Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Nov;86(22):12251-61.
doi: 10.1128/JVI.01407-12. Epub 2012 Sep 5.

Epstein-Barr virus latent membrane protein 1 regulates the function of interferon regulatory factor 7 by inducing its sumoylation

Gretchen L Bentz  1 , Julia Shackelford, Joseph S Pagano
Affiliations

Epstein-Barr virus latent membrane protein 1 regulates the function of interferon regulatory factor 7 by inducing its sumoylation

Gretchen L Bentz et al. J Virol. 2012 Nov.

Abstract

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) induces multiple signal transduction pathways during latent EBV infection via its C-terminal activating region 1 (CTAR1), CTAR2, and the less-studied CTAR3. One mechanism by which LMP1 regulates cellular activation is through the induction of protein posttranslational modifications, including phosphorylation and ubiquitination. We recently documented that LMP1 induces a third major protein modification by physically interacting with the SUMO-conjugating enzyme Ubc9 through CTAR3 and inducing the sumoylation of cellular proteins in latently infected cells. We have now identified a specific target of LMP1-induced sumoylation, interferon regulatory factor 7 (IRF7). We hypothesize that during EBV latency, LMP1 induces the sumoylation of IRF7, limiting its transcriptional activity and modulating the activation of innate immune responses. Our data show that endogenously sumoylated IRF7 is detected in latently infected EBV lymphoblastoid cell lines. LMP1 expression coincided with increased sumoylation of IRF7 in a CTAR3-dependent manner. Additional experiments show that LMP1 CTAR3-induced sumoylation regulates the expression and function of IRF7 by decreasing its turnover, increasing its nuclear retention, decreasing its DNA binding, and limiting its transcriptional activation. Finally, we identified that IRF7 is sumoylated at lysine 452. These data demonstrate that LMP1 CTAR3 does in fact function in intracellular signaling, leading to biologic effects. We propose that CTAR3 is an important signaling region of LMP1 that regulates protein function by sumoylation. We have shown specifically that LMP1 CTAR3, in cooperation with CTAR2, can limit the ability of IRF7 to induce innate immune responses by inducing the sumoylation of IRF7.

