Highly efficient CRISPR/ Cas9-mediated cloning and functional characterization of gastric cancer-derived Epstein-Barr virus strains

Teru Kanda, Yuki Furuse, Hitoshi Oshitani, Tohru Kiyono

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

The Epstein-Barr virus (EBV) is etiologically linked to approximately 10% of gastric cancers, in which viral genomes are maintained as multicopy episomes. EBV-positive gastric cancer cells are incompetent for progeny virus production, making viral DNA cloning extremely difficult. Here we describe a highly efficient strategy for obtaining bacterial artificial chromosome (BAC) clones of EBV episomes by utilizing a CRISPR/Cas9-mediated strand break of the viral genome and subsequent homology-directed repair. EBV strains maintained in two gastric cancer cell lines (SNU719 and YCCEL1) were cloned, and their complete viral genome sequences were determined. Infectious viruses of gastric cancer cell-derived EBVs were reconstituted, and the viruses established stable latent infections in immortalized keratinocytes. While Ras oncoprotein overexpression caused massive vacuolar degeneration and cell death in control keratinocytes, EBV-infected keratinocytes survived in the presence of Ras expression. These results implicate EBV infection in predisposing epithelial cells to malignant transformation by inducing resistance to oncogene-induced cell death.

Original languageEnglish
Pages (from-to)4383-4393
Number of pages11
JournalJournal of virology
Volume90
Issue number9
DOIs
Publication statusPublished - 2016 May 1

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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