Epstein-Barr virus immediate-early protein BRLF1 induces the lytic form of viral replication through a mechanism involving phosphatidylinositol-3 kinase activation

Abstract

Expression of the Epstein-Barr virus (EBV) immediate-early (IE) protein BRLF1 induces the lytic form of viral replication in most EBV-positive cell lines. BRLF1 is a transcriptional activator that binds directly to a GC-rich motif present in some EBV lytic gene promoters. However, BRLF1 activates transcription of the other IE protein, BZLF1, through an indirect mechanism which we previously showed to require activation of the stress mitogen-activated protein kinases. Here we demonstrate that BRLF1 activates phosphatidylinositol-3 (PI3) kinase signaling in host cells. We show that the specific PI3 kinase inhibitor, LY294002, completely abrogates the ability of a BRLF1 adenovirus vector to induce the lytic form of EBV infection, while not affecting lytic infection induced by a BZLF1 adenovirus vector. Furthermore, we demonstrate that the requirement for PI3 kinase activation in BRLF1-induced transcriptional activation is promoter dependent. BRLF1 activation of the SM early promoter (which occurs through a direct binding mechanism) does not require PI3 kinase activation, whereas activation of the IE BZLF1 and early BMRF1 promoters requires PI3 kinase activation. Thus, there are clearly two separate mechanisms by which BRLF1 induces transcriptional activation.

FIG. 1

BRLF1 expression is associated with Akt phosphorylation. Normal human fibroblasts were serum starved for 3 days and then either mock infected or infected with adenovirus vectors encoding β-galactosidase (Ad-LacZ), BZLF1 (Ad-Z), or BRLF1 (Ad-R). Phosphorylation of Akt was quantitated 2 days later by immunoblot analysis using an antibody that recognizes only the activated (phosphorylated on Ser 473 residue) form of Akt (top panels) or an antibody which recognizes total Akt (bottom panels). Two separate experiments are shown.

FIG. 2

BRLF1 induces Akt phosphorylation through a PI3 kinase-dependent pathway. Serum-starved normal human fibroblasts were infected with the various adenovirus vectors in the presence or absence of the PI3 kinase inhibitors, LY294002 (15 μM) or wortmannin (0.1 μM). The amount of phosphorylated Akt was quantitated 2 days later by immunoblot analysis using an antibody that recognizes only the phosphorylated form of Akt (top panel; NS is a nonspecific band) or an antibody that recognizes total Akt (bottom panel).

FIG. 3

PI3 kinase activation is required for BRLF1-induced, but not BZLF1-induced, lytic EBV infection. (A) EBV-positive D98/HE-R-1 cells were infected with the various adenovirus vectors (MOI of 50) in the presence or absence of the PI3 kinase inhibitor LY294002. Induction of the lytic form of EBV infection was quantitated by immunoblot analysis 1 day later using antibodies directed against the early lytic EBV protein BMRF1 or against the IE proteins BRLF1 (R) and BZLF1 (Z). Two separate experiments are shown. (B) The level of cellular BZLF1 expression was quantitated by FACS analysis in D98/HE-R-1 cells (top panel) infected with Ad-LacZ (MOI of 20) or Ad-Z (MOI of 1 or of 20) and in AGS-EBV cells (bottom panel). In AGS-EBV cells, only about 5% of cells express BZLF1. Isotype control antibodies and EBV-negative AGS cells were also used in the experiments described above to confirm which cells are the BZLF1-expressing cells (data not shown). (C) Procedure given for panel A was followed, except that D98/HE-R-1 cells were infected with adenovirus vectors using an MOI of 1. Abbreviations are the same as those given for panel A. −, absence of LY294002; +, presence of LY294002.

