Fig. 2

Clearance of EBV genomes reduces VM formation. a Schematic representation of the EBNA1 gene and its gRNA target sites. Primers for T7E1 assay are shown (see Supplementary Fig. 2a). b Western blot analysis of EBNA1 in NPC-EBV cells transduced with control lentivirus or lentivirus expressing two EBNA1 gRNAs. c CNE2-EBV cells carrying recombinant EBV-GFP virions were subjected to CRISPR/Cas9-mediated EBV targeting as in b, and the GFP expression was determined by flow cytometry. Mean ± SD, n = 2. d Relative EBV copy numbers in NPC-EBV cells infected with control lentivirus or lentivirus carrying EBNA1 gRNAs, as determined by real-time PCR using primers directed to BALF5 and EBNA1. Data are represented as the mean ± SD, n = 3, two-tailed paired t-test. e (left) Images of TW03-EBV cells transduced with control lentivirus or lentivirus expressing EBNA1 gRNAs grown in conventional 2D culture plates. Scale bars = 100 μm. (right) Proportions of sheet-like and thread-like cell types were determined by microscopy with 200× magnification in randomly chosen fields. Mean ± SD, n = 5, two-tailed unpaired t-test with Welch’s correction. f (top) Images of the cells as in e, grown on 3D Matrigel. Scale bars = 50 μm. (bottom) Quantification of the tube numbers. Tubes were counted under a microscope with 200× magnification. Mean ± SD, n = 5, two-tailed Mann–Whitney test. g Tube formation of CNE2-EBV cells transfected with empty vector or HA-tagged dominant-negative EBNA1 mutant (dnEBNA1). Mean ± SD, n = 5, two-tailed paired t-test. h (top) Images of xenografts formed by CNE2-EBV cells transduced with lentiviral vector or lentivirus expressing EBNA1 gRNA #1 or #2 at day 20 post-implantation. (bottom) Growth curves of xenografts formed by parental CNE2-EBV cells and CNE2-EBV cells subjected to EBNA1 deletion by gRNA #1 and #2. Mean ± SD, n = 4, two-tailed Mann–Whitney test. *p < 0.05, **p < 0.01 and ***p < 0.001