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. 2012 Jun;86(12):6999-7002.
doi: 10.1128/JVI.00194-12. Epub 2012 Apr 11.

Proteomic profiling of EBNA1-host protein interactions in latent and lytic Epstein-Barr virus infections

Natasha Malik-Soni  1 , Lori Frappier
Affiliations

Proteomic profiling of EBNA1-host protein interactions in latent and lytic Epstein-Barr virus infections

Natasha Malik-Soni et al. J Virol. 2012 Jun.

Erratum in

Abstract

The Epstein-Barr nuclear antigen 1 (EBNA1) protein of Epstein-Barr virus (EBV) is expressed in both latent and lytic modes of EBV infection and contributes to EBV-associated cancers. Using a proteomics approach, we profiled EBNA1-host protein interactions in nasopharyngeal and gastric carcinoma cells in the context of latent and lytic EBV infection. We identified several interactions that occur in both modes of infection, including a previously unreported interaction with nucleophosmin and RNA-mediated interactions with several heterogeneous ribonucleoproteins (hnRNPs) and La protein.

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Figures

Fig 1
Fig 1
Expression of SPA-tagged EBNA1 and lytic cycle induction. (A) C666, CNE2, and AGS cells were grown on coverslips and infected with adenovirus to deliver SPA-tagged EBNA1. Forty-eight hours after infection, cells were fixed and stained as previously described (24) using FLAG antibody (Bethyl; 1:800) and goat anti-rabbit Alexa Fluor 555 secondary antibody (Invitrogen; 1:100). Coverslips were mounted onto slides using ProLong Gold antifade medium containing DAPI (4′,6-diamidino-2-phenylindole) (Invitrogen). Images were obtained using the 40× oil objective on a Leica DM IRE2 inverted fluorescence microscope and processed using OpenLAB (version X.0) software. (B) C666, CNE2, and AGS cells were infected with the adenovirus expressing EBNA1-SPA (+V) and, 48 h later, lysed in 9 M urea and 5 mM Tris-HCl (pH 6.8) followed by brief sonication. Similar lysates were also generated of AGS cells 48 h posttransfection with pMZS3F.EBNA1 (22) expressing SPA-tagged EBNA1 (AGS tran) and of C666 cells (first lane). A total of 30 μg of each lysate was analyzed by Western blotting using K67 rabbit serum against EBNA1 (kindly supplied by Jaap Middeldorp). The positions of the native EBNA1 in C666 cells (EBNA1) and EBNA1-SPA are indicated. The latter is smaller than native EBNA1 because it lacks most of the nonessential Gly-Ala repeat region. (C) AGS-EBV cells were treated with 3 mM NaB and 20 ng/ml TPA and harvested 3 to 24 h later as indicated. Cells were lysed in 9 M urea 5 mM Tris-HCl (pH 6.8) and briefly sonicated. A total of 50 μg of total protein was analyzed by Western blotting using antibodies against BZLF1 (Santa Cruz; 1:1,000 dilution), BMRF1 (Chemicon; 1:5,000), VCA-p40 (1:500; kindly supplied by Jaap Middledorp), and actin (Santa Cruz; 1:5,000). (D) AGS-EBV cells grown on coverslips were fixed 16 h after NaB/TPA treatment, stained with antibody against BZLF1 (Santa Cruz; 1:50) followed by anti-mouse Alexa Fluor 488 (Invitrogen; 1:100), and counterstained with DAPI. Images were obtained as described for panel A.
Fig 2
Fig 2
Coimmunoprecipitations of cellular proteins with EBNA1. EBNA1-SPA or β-Gal-SPA were immunoprecipitated from 1 mg of C666 cell lysates using M2 anti-FLAG resin, before (lanes 3 and 4) and after (lanes 7 and 8) treatment of the lysate with 50 μl/ml RNase at 25°C for 30 min. Immunoprecipitated proteins (IP) and samples of the input (1% of lysate) were analyzed by Western blotting using antibodies against FLAG (top); hnRNPs A2/B1 (Santa Cruz; 1:1,000), D (Upstate; 1:1,000), or R (Abcam; 1:1,000); La (Novus; 1:1,000); nucleophosmin (B23; Santa Cruz; 1:3,000); or USP7 (Bethyl; 1:5,000) as indicated.
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References

    1. Chen GI, Gingras AC. 2007. Affinity-purification mass spectrometry (AP-MS) of serine/threonine phosphatases. Methods 42:298–305 - PubMed
    1. Chen MR, Yang JF, Wu CW, Middeldorp JM, Chen JY. 1998. Physical association between the EBV protein EBNA-1 and P32/TAP/hyaluronectin. J. Biomed. Sci. 5:173–179 - PubMed
    1. Cheung ST, et al. 1999. Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus. Int. J. Cancer 83:121–126 - PubMed
    1. Colombo E, Alcalay M, Pelicci PG. 2011. Nucleophosmin and its complex network: a possible therapeutic target in hematological diseases. Oncogene 30:2595–2609 - PubMed
    1. Deng Z, Norseen J, Wiedmer A, Riethman H, Lieberman PM. 2009. TERRA RNA binding to TRF2 facilitates heterochromatin formation and ORC recruitment at telomeres. Mol. Cell 35:403–413 - PMC - PubMed

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