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. 2004 Nov 2;101(44):15730-5.
doi: 10.1073/pnas.0402135101. Epub 2004 Oct 21.

Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-kappaB transcription factor pathway

Suzanne Stewart  1 , Christopher W Dawson, Kenzo Takada, John Curnow, Cary A Moody, John W Sixbey, Lawrence S Young
Affiliations

Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-kappaB transcription factor pathway

Suzanne Stewart et al. Proc Natl Acad Sci U S A. .

Abstract

Epstein-Barr virus (EBV)-associated malignancies display distinct patterns of virus latent gene expression that reflect the complex interplay between the virus and its host cell. In the EBV-associated epithelial tumor nasopharyngeal carcinoma (NPC), the virus-encoded latent membrane protein LMP2A is consistently expressed whereas the oncogenic LMP1 protein appears to be restricted to only a proportion of tumors. In an attempt to understand the contribution of LMP2A to the pathogenesis of NPC, we established carcinoma cell lines stably infected in vitro with either a wild-type recombinant EBV (rEBV) or a mutant rEBV in which LMP2A is deleted (rEBV-2A). An NPC-like pattern of EBV gene expression including LMP2A but not LMP1 was consistently observed in carcinoma cells infected with rEBV. However, carcinoma cells infected with rEBV-2A expressed high levels of LMP1 from the signal transducer and activator of transcription (STAT)-regulated L1-TR promoter. Consistent with this effect, basal STAT activity was reduced in rEBV-infected carcinoma cells, and this repression was relieved in the absence of LMP2A. This modulation of STAT activity correlated with the ability of LMP2A to inhibit the autocrine secretion of IL-6 from carcinoma cell lines. Exogenous IL-6 was able to induce expression of LMP1 by means of STAT3 activation both in rEBV-infected carcinoma cell lines and in the EBV-positive C666-1 NPC cell line. The LMP2A-mediated suppression of IL-6 was a consequence of NF-kappaB inhibition. These data reveal that LMP2A modulates two key transcription factor pathways in carcinoma cells and suggest that this finding may be important in the pathogenesis of EBV-associated tumors.

