This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kitamura, R. Articles by Yanagi, K. Search for Related Content PubMed PubMed Citation Articles by Kitamura, R. Articles by Yanagi, K.

 Previous Article  |  Next Article 

Journal of Virology, February 2006, p. 1979-1991, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1979-1991.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Nuclear Import of Epstein-Barr Virus Nuclear Antigen 1 Mediated by NPI-1 (Importin 5) Is Up- and Down-Regulated by Phosphorylation of the Nuclear Localization Signal for Which Lys379 and Arg380 Are Essential

Ryo Kitamura,^1,2, Toshihiro Sekimoto,³ Sayuri Ito,¹ Shizuko Harada,¹ Hideo Yamagata,² Hisao Masai,⁴ Yoshihiro Yoneda,³ and Kazuo Yanagi^1,*

Herpesvirus Laboratory, Department of Virology I, National Institute of Infectious Diseases, Tokyo 162-8640, Japan,¹ Environmental Science Division, School of Life Science, Tokyo University of Pharmacy and Life Science, Tokyo 192-0392, Japan,² Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan,³ Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan⁴

Received 12 March 2005/ Accepted 9 November 2005

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin NPI-1 (importin 5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin 1) bound only weakly and Qip1 (importin 3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.

Present address: Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan.

Present address: AIDS Research Center, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.

This article has been cited by other articles:

• Isegawa, Y., Miyamoto, Y., Yasuda, Y., Semi, K., Tsujimura, K., Fukunaga, R., Ohshima, A., Horiguchi, Y., Yoneda, Y., Sugimoto, N. (2008). Characterization of the Human Herpesvirus 6 U69 Gene Product and Identification of Its Nuclear Localization Signal. J. Virol. 82: 710-718 [Abstract] [Full Text]  
• Bian, X.-L., Rosas-Acosta, G., Wu, Y.-C., Wilson, V. G. (2007). Nuclear Import of Bovine Papillomavirus Type 1 E1 Protein Is Mediated by Multiple Alpha Importins and Is Negatively Regulated by Phosphorylation near a Nuclear Localization Signal. J. Virol. 81: 2899-2908 [Abstract] [Full Text]