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Journal of Virology, October 2006, p. 10064-10072, Vol. 80, No. 20
0022-538X/06/$08.00+0     doi:10.1128/JVI.00678-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Phosphorylation of MCM4 at Sites Inactivating DNA Helicase Activity of the MCM4-MCM6-MCM7 Complex during Epstein-Barr Virus Productive Replication

Ayumi Kudoh,1 Tohru Daikoku,1,{dagger} Yukio Ishimi,2 Yasushi Kawaguchi,3 Noriko Shirata,1 Satoko Iwahori,1 Hiroki Isomura,1 and Tatsuya Tsurumi1*

Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan,1 Department of Materials and Biological Sciences, Faculty of Science, Ibaraki University, Mito 310-8512, Japan,2 Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan3

Received 4 April 2006/ Accepted 25 July 2006

Induction of Epstein-Barr virus (EBV) lytic replication blocks chromosomal DNA replication notwithstanding an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. We report here that the phosphorylated form of MCM4, a subunit of the MCM complex essential for chromosomal DNA replication, increases with progression of lytic replication, Thr-19 and Thr-110 being CDK2/CDK1 targets whose phosphorylation inactivates MCM4-MCM6-MCM7 (MCM4-6-7) complex-associated DNA helicase. Expression of EBV-encoded protein kinase (EBV-PK) in HeLa cells caused phosphorylation of these sites on MCM4, leading to cell growth arrest. In vitro, the sites of MCM4 of the MCM4-6-7 hexamer were confirmed to be phosphorylated with EBV-PK, with the same loss of helicase activity as with CDK2/cyclin A. Introducing mutations in the N-terminal six Ser and Thr residues of MCM4 reduced the inhibition by CDK2/cyclin A, while EBV-PK inhibited the helicase activities of both wild-type and mutant MCM4-6-7 hexamers, probably since EBV-PK can phosphorylate MCM6 and another site(s) of MCM4 in addition to the N-terminal residues. Therefore, phosphorylation of the MCM complex by redundant actions of CDK and EBV-PK during lytic replication might provide one mechanism to block chromosomal DNA replication in the infected cells through inactivation of DNA unwinding by the MCM4-6-7 complex.

* Corresponding author. Mailing address: Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan. Phone and Fax: 81-52-764-2979. E-mail: ttsurumi{at}aichi-cc.jp.

{dagger} Present address: Department of Virology, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.

Journal of Virology, October 2006, p. 10064-10072, Vol. 80, No. 20
0022-538X/06/$08.00+0     doi:10.1128/JVI.00678-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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