PubMed Disclaimer

Figures

Fig 1
Fig 1
IRF7 is sumoylated endogenously in type III EBV LCLs. (A to C) KR4 cells were grown, and cell lysates were collected. (A) Nondenaturing immunoprecipitations were performed with SUMO-1, IRF7, or isotype control (anti-EBV EA-R p85) antibodies. Additional nondenaturing (B) or denaturing (C) immunoprecipitations were performed with SUMO-1 or isotype control antibodies. Western blot analyses were used to detect IRF7 expression. Duplicate experiments are shown. (D and E) BL41 EBV WT and BL41 EBV mut cells were grown, and cell lysates were collected. Denaturing immunoprecipitations were performed with 1 mg of cell lysates with SUMO-1, IRF7, or isotype control antibodies. Western blot analyses were used to detect IRF7 or SUMO-1 expression. Duplicate experiments are represented.
Fig 2
Fig 2
LMP1 expression increases the sumoylation of IRF7. (A) 293T cells were transfected as indicated and cultured for 48 h. Cell lysates were collected, and denaturing immunoprecipitations were performed with FLAG antibodies. Western blot analyses of the immunoprecipitates and whole-cell lysates (WCL) (2% of total lysates) were performed for detection of IRF7, sumoylated IRF7, and LMP1. (B) 293T cells were transfected as indicated and cultured for 48 h. Cell lysates were collected, followed by denaturing immunoprecipitations with c-Myc antibodies. (C and D) 293T cells were transfected as indicated, and WCLs were collected 48 h posttransfection. Western blot analyses of the immunoprecipitates and WCLs were performed to detect IRF7, sumoylated IRF7, LMP1, SENP1, and SENP2.
Fig 3
Fig 3
LMP1-induced sumoylation of IRF7 is dependent on CTAR3. (A) 293T cells were transfected as indicated and cultured for 48 h. Cell lysates were collected, and denaturing immunoprecipitations were performed with c-Myc antibodies. Western blot analyses of the immunoprecipitates and WCLs were used to detect IRF7, sumoylated IRF7, and LMP1. (B) 293T cells were transfected as indicated and cultured for 48 h. Cell lysates were collected, and nondenaturing immunoprecipitations were performed with FLAG antibodies, followed by Western blot analyses as described above. (C) 293T cells were transfected with GFP-IRF7, Myc–SUMO-1, and the indicated FLAG-LMP1 constructs. Cell lysates were collected 48 h posttransfection, and Western blot analyses were used to detect IRF7, sumoylated IRF7, and LMP1. Actin served as a loading control.
Fig 4
Fig 4
LMP1-induced sumoylated IRF7 localizes to the nucleus. (A) 293T cells were transfected with FLAG-LMP1 or FLAG-LMP1dCTAR3 along with GFP-IRF7, Myc–SUMO-1, or the vector control and cultured for 48 h. Cytoplasmic (CE) and nuclear (NE) extracts were collected, and Western blot analyses were performed for detection of IRF7, sumoylated IRF7, and LMP1. PARP and GAPDH were used as loading controls. (B) 293T cells were transfected with FLAG-LMP1 or FLAG-LMP1dCTAR3 along with GFP-IRF7 or the vector control and cultured for 24 h. Cells were fixed and examined by immunofluorescence microscopy (magnification, ×100).
Fig 5
Fig 5
LMP1-induced sumoylation of IRF7 inhibits IRF7 turnover. 293T cells were transfected as indicated, and 18 h before harvesting, cells were treated with DMSO, cycloheximide (CHX) (75 μg/ml), or MG132 (50 μM). Cell lysates were harvested 48 h posttransfection and immunoblotted to determine IRF7 and LMP1 levels. Actin was used as a loading control. Densitometry was used to determine relative protein levels (normalized to actin). Data are shown as means ± SD.
Fig 6
Fig 6
LMP1-induced sumoylation of IRF7 limits its transcriptional activity. (A and B) 293T cells were transfected as indicated along with ISRE-luciferase and beta-galactosidase and assayed 24 h posttransfection. Firefly luciferase activity was normalized to beta-galactosidase expression, and fold changes in ISRE activity were calculated. Data are shown as means ± SD. (C) 293T cells were transfected as indicated, and total RNA was harvested 24 h posttransfection. RT-PCR was carried out to determine IFN-β and GAPDH mRNA levels, and fold changes in IFN-β expression (relative to GADPH expression) were determined. Data are shown as means ± SD.
Fig 7
Fig 7
LMP1-induced sumoylation of IRF7 inhibits its association with chromatin. (A and B) 293T cells were transfected as indicated. WCLs and chromatin fractions were collected 48 h posttransfection (15), and Western blot analyses were used to detect IRF7 expression. Densitometry was performed, and IRF7 expression in the chromatin fractions was normalized to IRF7 expression in the WCLs. Fold changes in IRF7 chromatin association were determined. Data are shown as means ± SD.
Fig 8
Fig 8
LMP1-induced sumoylation of IRF7 occurs at IRF7 lysine 452. 293T cells were transfected as indicated. (A) Cell lysates were collected 48 h posttransfection. Denaturing immunoprecipitations were performed with c-Myc antibodies. Western blot analyses were performed on the immunoprecipitants and whole-cell lysates to determine IRF7, sumoylated IRF7, IRF7 K452R, sumoylated IRF7 K452R, and LMP1 expression. (B) Cytoplasmic (CE) and nuclear (NE) extracts were collected, and Western blot analyses were used to detect IRF7 K452R and LMP1. Histone H1 and GAPDH were used as loading controls. (C) Eighteen hours before harvesting, cells were treated with DMSO or CHX (75 μg/ml). Cell lysates were harvested 48 h posttransfection and immunoblotted to determine IRF7/IRF7 K452R and actin levels. Densitometry was used to determine relative protein levels, and the fold change in relative IRF7/IRF7 K452R expression was determined. (D) Reporter assays were performed as described for Fig. 6. (E) WCLs and chromatin fractions were collected, and fold changes in IRF7 chromatin association were determined as described for Fig. 7. Data are shown as the means ± SD.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bailey D, O'Hare P. 2004. Characterization of the localization and proteolytic activity of the SUMO-specific protease, SENP1. J. Biol. Chem. 279:692–703 - PubMed
    1. Bentz GL, Whitehurst CB, Pagano JS. 2011. Epstein-Barr virus latent membrane protein 1 (LMP1) C-terminal-activating region 3 contributes to LMP1-mediated cellular migration via its interaction with Ubc9. J. Virol. 85:10144–10153 - PMC - PubMed
    1. Brennan P, Floettmann JE, Mehl A, Jones M, Rowe M. 2001. Mechanism of action of a novel latent membrane protein-1 dominant negative. J. Biol. Chem. 276:1195–1203 - PubMed
    1. Calender A, et al. 1987. Epstein-Barr virus (EBV) induces expression of B-cell activation markers on in vitro infection of EBV-negative B-lymphoma cells. Proc. Natl. Acad. Sci. U. S. A. 84:8060–8064 - PMC - PubMed
    1. Calender A, Cordier M, Billaud M, Lenoir GM. 1990. Modulation of cellular gene expression in B lymphoma cells following in vitro infection by Epstein-Barr virus (EBV). Int. J. Cancer 46:658–663 - PubMed

Publication types