FIG. 3

PI3 kinase activation is required for BRLF1-induced, but not BZLF1-induced, lytic EBV infection. (A) EBV-positive D98/HE-R-1 cells were infected with the various adenovirus vectors (MOI of 50) in the presence or absence of the PI3 kinase inhibitor LY294002. Induction of the lytic form of EBV infection was quantitated by immunoblot analysis 1 day later using antibodies directed against the early lytic EBV protein BMRF1 or against the IE proteins BRLF1 (R) and BZLF1 (Z). Two separate experiments are shown. (B) The level of cellular BZLF1 expression was quantitated by FACS analysis in D98/HE-R-1 cells (top panel) infected with Ad-LacZ (MOI of 20) or Ad-Z (MOI of 1 or of 20) and in AGS-EBV cells (bottom panel). In AGS-EBV cells, only about 5% of cells express BZLF1. Isotype control antibodies and EBV-negative AGS cells were also used in the experiments described above to confirm which cells are the BZLF1-expressing cells (data not shown). (C) Procedure given for panel A was followed, except that D98/HE-R-1 cells were infected with adenovirus vectors using an MOI of 1. Abbreviations are the same as those given for panel A. −, absence of LY294002; +, presence of LY294002.

FIG. 3

PI3 kinase activation is required for BRLF1-induced, but not BZLF1-induced, lytic EBV infection. (A) EBV-positive D98/HE-R-1 cells were infected with the various adenovirus vectors (MOI of 50) in the presence or absence of the PI3 kinase inhibitor LY294002. Induction of the lytic form of EBV infection was quantitated by immunoblot analysis 1 day later using antibodies directed against the early lytic EBV protein BMRF1 or against the IE proteins BRLF1 (R) and BZLF1 (Z). Two separate experiments are shown. (B) The level of cellular BZLF1 expression was quantitated by FACS analysis in D98/HE-R-1 cells (top panel) infected with Ad-LacZ (MOI of 20) or Ad-Z (MOI of 1 or of 20) and in AGS-EBV cells (bottom panel). In AGS-EBV cells, only about 5% of cells express BZLF1. Isotype control antibodies and EBV-negative AGS cells were also used in the experiments described above to confirm which cells are the BZLF1-expressing cells (data not shown). (C) Procedure given for panel A was followed, except that D98/HE-R-1 cells were infected with adenovirus vectors using an MOI of 1. Abbreviations are the same as those given for panel A. −, absence of LY294002; +, presence of LY294002.

FIG. 4

PI3 kinase activation is not required for BRLF1 activation of the early SM promoter. EBV-positive D98/HE-R-1 cells were infected with the various adenovirus vectors in the presence or absence of the PI3 kinase inhibitor LY294002. Induction of the early SM EBV gene was quantitated by immunoblot analysis using an antibody directed against the early lytic EBV protein SM.

FIG. 5

BRLF1 is both a nuclear and cytoplasmic protein. Normal human fibroblasts were infected with Ad-LacZ or Ad-R. Immunocytochemistry was performed 2 days later using a BRLF1 monoclonal antibody and confocal microscopy. An isotype control primary antibody produced no visible staining (data not shown). Cells containing primarily nuclear BRLF1 (red arrows) versus those containing diffuse BRLF1 (white arrows) are indicated.

FIG. 6

PI3 kinase activation is required for anti-Ig-induced lytic EBV infection in Akata cells. Akata cells were treated with 100 μg of anti-IgG/ml for 24 h in the absence (−) or presence (+) of the PI3 kinase inhibitor LY294002. The level of BMRF1 expression in cells was determined by FACS analysis using a BMRF1-specific antibody. Similar results were obtained in two separate experiments.

FIG. 7

Activated RAS is required for BRLF1- and BZLF1-induced lytic EBV infection. D98/HE-R-1 cells were transfected with a control vector (vector) or BRLF1 (R) and BZLF1 (Z) expression vectors in the presence or absence of a dominant-negative RAS mutant (RAS17N). The level of BMRF1 induction (top panel) was analyzed 2 days after transfection by immunoblot analysis. The level of transfected BRLF1 (R) (or BRLF1 induced by BZLF1 from the endogenous viral genome) is shown in the bottom panel.