Characterization of a new CCCTC-binding factor binding site as a dual regulator of Epstein-Barr virus latent infection

Sun Hee Lee; Kyoung-Dong Kim; Miyeon Cho; Sora Huh; Seong Ho An; Donghyun Seo; Kyuhyun Kang; Minhee Lee; Hideki Tanizawa; Inuk Jung; Hyosun Cho; Hyojeung Kang

doi:10.1371/journal.ppat.1011078

Characterization of a new CCCTC-binding factor binding site as a dual regulator of Epstein-Barr virus latent infection

PLoS Pathog. 2023 Jan 25;19(1):e1011078. doi: 10.1371/journal.ppat.1011078. eCollection 2023 Jan.

Authors

Affiliations

¹ College of Pharmacy, Vessel-Organ Interaction Research Center, Research Institute of Pharmaceutical Science, Kyungpook National University, Daegu, Korea.
² Department of Systems Biotechnology, Chung-Ang University, Anseong, Korea.
³ Institute of Molecular Biology, University of Oregon, Eugene, Oregon, United States of America.
⁴ Department of Computer Science and Engineering, Kyungpook National University, Daegu, Korea.
⁵ Duksung Innovative Drug Center, College of Pharmacy, Duksung Women's University, Seoul, Korea.

Abstract

Distinct viral gene expression characterizes Epstein-Barr virus (EBV) infection in EBV-producing marmoset B-cell (B95-8) and EBV-associated gastric carcinoma (SNU719) cell lines. CCCTC-binding factor (CTCF) is a structural chromatin factor that coordinates chromatin interactions in the EBV genome. Chromatin immunoprecipitation followed by sequencing against CTCF revealed 16 CTCF binding sites in the B95-8 and SNU719 EBV genomes. The biological function of one CTCF binding site (S13 locus) located on the BamHI A right transcript (BART) miRNA promoter was elucidated experimentally. Microscale thermophoresis assay showed that CTCF binds more readily to the stable form than the mutant form of the S13 locus. EBV BART miRNA clusters encode 22 miRNAs, whose roles are implicated in EBV-related cancer pathogenesis. The B95-8 EBV genome lacks a 11.8-kb EcoRI C fragment, whereas the SNU719 EBV genome is full-length. ChIP-PCR assay revealed that CTCF, RNA polymerase II, H3K4me3 histone, and H3K9me3 histone were more enriched at S13 and S16 (167-kb) loci in B95-8 than in the SNU719 EBV genome. 4C-Seq and 3C-PCR assays using B95-8 and SNU719 cells showed that the S13 locus was associated with overall EBV genomic loci including 3-kb and 167-kb region in both EBV genomes. We generated mutations in the S13 locus in bacmids with or without the 11.8-kb BART transcript unit (BART(+/-)). The S13 mutation upregulated BART miRNA expression, weakened EBV latency, and reduced EBV infectivity in the presence of EcoRI C fragment. Another 3C-PCR assay using four types of BART(+/-)·S13(wild-type(Wt)/mutant(Mt)) HEK293-EBV cells revealed that the S13 mutation decreased DNA associations between the 167-kb region and 3-kb in the EBV genome. Based on these results, CTCF bound to the S13 locus along with the 11.8-kb EcoRI C fragment is suggested to form an EBV 3-dimensional DNA loop for coordinated EBV BART miRNA expression and infectivity.

Copyright: © 2023 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Binding Sites
CCCTC-Binding Factor / genetics
Chromatin
Epstein-Barr Virus Infections* / genetics
HEK293 Cells
Herpesvirus 4, Human / genetics
Histones / genetics
Humans
Latent Infection*
MicroRNAs* / genetics

Substances

CCCTC-Binding Factor
Histones
MicroRNAs
Chromatin

Grants and funding

This work was supported by 1) grants from the National Research Foundation of Korea (2018R1D1A3B07045094 (HK), 2019R1I1A3A01059629 (HK), 2022R1C1C2004274 (SHL), 2022R1I1A2066092 (HK)), 2) a grant from the Priority Research Centers Program through the National Research Foundation funded by the Korean Ministry of Education, Science, and Technology (2016R1A6A1A03007648 (HC)), 3) grants from the National Research Foundation of Korea grant funded by the Korean Government (MSIT) (2019R1F1A1061826 (KDK), 2020R1A5A2017323 (HK)), and 4) a grant from the 4TH BK21 project (Educational Research Group for Platform development of management of emerging infectious disease) funded by the Korean ministry of education (5199990614732 (HK)). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.