title: Highly Efficient CRISPR/Cas9-Mediated Cloning and Functional Characterization of Gastric Cancer-Derived Epstein-Barr Virus Strains.
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.
  
  
  OBJECTIVE
  Recent progress in DNA-sequencing technology has accelerated EBV whole-genome sequencing, and the repertoire of sequenced EBV genomes is increasing progressively. Accordingly, the presence of EBV variant strains that may be relevant to EBV-associated diseases has begun to attract interest. Clearly, the determination of additional disease-associated viral genome sequences will facilitate the identification of any disease-specific EBV variants. We found that CRISPR/Cas9-mediated cleavage of EBV episomal DNA enabled the cloning of disease-associated viral strains with unprecedented efficiency. As a proof of concept, two gastric cancer cell-derived EBV strains were cloned, and the infection of epithelial cells with reconstituted viruses provided important clues about the mechanism of EBV-mediated epithelial carcinogenesis. This experimental system should contribute to establishing the relationship between viral genome variation and EBV-associated diseases.
authors.affiliation: Division of Microbiology and Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan tkanda@aichi-cc.jp.
authors.forename: Teru
authors.initials: T
authors.lastname: Kanda
authors.affiliation: Department of Virology, Tohoku University School of Medicine, Sendai, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Furuse
authors.affiliation: Department of Virology, Tohoku University School of Medicine, Sendai, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Oshitani
authors.affiliation: Division of Carcinogenesis and Cancer Prevention, National Cancer Center Research Institute, Tokyo, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Kiyono
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D045744:Cell Line, Tumor; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D003588:Cytopathogenic Effect, Viral; D004279:DNA, Viral; D020031:Epstein-Barr Virus Infections / Q000150:complications / Q000821:virology; D018390:Gene Targeting / Q000379:methods; D016679:Genome, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013274:Stomach Neoplasms / Q000209:etiology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004279:DNA, Viral; D014764:Viral Proteins; D018631:ras Proteins