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A comprehensive single cell data analysis of in lymphoblastoid cells reveals the role of Super-enhancers in maintaining EBV latency

Bingyu Yan, Chong Wang, Srishti Chakravorty, Zonghao Zhang, Simran D. Kadadi, Yuxin Zhuang, Isabella Sirit, Yonghua Hu, Minwoo Jung, Subhransu Sahoo, Luopin Wang, Kunming Shao, Nicole L. Anderson, Jorge L. Trujillo-Ochoa, Xing Liu, Matthew R. Olson, Behdad Afzali, View ORCID ProfileBo Zhao, View ORCID ProfileMajid Kazemian
doi: https://doi.org/10.1101/2022.08.10.503552
Bingyu Yan
1Department of Biochemistry, Purdue University, West Lafayette IN, USA
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Chong Wang
2Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
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Srishti Chakravorty
1Department of Biochemistry, Purdue University, West Lafayette IN, USA
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Zonghao Zhang
3Department of Agricultural and Biological Engineering, Purdue University, West Lafayette IN, USA
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Simran D. Kadadi
4Department of Computer Science, Purdue University, West Lafayette IN, USA
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Yuxin Zhuang
1Department of Biochemistry, Purdue University, West Lafayette IN, USA
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Isabella Sirit
5Department of Biological Sciences, Purdue University, West Lafayette IN, USA
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Yonghua Hu
5Department of Biological Sciences, Purdue University, West Lafayette IN, USA
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Minwoo Jung
4Department of Computer Science, Purdue University, West Lafayette IN, USA
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Subhransu Sahoo
1Department of Biochemistry, Purdue University, West Lafayette IN, USA
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Luopin Wang
4Department of Computer Science, Purdue University, West Lafayette IN, USA
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Kunming Shao
3Department of Agricultural and Biological Engineering, Purdue University, West Lafayette IN, USA
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Nicole L. Anderson
5Department of Biological Sciences, Purdue University, West Lafayette IN, USA
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Jorge L. Trujillo-Ochoa
6Immunoregulation Section, Kidney Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda, MD, USA
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Xing Liu
1Department of Biochemistry, Purdue University, West Lafayette IN, USA
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Matthew R. Olson
5Department of Biological Sciences, Purdue University, West Lafayette IN, USA
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Behdad Afzali
6Immunoregulation Section, Kidney Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda, MD, USA
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Bo Zhao
2Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
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  • For correspondence: bzhao@bwh.harvard.edu kazemian@purdue.edu
Majid Kazemian
1Department of Biochemistry, Purdue University, West Lafayette IN, USA
4Department of Computer Science, Purdue University, West Lafayette IN, USA
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  • ORCID record for Majid Kazemian
  • For correspondence: bzhao@bwh.harvard.edu kazemian@purdue.edu
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Abstract

We probed the lifecycle of EBV on a cell-by-cell basis using single cell RNA sequencing (scRNA-seq) data from nine publicly available lymphoblastoid cell lines (LCL). While the majority of LCLs comprised cells containing EBV in the latent phase, two other clusters of cells were clearly evident and were distinguished by distinct expression of host and viral genes. Notably, both were high expressors of EBV LMP1/BNLF2 and BZLF1 compared to another cluster that expressed neither gene. The two novel clusters differed from each other in their expression of EBV lytic genes, including glycoprotein gene GP350. The first cluster, comprising GP350−LMP1hi cells, expressed high levels of HIF1A and was transcriptionally regulated by HIF1-α. Treatment of LCLs with Pevonedistat, a drug that enhances HIF1-α signaling, markedly induced this cluster. The second cluster, containing GP350+LMP1hi cells, expressed EBV lytic genes. Host genes that are controlled by super-enhancers (SEs), such as transcription factors MYC and IRF4, had the lowest expression in this cluster. Functionally, the expression of genes regulated by MYC and IRF4 in GP350+LMP1hi cells were lower compared to other cells. Indeed, induction of EBV lytic reactivation in EBV+ AKATA reduced the expression of these SE-regulated genes. Furthermore, CRISPR-mediated perturbation of the MYC or IRF4 SEs in LCLs induced the lytic EBV gene expression, suggesting that host SEs and/or SE target genes are required for maintenance of EBV latency. Collectively, our study revealed EBV associated heterogeneity among LCLs that may have functional consequence on host and viral biology.

Importance Epstein-Barr virus (EBV) establishes a life-long latency program within host cells. As such, EBV immortalized lymphoblastoid cells (LCLs) often carry the latent EBV genome and only a small percentage of LCLs containing lytic EBV. However, the cellular programs that distinguish latent from lytic cells and the heterogeneity of cells in latent or lytic phases remains poorly explored. To explore these unknowns, we reanalyzed publicly available single cell RNA-seq data from nine LCLs. This approach permitted the simultaneous study of cells in both latent and lytic phases. We identified three cell populations with distinct lytic/latent activity and further characterized the transcriptomes of these cells. We also identified a new role of super-enhancers in regulating EBV lytic replication. Collectively, our studies revealed EBV associated heterogeneity among LCLs that contribute to EBV life cycle and biology.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted August 12, 2022.
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A comprehensive single cell data analysis of in lymphoblastoid cells reveals the role of Super-enhancers in maintaining EBV latency
Bingyu Yan, Chong Wang, Srishti Chakravorty, Zonghao Zhang, Simran D. Kadadi, Yuxin Zhuang, Isabella Sirit, Yonghua Hu, Minwoo Jung, Subhransu Sahoo, Luopin Wang, Kunming Shao, Nicole L. Anderson, Jorge L. Trujillo-Ochoa, Xing Liu, Matthew R. Olson, Behdad Afzali, Bo Zhao, Majid Kazemian
bioRxiv 2022.08.10.503552; doi: https://doi.org/10.1101/2022.08.10.503552
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A comprehensive single cell data analysis of in lymphoblastoid cells reveals the role of Super-enhancers in maintaining EBV latency
Bingyu Yan, Chong Wang, Srishti Chakravorty, Zonghao Zhang, Simran D. Kadadi, Yuxin Zhuang, Isabella Sirit, Yonghua Hu, Minwoo Jung, Subhransu Sahoo, Luopin Wang, Kunming Shao, Nicole L. Anderson, Jorge L. Trujillo-Ochoa, Xing Liu, Matthew R. Olson, Behdad Afzali, Bo Zhao, Majid Kazemian
bioRxiv 2022.08.10.503552; doi: https://doi.org/10.1101/2022.08.10.503552

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