J Korean Med Sci.  2006 Jun;21(3):379-384. 10.3346/jkms.2006.21.3.379.

Colocalization of Interferon Regulatory Factor 7 (IRF7) with Latent Membrane Protein 1 (LMP1) of Epstein-Barr Virus

• Kim IW
• Park HS

• KMID: 1778415
• DOI: http://doi.org/10.3346/jkms.2006.21.3.379

Abstract

Interferon regulatory factor 7 (IRF7) is one of the transcriptional factors for the activation of type I Interferon (IFN) genes. It is known that IRF7 and the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) are highly expressed in EBV type III latency cells, and LMP1 induces mRNA expression of IRF7. In this study, the expression pattern of endogenous IRF7 was observed in several B cell lines with or without EBV infection by immunofluorescence staining. IRF7 was localized in the cytoplasm of EBV-negative B cells and EBV type I latency B cell lines. However, IRF7 was located both in the cytoplasm and nucleus of EBV type III latency cell lines. In the Jijoye cell (type III latency cell), IRF7 was colocalized with LMP1 in the cytoplasm in a capping configuration, and their interaction was confirmed by co-immunoprecipitation of LMP1 and IRF7. This colocalization was confirmed by co-transfection of IRF7 and LMP1 plasmids in EBV-negative B cells. These results suggest that the IRF7 and LMP1 interact with each other, and this may relate to the mechanism whereby LMP1 exerts functional effects in B-lymphocytes.

Keyword

Interferon Regulatory Factor-7; Epstein-Barr Virus; Herpesvirus 4, Human; Latent Membrane Protein 1; EBV-associated membrane antigen; Epstein-Barr virus

MeSH Terms

Viral Matrix Proteins/*biosynthesis/metabolism
Trans-Activation (Genetics)
Signal Transduction
RNA, Messenger/metabolism
Plasmids/metabolism
Microscopy, Fluorescence
Interferon Regulatory Factor-7/*biosynthesis
Immunoprecipitation
Humans
Herpesvirus 4, Human/metabolism
*Gene Expression Regulation
Cytoplasm/metabolism
Cell Line, Tumor
B-Lymphocytes/metabolism/virology

Figure

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  Fig. 1 The immunofluorescence pattern of endogenous IRF7 in various B lymphoblastoid cell lines. Immunofluorescence assays (IFA) were performed with a rabbit polyclonal IRF7 antibody, with subsequent reaction with donkey anti-rabbit IgG-FITC in DG75 (A), Akata (B), Sav I (C), Sav III (D) and Jijoye (E, F) cell lines. IRF7 was captured with a Axioscope fluorescence microscope using a Scion software program.

• 

  Fig. 2 Colocalization of LMP1 and IRF7. The LMP1 and IRF7 proteins are double immunostained with mouse monoclonal LMP1 antibody and rabbit polyclonal IRF7 antibody, and subsequently reacted with donkey anti-mouse IgG-rhodamine and donkey anti-rabbit IgG-FITC, respectively. Panel (A) and (B) are Jijoye, (C) is Sav III, and (D) is DG75 cell line co-transfected with LMP1 and IRF7 plasmids. Fluoresecence was captured with a confocal microscope. Red and green color represent of LMP1 and IRF7, respectively; yellow indicates colocalization of both proteins.

• 

  Fig. 3 In vivo association of LMP1 with IRF7 in Jijoye. Cell lysates of Jijoye were subjected to immunoprecipitation with normal mouse serum (lane 1), LMP1 antibody (lane 2), normal rabbit serum (lane 3), or IRF7 antibody (lane 4). The immunocomplexes were analyzed by SDS-PAGE on 10% gel and subjected to western blot analysis with mouse LMP1 antibody (upper panel) or rabbit IRF7 antibody (lower panel).

• 

  Fig. 4 Interaction of LMP1 and IRF7 in transfected DG75. DG75 cells were transfected with pcDNA3 (lane 1), FLAG-pcLMP1 (lane 2), pcIRF7 (lane 3) or FLAG-pcLMP1 plus pcIRF7 (lane 4). Cell lysates were immunopurified with M2 FLAG antibody (A) or IRF7 antibody (B) and then subjected to western blot analysis with mouse LMP1 antibody (upper panels) or rabbit IRF7 antibody (lower panels).

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