to enhancers occupied by multiple EBNAs or NF-κB subunits. Incorporating these assays, we generated a comprehensive EBV regulome in LCLs.IMPORTANCE Epstein-Barr virus (EBV) immortalization of resting B lymphocytes (RBLs) is a useful model system to study EBV oncogenesis. By incorporating transcriptome sequencing (RNA-seq), chromatin immune precipitation followed by deep sequencing (ChIP-seq), chromatin interaction analysis followed by paired-end tag sequencing (ChIA-PET), and genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen, we identified key pathways that EBV usurps to enable B cell growth and transformation. Multiple layers of regulation could be achieved by cooperations between multiple EBV transcription factors binding to the same enhancers. EBV manipulated the expression of most cell genes essential for lymphoblastoid cell line (LCL) growth and survival. In addition to proteins, long noncoding RNAs (lncRNAs) regulated by EBV also contributed to LCL growth and survival. The data presented in this paper not only allowed us to further define the molecular pathogenesis of EBV but also serve as a useful resource to the EBV research community. SN - 1098-5514 UR - https://www.unboundmedicine.com/medline/citation/31019051/RNA_Sequencing_Analyses_of_Gene_Expression_during_Epstein_Barr_Virus_Infection_of_Primary_B_Lymphocytes_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/31019051/ DB - PRIME DP - Unbound Medicine ER -