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Highly Efficient CRISPR/Cas9-Mediated Cloning and Functional Characterization of Gastric Cancer-Derived Epstein-Barr Virus Strains
- Source :
- Journal of Virology. 90:4383-4393
- Publication Year :
- 2016
- Publisher :
- American Society for Microbiology, 2016.
-
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. IMPORTANCE 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.
- Subjects :
- 0301 basic medicine
Chromosomes, Artificial, Bacterial
Epstein-Barr Virus Infections
Herpesvirus 4, Human
Molecular Sequence Data
Immunology
Genome, Viral
Biology
Virus Replication
medicine.disease_cause
Microbiology
Genome
Transformation and Oncogenesis
Virus
Malignant transformation
Viral Proteins
03 medical and health sciences
0302 clinical medicine
Cytopathogenic Effect, Viral
Stomach Neoplasms
Cell Line, Tumor
hemic and lymphatic diseases
Virology
medicine
Humans
CRISPR
Cloning, Molecular
Phylogeny
Cloning
Bacterial artificial chromosome
Base Sequence
Computational Biology
Molecular Sequence Annotation
Sequence Analysis, DNA
Epstein–Barr virus
Molecular biology
030104 developmental biology
030220 oncology & carcinogenesis
Insect Science
DNA, Viral
Gene Targeting
ras Proteins
CRISPR-Cas Systems
Carcinogenesis
Sequence Alignment
Plasmids
Subjects
Details
- ISSN :
- 10985514 and 0022538X
- Volume :
- 90
- Database :
- OpenAIRE
- Journal :
- Journal of Virology
- Accession number :
- edsair.doi.dedup.....4131b74c17ac96faad0129c540875a8b