Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection
- Center for Virology, Duke University School of Medicine, United States
- Imperial College London, United Kingdom
- Harvard Medical School, United States
- University of Wisconsin School of Medicine and Public Health, United States
- Duke University, United States
Figures
Figure 1 with 1 supplement
BH3 profiling reveals two distinct stages of mitochondrial priming after EBV infection.
(A) Schematic of the BH3 profiling technique, which involves first permeabilizing the outer membrane followed by incubation with BH3-only peptides to induce depolarization of the mitochondrial …
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Figure 1—source data 1
Source data for individual responses to BH3 peptides of uninfected, early-infected, and late-infected B cells.
- https://doi.org/10.7554/eLife.22509.004
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Figure 1—source data 2
Source data for individual responses to Bim 1, Puma 10, Bad, and Bmf BH3 peptides of uninfected, early-infected, and late-infected B cells.
- https://doi.org/10.7554/eLife.22509.005
Figure 1—figure supplement 1
Varying sensitivity to select BH3-only peptides reveals differences in apoptotic regulation during early- and late-infection with EBV.
Paired t-test analysis of the mitochondrial depolarizations from uninfected B cells, hyper-proliferating infected cells (Prolif), and LCLs when treated with 1 µM Bim, 10 µM Puma, and 100 µM of Bad …
Figure 2
EBV Infection promotes potent resistance to BCL-2 antagonists.
(A) Schematic of drug treatment time course. (B) Dose-response curves generated from treating 3–5 human donors with the BCL-2, -xL, and –w inhibitor ABT-737. Percent survival is the percent of …
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Figure 2—source data 1
Source data for cell counts and apoptosis assays performed with BH3 mimetics targeting BCL-2.
- https://doi.org/10.7554/eLife.22509.008
Figure 3 with 1 supplement
MCL-1 collaborates with BCL-2 to protect EBV-infected proliferating B cells early after infection while BFL-1 additionally protects LCLs late after infection.
(A) Quantitative PCR (qPCR) of MCL-1, BCL-2, and BFL-1 mRNA levels post EBV infection. Average plus SEM of three human donors is plotted. (B) Immunoblot analysis of MCL-1 isoforms, BCL-2, EBNA2, …
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Figure 3—source data 1
Source data for protein and mRNA expression levels of anti-apoptotic members, apoptotic assays, and characterization of a BFL1-CRISPR mutant.
- https://doi.org/10.7554/eLife.22509.010
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Figure 3—source data 2
Source data for Figure 3—figure supplement 1.
Source data for mRNA levels and apoptosis assays for cells treated with flavoporidol.
- https://doi.org/10.7554/eLife.22509.011
Figure 3—figure supplement 1
Flavopiridol sensitizes EBV-infected early-proliferating B cells to ABT-737.
(A) qPCR of one donor LCL in triplicate shows that treatment with 100 nM flavopiridol reduces MCL-1 mRNA levels over time without affecting mRNA levels of BCL-2 or BFL-1. (B) Representative …
Figure 4
Resistance to BCL-2 antagonism is virus specific.
(A) Flow cytometry plot of proliferating (Prolif) EBV-infected PBMCs. (B) Same as in (A), but treated with the TLR9-ligand CpG DNA. (C) Same as in (A), but treated with soluble recombinant CD40L and …
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Figure 4—source data 1
Source data for cell counts and apoptotic assays of uninfected, mitogen-stimulated B cells.
- https://doi.org/10.7554/eLife.22509.014
Figure 5 with 1 supplement
EBV-induced resistance to BCL-2 antagonism is mediated by EBNA3A.
(A) Schematic to show the genetic differences between prototypical transforming strain of EBV (B95-8) and P3HR1 (EBNA2-deleted) strain. (B) Dose-response curves to assess ABT-737 sensitivity in …
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Figure 5—source data 1
Source data for cell counts performed with cells infected with EBV mutant strains and mRNA levels of EBV viral transcripts.
- https://doi.org/10.7554/eLife.22509.016
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Figure 5—source data 2
Source data for Figure 5—figure supplement 1.
Source data for cell counts
- https://doi.org/10.7554/eLife.22509.017
Figure 5—figure supplement 1
ABT-737 resistance is gained within two days post infection.
(A) Growth curve showing the effect of ABT-737 addition on proliferating EBV-infected B cells on different days of addition. Data is shown as the SEM of two matched human donors normalized to the …
Figure 6 with 1 supplement
EBV EBNA3A is required for MCL-1 mitochondrial localization and BFL-1 transcription.
(A) BH3 profile shows increased sensitivity to Bad and Bmf peptides in EBNA3A-deleted LCLs compared to wildtype LCLs, indicative of a BCL-2 dependence. Three technical replicates with seven repeated …
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Figure 6—source data 1
Source data for individual responses to BH3 peptides, mRNA and protein levels.
- https://doi.org/10.7554/eLife.22509.020
Figure 6—figure supplement 1
Additional validation of BFL-1 mRNA regulation by EBNA3A.
(A) qPCR of three individual clones △3A LCLs and their matching 3A-revertant counterparts showing BFL-1 mRNA expression. (B) Two clones of an EBNA3A-regulatable cell line with EBNA3A under control …
Additional files
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Supplementary file 1
Antibodies used for western blot and chromatin immunoprecipitation are included below.
- https://doi.org/10.7554/eLife.22509.022
