Epstein-Barr virus provides a new paradigm: a requirement for the immediate inhibition of apoptosis

Abstract

DNA viruses such as herpesviruses are known to encode homologs of cellular antiapoptotic viral Bcl-2 proteins (vBcl-2s), which protect the virus from apoptosis in its host cell during virus synthesis. Epstein-Barr virus (EBV), a human tumor virus and a prominent member of gamma-herpesviruses, infects primary resting B lymphocytes to establish a latent infection and yield proliferating, growth-transformed B cells in vitro. In these cells, 11 viral genes that contribute to cellular transformation are consistently expressed. EBV also encodes two vBcl-2 genes whose roles are unclear. Here we show that the genetic inactivation of both vBcl-2 genes disabled EBV's ability to transform primary resting B lymphocytes. Primary B cells infected with a vBcl-2-negative virus did not enter the cell cycle and died of immediate apoptosis. Apoptosis was abrogated in infected cells in which vBcl-2 genes were maximally expressed within the first 24 h postinfection. During latent infection, however, the expression of vBcl-2 genes became undetectable. Thus, both vBcl-2 homologs are essential for initial cellular transformation but become dispensable once a latent infection is established. Because long-lived, latently infected memory B cells and EBV-associated B-cell lymphomas are derived from EBV-infected proapoptotic germinal center B cells, we conclude that vBcl-2 genes are essential for the initial evasion of apoptosis in cells in vivo in which the virus establishes a latent infection or causes cellular transformation or both.

Figure 1. Construction and Genealogy of EBV Mutants and Their Efficiency of B-Cell Growth Transformation

(A) Construction of the loss of function mutation in BALF1 and BHRF1. (B) Wild-type (WT) 2089 EBV is based on the genome of the EBV strain B95.8. The BALF1 and BHRF1 genes were inactivated through insertional mutagenesis with antibiotic resistance genes. Two vBcl-2 mutants 2636 and 2765 were constructed independently on the basis of the singly inactivated viruses, and the BALF1⁻/BHRF1⁻ mutant 2636 was reverted to BALF1⁺/BHRF1⁻. (C) The numbers indicate the number of GRUs of the virus stocks, which were required to yield one clonal LCL for each virus noted in (B). (D) Efficiency of B-cell growth transformation. The different singly and dually vBcl-2⁻ EBV virus mutants were compared with wild-type 2089 EBV for their efficiencies to growth transform primary B cells in limiting dilution assays. Forty-eight wells with 10⁵ target B cells per well were infected with each virus dilution, and wells with proliferating cells were recorded 6 wk p.i. The data are graphed to identify the number of GRUs of the different virus isolates required to yield one LCL. The horizontal line at 30 wells positive for 48 wells plated identifies for a Poisson distribution the required number of GRUs shown on the x-axis. Infection of primary B cells with the vBcl-2⁻ mutants 2636 and 2765 did not yield stable LCLs even with up to 10⁴ GRUs per 10⁵ cells per well. (E) Comparison of the efficiency of wild-type 2089 EBV and the BALF1⁻/BHRF1⁻ mutant 2636 to yield stable, clonal LCLs in limiting dilution assays with primary B cells or activated B blasts as target cells. Activated B blasts readily gave rise to LCL clones with the BALF1⁻/BHRF1⁻ mutant 2636 in contrast to the situation in (D), although about 200 GRUs were statistically required to establish clonal lines, which all could be further expanded and characterized (data not shown).

