The conserved domain CR2 of Epstein-Barr virus nuclear antigen leader protein is responsible not only for nuclear matrix association but also for nuclear localization.
There is a growing body of evidence for the importance of the nuclear matrix in various nuclear events including gene expression and DNA replication. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a nuclear matrix-associated protein that has been suggested to play an important role in EBV-induced transformation. To define the biological significance of the association of EBNA-LP with the nuclear matrix, we mapped the domain of EBNA-LP responsible for nuclear matrix association and investigated the functions of the EBNA-LP mutant mutagenized by substitution of alanines for the cluster of arginine residues in the mapped region. The results of the present study were as follows. (i) Transiently expressed EBNA-LP in COS-7 or BOSC23 cells was associated with the nuclear matrix, similarly to that in EBV-infected B cells. (ii) Mutational analysis of EBNA-LP revealed that a 10-amino acid segment of EBNA-LP is critical for nuclear matrix association of the protein. Interestingly, the identified region overlapped with the region CR2 of EBNA-LP conserved among a subset of primate gammaherpesviruses. The identified segment is referred to as EBNA-LP NMTS (nuclear matrix targeting signal). (iii) The EBNA-LP mutant with the arginine to alanine substitutions in NMTS was no longer localized not only to the nuclear matrix but also to the nucleus. (iv) The EBNA-LP mutant lacked its ability to coactivate EBNA-2-dependent transactivation. These results indicated that EBNA-LP needs to be localized in the nucleus and/or associated with the nuclear matrix through CR2 to elicit its function such as the coactivation of the EBNA-2-dependent transcriptional activation.
Yokoyama
A
A
Department of Tumor Virology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, 1-5-45, Yushima, Bunkyo-ku, 113-8510, Japan.
Kawaguchi
Y
Y
Kitabayashi
I
I
Ohki
M
M
Hirai
K
K
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002472:Cell Transformation, Viral; D002522:Chlorocebus aethiops; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015530:Nuclear Matrix / Q000378:metabolism*; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
C402641:EBNA-2 protein, Human herpesvirus 4; C402640:EBNA-LP protein, Human herpesvirus 4; D019309:Epstein-Barr Virus Nuclear Antigens; D014764:Viral Proteins; D001120:Arginine; D000409:Alanine