Article Figures & Data
Figures
- FIG. 1.
BSAP binding sites in Wp. (A) Map of the Wp440 reporter construct showing the UAS1 and UAS2 regulatory sequences (black boxes) containing binding sites for known transcription factors, and the Wp mutants Δ1 (with UAS1 deleted), Δ2 (with UAS2 deleted), Δ1+Δ2 (double deletion), and BSAPm (mutations introduced into BSAP sites B and D as shown). (B) Electrophoretic mobility shift assay with a BSAP binding H2B2.1 probe. Radiolabeled probe was incubated alone (probe), with T cell (Jurkat) or B cell (DG75) nuclear extract, or with in vitro-translated BSAP (IVT BSAP) protein. Competition assays were carried out with Wp BSAP site binding sequences (B and D) and with their mutated derivatives (Bm and Dm). Supershift assays were performed with a BSAP-specific antibody (BSAP Ab) and an irrelevant YY1 antibody (YY1 Ab). (C) Wp reporter activity assayed by quantifying luciferase expression (arbitrary units) in BJAB and K562 cell lines transiently transfected with the constructs shown in A. (D) ChIP assays to examine BSAP and EBNA1 binding activities in two different LCLs. Immunoprecipitates were analyzed by Q-PCR for cellular (CD19) and EBV (Wp UAS1, oriP, and Qp) DNA sequences, and in each case, the results obtained (black bars) are shown relative to those obtained with a control antibody (open bars). Assays were performed in duplicate, and the amount of DNA present in immunoprecipitates is expressed as a percentage of input DNA values.
- FIG. 2.
Delivery of recombinant viral genomes to the nucleus. (A) Schematic representation of the 11W recombinant 2089 and the panel of 2W wild-type and 2W mutant recombinant genomes, all of which carry the genes for hygromycin (HYG) resistance and green fluorescent protein (GFP); also shown are the BamHI W repeats (gray boxes), terminal repeats (TR), and origin of latent replication (oriP). (B) Detection of viral genomes (green spots) within B-cell nuclei (blue DAPI stains) by FISH using a fluorescein-labeled EBV DNA cosmid probe. Upper panels show hybridization to uninfected B cells and to the EBV-positive Namalwa and Raji cell lines. Lower panels show EBV genomes detected in primary B cells 24 h p.i. with the indicated 2W recombinant viruses (50 MOI). (C) Data showing the percentages of B cells acquiring detectable EBV genomes within 24 h of exposure to the indicated 2W recombinant viruses and to the 11W virus (all at 50 MOI).
- FIG. 3.
EBV latent gene transcription in primary B cells infected with recombinant viruses. Wp and EBNA2 transcripts (assayed at 24 h p.i.) and Cp transcripts (at 48 h p.i.) were quantified by Q-RT-PCR in B cells exposed to the indicated viruses (50 MOI) alone (A) or in combination with the EBNA2KO virus (E2KO) (B). The exon structures of the transcripts are shown on the left together with the relative positions of the primers (open boxes) and probes (black boxes) used in the Q-RT-PCR assays. Values are expressed relative to those seen in cells infected with the 2W wild-type virus alone.
- FIG. 4.
EBV latent antigen expression in B cells infected with recombinant viruses. (A) Immunofluorescence staining for EBNA-LP, EBNA2, and LMP1 in primary B cells either unexposed (mock) or exposed to 2W wild-type or BSAPm virus (50 MOI), in each case with or without the EBNA2KO virus (50 MOI), and stained 4 days p.i.; blue DAPI staining shows all nuclei in the field. (B) Summary of results showing percentages of cells positive for EBNA-LP, EBNA2, and LMP1 at day 4 p.i.
- FIG. 5.
Transformation efficiencies of recombinant viruses. (A) Bright-field images of B-cell cultures 5 and 28 days p.i. with the indicated recombinant viruses (50 MOI), with or without coinfection with the EBNA2KO virus (50 MOI). (B) FACS analysis of cell proliferation using CFSE-labeled B cells infected as described above in the presence of the EBNA2KO virus and cultured for 4 and 8 days p.i.; cell proliferation is seen as a decrease in CFSE staining. d4, day 4; d8, day 8. (C) Number of wells from a 96-well plate showing transformation within 8 weeks following exposure to the 2W wild-type and BSAPm viruses at a range of MOIs, with or without EBNA2KO virus (E2KO; 50 MOI).
- FIG. 6.
EBV latent antigen expression in AdAH epithelial cells infected with recombinant viruses by transfer infection. (A) Immunofluorescence staining for EBNA1, LMP1, and BZLF1 in AdAH cells cultured for 4 days p.i. with the indicated viruses (50 MOI). (B) Summary of results showing percentages of cells positive for EBNA1, LMP1, and BZLF1 at day 4 p.i.
Tables
- TABLE 1.
Real-time PCR primer/probe combinations used to amplify immunoprecipitates from ChIP assays
Target Primer or probe and sequencea Concn EBV sequence coordinates CD19 5′ primer, 5′-TGGAGAATGGGGCCTGAG 300 nM 3′ primer, 5′-AGGTGGCATGGTGGTCAGAC 300 nM Probe, 5′-CAGCATCCCCTGCGCGAAGCT 200 nM Wp 5′ primer, 5′-GAATGGGCGCCATTTTGTC 300 nM 14079-14098 3′ primer, 5′-GGCTTATTCCTCTTTTCCCCTCTA 300 nM 14200-14177 Probe, 5′-AGATAGCAGCAGCGCAGCCAACCATA 200 nM 14175-14150 oriP 5′ primer, 5′-ATGTAAATAAAACCGTGACAGCTCAT 1 μM 8957-8982 3′ primer, 5′-TTACCCAACGGGAAGCATATG 1 μM 9038-9058 Probe, 5′-TGGGAGATATCGCTGTTCCTTAGGACCC 200 nM 8987-9014 Qp 5′ primer, 5′-TTGAAAAGGCGCGGGATA 1 μM 62420-62437 3′ primer, 5′-TCCCAGCTGCCCAAAATG 1 μM 62503-62486 Probe, 5′-TAAGGATAGCATGTATTACCCGCCATCCG 200 nM 62477-62449 ↵ a The TaqMan probes were labeled with a 6-carboxyfluorescein phosphoramidite reporter dye at the 5′ end and 6-carboxytetramethyl-rhodamine at the 3′ end.