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Figures

Fig. 1.
Fig. 1.
Expression of EBNA1 and LMP1 in EBV-infected AGS, Ad/AH, and HONE-1 cell lines. Immunoblot analysis demonstrates EBNA1 (A) and LMP1 (C) expression in cells infected with rEBV- and rEBV-2A-infected cell lines. The LCL X50-7 was used as a positive control for both EBNA1 and LMP1. EBNA1 reference serum (AM) was used to detect EBNA1, and CS1-3 was used to detect LMP1. (B) Immunofluorescence staining using CS1-3 demonstrates LMP1 expression in cell lines infected with rEBV-2A (Bottom). LMP1 is absent from parental cells (Top) and cells infected with wild-type rEBV (Middle).
Fig. 2.
Fig. 2.
Cells infected with rEBV-2A show increased transcriptional activity from the alternative LMP1 promoter L1-TR and EBNA1 Qp promoter. (A) Transcription of LMP1 from the alternative L1-TR (3.7 kb) is significantly elevated in cells infected with rEBV-2A as demonstrated by RT-PCR and Southern blotting. LMP1 expression in the X50-7 LCL was predominantly from the 2.8-kb promoter, and low-level L1-TR activity was detected in both X50-7 and Akata virus-transformed LCLs (data not shown). Uninfected parental cells served as a negative control. (B) Cells infected with rEBV-2A display increased transcription from the EBNA1 Qp promoter as detected using RT-PCR and Southern blotting. RAEL, a type 1 latently infected Burkitt's lymphoma cell line that expresses EBNA1 transcribed from the Qp promoter, served as a positive control. (C) EMSA demonstrates that LMP2A deletion results in derepression of STAT activity in nuclear extracts isolated from AGS and Ad/AH EBV-infected cells. The amount of activated STAT was determined by measuring its binding to a 32P-labeled generic STAT probe. Parental cells lines show constitutive levels of STAT activity comparable to that of cells infected with rEBV-2A. Basal STAT activity is reduced in cells infected with rEBV. IL-6 stimulated C666-1 NPC cells served as a positive control for STAT activation. (D) EMSA analysis of nuclear extracts isolated from HONE-1 parental and rEBV-infected cell lines confirms that LMP2A deletion results in derepression of STAT3 activity. IL-6-stimulated HONE-1 parental cells serve as a positive control for STAT3 activation. (E) Immunoblotting for phosphorylation of STAT3 confirms the ability of LMP2A to suppress STAT3 activity. Total STAT3 served as a loading control.
Fig. 3.
Fig. 3.
IL-6 secretion is inhibited by LMP2A and controls LMP1 expression. (A) The ability of LMP2A to down-regulate IL-6 secretion in rEBV-infected HONE-1 and Ad/AH cell lines was determined by using an IL-6 ELISA. The histograms are representative of at least three separate experiments, and the mean ± SE of triplicate determinations is shown. (B) Addition of human recombinant IL-6 to the HONE-1 rEBV-infected cell line induces LMP1 expression as demonstrated by immunoblot analysis. (C) Addition of human recombinant IL-6 to the C666-1 NPC cell line induces LMP1 expression and phosphorylation of STAT3 as demonstrated by immunoblot analysis. Total STAT3 served as a loading control.
Fig. 4.
Fig. 4.
Confirmation that LMP2A regulates LMP1 expression. (A) Transient delivery of LMP2A represses LMP1 expression in rEBV-2A-infected HONE-1 cells. HONE-1 cells infected with rEBV-2A were transiently transfected with a vector (pSG-LMP2A) expressing HA-tagged LMP2A or with a control vector (pSG5), and, after 48 h, the protein expression was assessed by immunoblotting with antibodies to HA for detection of tagged LMP2A, LMP1, phospho-STAT3, and total STAT3. LMP2A expression resulted in decreased expression of both LMP1 and phospho-STAT3. pSG5 control transfected cells were used as a negative control. (B) HONE-1 rEBV-infected cells were transiently transfected with siRNA targeted to LMP2A or with a control scrambled siRNA. This treatment resulted in a 50% reduction in the levels of LMP2A relative to control (Upper) and a 6-fold increase in LMP1 relative to control (Lower) at 48 h posttransfection. Data shown are representative of two separate experiments with triplicate determination in each experiment, and data are expressed as the mean ± SE.
Fig. 5.
Fig. 5.
LMP2A negatively regulates IL-6 secretion by means of inhibition of NF-κB. (A) EMSA demonstrates that infection of HONE-1 cells with rEBV inhibits NF-κB activity and that this effect is relieved in cells infected with rEBV-2A. Nuclear extracts isolated from HONE-1 cells or HONE-1 cells infected with rEBVs were analyzed for basal NF-κB activity. Tumor necrosis factor α-stimulated HONE-1 parental cells were used as a positive control. The presence of p65 and p50 subunits in the NF-κB complexes was confirmed by using antibodies to these subunits. (B) Blockade of NF-κB activity in rEBV-2A-infected HONE-1 cells with the RAd-IκBαSS/AA virus inhibited IL-6 secretion in a dose-dependent manner as determined by IL-6 ELISA. Data are representative of two separate experiments with triplicate determination, and the mean ± SE were all <5 pg/ml and thus unable to be accurately depicted on the histograms. (C) Transient transfection of the HONE-1 cell panel with an IL-6 reporter plasmid (p-IL-6-luc651) confirms that LMP2A modulates IL-6 expression. Thus, IL-6 promoter acting was similar in parental HONE-1 cells and rEBV-2A-infected HONE-1 cells but significantly reduced in rEBV-infected HONE-1 cells. Data are representative of three experiments and are presented as the mean ± SE of triplicate determinations. (D) NF-κB activity was inhibited in HONE-1 parent cells transiently transfected with a LMP2A-expressing plasmid as determined by EMSA. The pSG5 control vector was used as a negative control. Tumor necrosis factor α-stimulated HONE-1 parent cells served as a positive control for NF-κB activity.
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