Abstract
The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Antibodies, Monoclonal / immunology
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Antibody Specificity / immunology
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Arginine / analogs & derivatives
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Arginine / metabolism
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Cell Line
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DNA / metabolism
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Epstein-Barr Virus Infections / metabolism
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Epstein-Barr Virus Nuclear Antigens / chemistry
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Epstein-Barr Virus Nuclear Antigens / immunology
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Epstein-Barr Virus Nuclear Antigens / metabolism*
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Gene Expression Regulation, Viral
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Herpesvirus 4, Human / genetics
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Herpesvirus 4, Human / physiology
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Heterogeneous-Nuclear Ribonucleoprotein K / metabolism*
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Humans
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Immunoprecipitation
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Methylation
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Molecular Sequence Data
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Multiprotein Complexes / metabolism
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Mutant Proteins / metabolism
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Promoter Regions, Genetic / genetics
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Protein Binding
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Protein Transport
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Repetitive Sequences, Amino Acid
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Transfection
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Viral Matrix Proteins / genetics
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Viral Matrix Proteins / metabolism*
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Viral Proteins / chemistry
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Viral Proteins / immunology
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Viral Proteins / metabolism*
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snRNP Core Proteins / metabolism
Substances
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Antibodies, Monoclonal
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EBNA-2 protein, Human herpesvirus 4
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EBV-associated membrane antigen, Epstein-Barr virus
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Epstein-Barr Virus Nuclear Antigens
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Heterogeneous-Nuclear Ribonucleoprotein K
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Multiprotein Complexes
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Mutant Proteins
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SNRPD3 protein, human
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Viral Matrix Proteins
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Viral Proteins
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snRNP Core Proteins
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symmetric dimethylarginine
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N,N-dimethylarginine
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DNA
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Arginine
Grants and funding
This work was supported by Deutsche Forschungsgemeinschaft through grant KR2218/2-1 to EK, OS290/3-2 to AO-L and DHO and GR950/12-1 to FG and the BioImaging Network Munich to HL. WD was supported by the Chinese Scholarship Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.