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

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.