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. 2024 Nov 8;169(12):241.
doi: 10.1007/s00705-024-06172-7.

Use of antibodies against Epstein-Barr virus nuclear antigen 1 for detection of cellular proteins with monomethylated arginine residues that are potentially involved in viral transformation

Christian Graesser  1 , Ruth Nord  1 , Heinrich Flaswinkel  2 , Elisabeth Kremmer  2 , Eckart Meese  3 , Karolina Magdalena Caban  4 , Thomas Fröhlich  4 , Friedrich A Grässer  1 , Martin Hart  5
Affiliations

Use of antibodies against Epstein-Barr virus nuclear antigen 1 for detection of cellular proteins with monomethylated arginine residues that are potentially involved in viral transformation

Christian Graesser et al. Arch Virol. .

Abstract

Epstein-Barr virus nuclear antigen 1 (EBNA1) contains two arginine-glycine (RG) repeats that contain symmetric/asymmetric dimethylarginine (SDMA/ADMA) and monomethylarginine (MMA) residues. We generated mouse monoclonal antibodies directed against a monomethylated GRGRGG-containing repeat located between amino acids 328 and 377 of EBNA1. In addition to detecting MMA-modified EBNA1, we also had the goal of identifying cellular proteins that bind to MMA-modified EBNA1 in EBV-positive Raji cells. Furthermore, we hypothesized that antibodies against MMA-modified EBNA1 might also recognize cell factors that use an MMA-modified surface structure similar to that of EBNA1 to bind to their common targets. Using a combination of immunoprecipitation and mass spectrometry, we identified a number of such cellular proteins, including SNRPD1-3, ALY/REF, RPS15, DIDO1, LSM12, LSM14A, DAP3, and CPSF1. An NACA complex protein that was shown previously to bind to the glycine-alanine repeat of EBNA1 was also identified. The proteins identified in this study are involved in splicing, tumorigenesis, transcriptional activation, DNA stability, and RNA processing or export.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
Characterization of mouse monoclonal antibodies (mAbs) directed against MMA-modified EBNA1 via Western blotting. (A) Extracts of the BL cell line Raji (EBV-positive) were precipitated with the mAbs 7C10, 5D7, and 1H7 as well as the control antibody “anti-ICP8” (isotype IgG control). Bound EBNA1 was analyzed by SDS-PAGE and visualized by immunoblotting with the rat monoclonal antibody 1H4 and the appropriate horseradish-peroxidase-conjugated mouse anti-rat IgG secondary antibody using enhanced chemiluminescence (ECL). (B) Extracts of the indicated cell lines were analyzed by Western blotting. EBNA1 was visualized by ECL after immunoblotting using the rat mAb 1H4 and the appropriate secondary antibodies. The cell lines expressed EBNA1 proteins of different sizes due to strain variability. (C) Extracts of the indicated cell lines were used for precipitation with mAb 5C7 or the isotype control antibody “anti-ICP8”. The precipitated EBNA1 was visualized by immunoblotting with mAb 1H4
Fig. 2
Fig. 2
Peptide release of EBNA1 and its interactors. (A) Extracts of Raji cells were subjected to immunoprecipitation using the mouse mAb 5C7. The resin was incubated with the peptide RGRGRGG containing either MM-modified arginine residues (“Release Me+”) or unmodified arginine residues (“Release Me-”). The released EBNA1 was visualized by ECL after immunoblotting using the rat mAb 1H4. A 10-µL aliquot of the cell extract was run in an adjacent lane. (B) Volcano plot analysis of mass-spectrometry-based label-free quantification (LFQ) values of proteins released with the MM-modified and unmodified RGRGRGG peptides. Proteins that were more abundant in the release experiment with the MM-modified peptide are highlighted in red. (C) Overrepresentation analysis of this set of proteins was performed with WebGestalt (www.webgestalt.org). The volcano plot includes the enriched functional categories. The size and color of the dots in the scale indicate the sizes of the gene sets
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