The C-mer gene is induced by Epstein-Barr virus immediate-early protein BRLF1

Abstract

BRLF1 (R) is one of two Epstein-Barr virus (EBV) immediate-early proteins that mediate the switch from the latent to the lytic form of viral replication. In this report, we show that R induces expression of the cellular C-mer gene in a variety of cell lines. C-mer expression was detected in lymphoblastoid cells immortalized with wild-type EBV but not in lymphoblastoid cells immortalized with an EBV that had BRLF1 deleted. Oral hairy leukoplakia tongue tissue, which contains the lytic form of EBV replication, also has enhanced C-mer expression. C-mer is a receptor tyrosine kinase activated by the ligand Gas6. C-mer is required for phagocytosis of apoptotic debris by monocytes/macrophages and retinal pigment epithelial cells and is capable of producing an antiapoptotic signal. Modulation of the C-mer signal transduction cascade by a variety of different approaches did not alter the ability of R to induce lytic EBV gene transcription. Therefore, C-mer activation may be important for some other aspect of lytic EBV infection.

FIG. 1.

An R adenovirus vector increases C-mer mRNA expression. HeLa, RPE, and 293 cells and TIK were infected with AdR or AdLacZ (MOI of 20); RNA was isolated 24 h postinfection, and Northern blot analysis was performed to detect C-mer or GAPDH mRNA expression.

FIG. 2.

Transfected R increases C-mer mRNA expression. HeLa and 293 cells were transfected with a vector expressing R (SG5-R) or a control vector (SG5); RNA was isolated 24 h posttransfection, and Northern blot analysis was performed to detect C-mer and GAPDH expression.

FIG. 3.

C-mer is activated by lytic EBV induction in B cells. EBV-positive or EBV-negative Akata Burkitt lymphoma cells were treated with or without anti-IgG (0.1 mg/ml) for 24 h to induce the lytic form of EBV infection (left panel). Total RNA was harvested, and RT-PCR analysis was performed to detect C-mer or β₂-microglobin RNA expression. C-mer expression was also examined in LCLs derived from the same donor with wild-type virus (WT) or virus having BRLF1 deleted (R-KO) (right panel). Samples without RT did not produce detectable C-mer expression (data not shown).

FIG. 4.

R increases C-mer protein expression. HeLa cells and 293 cells were infected with AdR or AdLacZ (MOI of 20), and protein extracts were harvested 24 h postinfection. Protein extracts were also obtained from LCLs derived from the same donor with wild-type virus (WT) or virus having BRLF1 deleted (R-KO). HeLa and LCL extracts were immunoprecipitated (IP) and immunoblotted (IB) with a C-mer polyclonal antibody. 293 cell extracts were first pulled down by using WGA-Sepharose beads and then immunoblotted with a C-mer antibody.

FIG. 5.

Gas6 phosphorylates C-mer induced by R in HeLa cells. HeLa cells infected with AdLacZ or AdR (MOI of 20) for 24 h were treated with recombinant Gas6 for 0, 10, 30, 60, and 90 min. Cell extracts were immunoprecipitated (IP) with a C-mer antibody and then immunoblotted (IB) with the same C-mer antibody to measure total C-mer protein or immunoblotted with a p-Tyr antibody to measure phosphorylated C-mer. The AdLacZ-infected cells had no detectable C-mer protein (data not shown).

FIG. 6.

C-mer expression is detected in OHL tongue tissue. Tongue biopsy specimens from healthy individuals (bottom panel) or OHL patients (top and middle panels) were incubated with either a C-mer monoclonal antibody (MAB891; R & D Systems; top and lower panels) or phosphate-buffered saline (middle panel), followed by incubation with an anti-mouse antibody conjugated with fluorescein isothiocyanate. C-mer expression was examined using fluorescence microscopy (left panels). 4′,6′-Diamidino-2-phenylindole (DAPI) staining was also performed on all specimens (right panels).

FIG. 7.

Modulation of C-mer activity does not affect R-mediated lytic gene expression. EBV-positive D98/HE-R-1 cells were transfected with the SG5 or SG5-R vectors, in combination with either a control vector, a vector that inhibits C-mer signaling in the presence of EGF (Mer-KD), or a vector that activates a C-mer-like pathway in the presence of EGF (C-mer). EGF was added 24 h after transfection to some conditions, and cells were harvested 48 h posttransfection for immunoblot analysis to detect lytic EBV gene expression.

FIG. 8.

Gas6 does not affect R-mediated EBV lytic gene expression. EBV-positive 293 cells were transfected with the control vector, SG5, or SG5-R. Human recombinant Gas6 (150 nM) was added 24 h posttransfection, and cells were harvested 48 h posttransfection for immunoblot analysis to detect EBV lytic gene expression.