Figure 2. Detection of EBNA2, BALF1, and BHRF1 Transcripts by RT-PCR Analysis

(A). Primary B cells were infected with B95.8 EBV, and RNA was prepared at various time points. The expression of BALF1, BHRF1, and HPRT (as control) was analyzed by RT-PCR. (B). Primary B cells were infected with wild-type 2089 EBV, the BZFL1⁻ mutant 2809, or the BALF1⁻/BHRF1⁻ mutant 2636 (MOI 0.1), and the expression of EBNA2 was determined by RT-PCR. EBNA2 mRNA was detectable in comparable amounts in cells infected with either virus stock on day 1 and on day 5 p.i. (C). Primary B cells were infected with wild-type 2089 EBV, the BZFL1⁻ mutant 2809, or the EBNA2⁻ mutant 2491. Similar to (A), vBcl-2 transcripts were detectable in comparable amounts in cells infected with either virus stock on day 1 and on day 5 p.i. but at a reduced level on day 5. (D). BALF1 and BHRF1 transcript levels in latently infected and lytically induced cell lines. Neither BALF1 nor BHRF1 are detectably expressed in the latently infected, EBV-positive Akata cell line [62] or in 293HEK cells stably transfected with wild-type 2089 EBV. Both mRNAs are easily detectable in B95.8 cells, which spontaneously support productive infection, or in lytically induced 293HEK cells carrying the maxi-EBV 2089). (E) Schematic overview of the PCR primers and their location used to detect EBNA2-, BALF1-, or BHRF1-specific cDNAs. The PCR product indicative of correctly spliced EBNA2 transcripts is 426 bp in length; the BALF1 PCR product is 443 bp in length. BHRF1-specific PCR products are expected to be 551 bp and 991 bp in length, representing the spliced and unspliced transcripts, respectively.

Figure 3. Cell Cycle Analysis of Uninfected Primary B Cells (PBls) or PBls Infected with EBNA2⁻ Virus, BALF1⁻/BHRF1⁻ Mutant 2636, or Wild-Type 2089 EBV

Primary B cells (10⁵) per time point were infected with a viral dose such that about 50% of the cells were infected or left uninfected, and their cell cycle profiles were determined by BrdU incorporation and FACS analysis. Only wild-type 2089 EBV induced cell cycle progression, whereas uninfected cells as well as cells infected with the two mutants became apoptotic as indicated by their subG₁ DNA content. (A) shows the compiled data of the primary results, which are illustrated in (B). One representative experiment out of three is shown.

Figure 4. Apoptosis of Primary B Cells

Primary B cells were infected with the EBNA2⁻ mutant 2491, the BALF1⁻/BHRF1⁻ mutant 2636, or wild-type 2089 EBV, at an MOI of 0.1, or the cells were left uninfected. Cells were analyzed by FACS at days 1, 3, 5, and 8 p.i. (A) Total cellular events were collected until 3 × 10³ BD CaliBRITE beads as a volume standard were counted. These beads, which are indicated by red circles in the FACS diagrams in (A) and (C), were used as an internal volume reference corresponding to 3 × 10⁴ cells plated initially. (B) Uninfected cells that fulfilled the criteria of lymphocytes according to their forward (FSC) and sideward (SSC) scatter characteristics were gated as indicated. One day after B-cell preparation, lymphocytes were present in the lymphocyte gate as expected, but only a few lymphocytes were still present in this gate 8 d after cell preparation when the cells had been left uninfected. Similarly, EBV-infected lymphocytes were selected according to the same scatter criteria (data not shown). (C) EBV-infected GFP⁺ cells were gated as indicated. The example shows an uninfected and a 2491 EBNA2⁻ EBV-infected B-cell sample with 3 × 10³ BD CaliBRITE beads added 1 d p.i. (D) Uninfected primary B cells within the lymphocyte gate or EBV-infected GFP⁺ lymphocytes were analyzed by FACS for Annexin-V binding and PI staining at different time points p.i. The absolute numbers of Annexin-V⁻/PI⁻/GFP⁺ cells allowed the calculation of surviving cells at each time point p.i. Uninfected cells indicate the rate and kinetics of spontaneous apoptosis of primary B cells ex vivo. Primary B cells infected with the BALF1⁻/BHRF1⁻ mutant 2636 died as rapidly as uninfected cells. Only B cells infected with the EBNA2⁻ mutant 2491, which is vBcl-2⁺, survived considerably longer. Primary B cells infected with wild-type 2089 EBV as a positive control rapidly increased in the number of Annexin-V⁻/PI⁻/GFP⁺ cells. (E) Summary of the primary data shown in (D). Annexin-V⁻/PI⁻ cells (uninfected control to determine spontaneous programmed cell death), or Annexin-V⁻/PI⁻/GFP⁺ (infected) cells were set to 100% at day 1, and the percentile of Annexin-V⁻/PI⁻ cells at each time point was calculated. One representative experiment out of three is shown.