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. 2002 Dec;76(24):12543-52.
doi: 10.1128/jvi.76.24.12543-12552.2002.

The Epstein-Barr virus immediate-early protein BZLF1 induces expression of E2F-1 and other proteins involved in cell cycle progression in primary keratinocytes and gastric carcinoma cells

Amy Mauser  1 , Elizabeth Holley-Guthrie, Adam Zanation, Wendall Yarborough, William Kaufmann, Aloysius Klingelhutz, William T Seaman, Shannon Kenney
Affiliations

The Epstein-Barr virus immediate-early protein BZLF1 induces expression of E2F-1 and other proteins involved in cell cycle progression in primary keratinocytes and gastric carcinoma cells

Amy Mauser et al. J Virol. 2002 Dec.

Abstract

The Epstein-Barr virus (EBV) immediate-early protein BZLF1 mediates the switch between the latent and lytic forms of EBV infection and has been previously shown to induce a G(1)/S block in cell cycle progression in some cell types. To examine the effect of BZLF1 on cellular gene expression, we performed microarray analysis on telomerase-immortalized human keratinocytes that were mock infected or infected with a control adenovirus vector (AdLacZ) or a vector expressing the EBV BZLF1 protein (AdBZLF1). Cellular genes activated by BZLF1 expression included E2F-1, cyclin E, Cdc25A, and a number of other genes involved in cell cycle progression. Immunoblot analysis confirmed that BZLF1 induced expression of E2F-1, cyclin E, Cdc25A, and stem loop binding protein (a protein known to be primarily expressed during S phase) in telomerase-immortalized keratinocytes. Similarly, BZLF1 increased expression of E2F-1, cyclin E, and stem loop binding protein (SLBP) in primary tonsil keratinocytes. In contrast, BZLF1 did not induce E2F-1 expression in normal human fibroblasts. Cell cycle analysis revealed that while BZLF1 dramatically blocked G(1)/S progression in normal human fibroblasts, it did not significantly affect cell cycle progression in primary human tonsil keratinocytes. Furthermore, in EBV-infected gastric carcinoma cells, the BZLF1-positive cells had an increased number of cells in S phase compared to the BZLF1-negative cells. Thus, in certain cell types (but not others), BZLF1 enhances expression of cellular proteins associated with cell cycle progression, which suggests that an S-phase-like environment may be advantageous for efficient lytic EBV replication in some cell types.

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Figures

FIG. 1.
FIG. 1.
BZLF1 induces E2F-1 in a cell type-dependent manner. (A) Telomerase-immortalized human keratinocytes and normal human fibroblasts were infected with AdLacZ or AdBZLF1. At 48 h postinfection, the levels of E2F-1, BZLF1, and β-actin were quantitated by immunoblot analysis. (B) AGS gastric carcinoma cells and primary tonsil keratinocytes were infected as in panel A, and the level of E2F-1 was determined by immunoblot analysis. NS, nonspecific cross-reacting band. (C) FACS analysis was performed to compare the level of BZLF1 expression in Ad-BZLF1-infected AGS cells or telomerase-immortalized keratinocytes to that in the constitutively BZLF1-positive population of EBV-AGS cells. Neg., negative; Pos., positive.
FIG. 1.
FIG. 1.
BZLF1 induces E2F-1 in a cell type-dependent manner. (A) Telomerase-immortalized human keratinocytes and normal human fibroblasts were infected with AdLacZ or AdBZLF1. At 48 h postinfection, the levels of E2F-1, BZLF1, and β-actin were quantitated by immunoblot analysis. (B) AGS gastric carcinoma cells and primary tonsil keratinocytes were infected as in panel A, and the level of E2F-1 was determined by immunoblot analysis. NS, nonspecific cross-reacting band. (C) FACS analysis was performed to compare the level of BZLF1 expression in Ad-BZLF1-infected AGS cells or telomerase-immortalized keratinocytes to that in the constitutively BZLF1-positive population of EBV-AGS cells. Neg., negative; Pos., positive.
FIG. 2.
FIG. 2.
BZLF1 increases the expression of E2F-1-responsive genes in some cell types. (A) Telomerase-immortalized keratinocytes or normal human fibroblasts were mock infected or infected with the AdLacZ or AdBZLF1 vector. Immunoblot analysis was performed 2 days later to quantitate expression of cyclin E, SLBP, or Cdc25A, as indicated. (B) AGS and primary tonsil keratinocytes were infected with the AdLacZ or AdBZLF1 vector, and immunoblot analysis was performed 2 days later to quantitate expression of cyclin E and SLBP as indicated.
FIG. 3.
FIG. 3.
BZLF1 has cell type-dependent effects on cell cycle progression. Normal human fibroblasts, primary keratinocytes, telomerase-immortalized keratinocytes, or AGS cells were infected with the AdLacZ or AdBZLF1 vector. Two days later, the cells were labeled for 1 h with 10 μM BrdU, harvested, fixed, and stained with propidium iodide. BrdU incorporation was quantitated by FACS analysis using a BrdU monoclonal antibody and a fluorescein isothiocyanate-labeled secondary antibody.
FIG. 4.
FIG. 4.
BZLF1 affects p53 level in a cell type-dependent manner. Primary or telomerase-immortalized keratinocytes and AGS cells or normal human fibroblasts were mock infected or infected with AdLacZ or AdBZLF1. Immunoblot analysis was performed 48 h after infection to quantitate the level of cellular p53.
FIG. 5.
FIG. 5.
BZLF1 induction of E2F-1 is not associated with apoptosis. Telomerase-immortalized human keratinocytes were mock infected or infected with AdLacZ or AdBZLF1. At 48 h postinfection, the binding of Annexin V-fluorescein isothiocyanate was quantitated by FACS analysis.
FIG. 6.
FIG. 6.
The BZLF1-positive subset of AGS-EBV cells has an increase in cells in S phase. The cell cycle stages of the BZLF1-positive cells and BZLF1-negative AGS-EBV cells were compared using propidium iodine staining and Mod Fit LT analysis. The percentages of BZLF1-positive and BZLF1-negative AGS-EBV cells in each stage of the cell cycle are shown (average of three separate experiments). The cell cycle distribution of at least 3,000 BZLF1-positive AGS cells was examined in each experiment.
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