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Journal of Virology, January 2003, p. 851-861, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.851-861.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Reactivation of Lytic Replication from B Cells Latently Infected with Epstein-Barr Virus Occurs with High S-Phase Cyclin-Dependent Kinase Activity while Inhibiting Cellular DNA Replication

Ayumi Kudoh,^1,2,3 Masatoshi Fujita,^1, Tohru Kiyono,^1, Kiyotaka Kuzushima,^1, Yutaka Sugaya,¹ Shunji Izuta,² Yukihiro Nishiyama,³ and Tatsuya Tsurumi^1*

Division of Virology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464-8681,¹ Graduate School of Science and Technology, Faculty of Science, Kumamoto University, Kumamoto 860-8555,² Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466-8550, Japan³

Received 10 June 2002/ Accepted 8 October 2002

Productive infection and replication of herpesviruses usually occurs in growth-arrested cells, but there has been no direct evidence in the case of Epstein-Barr virus (EBV), since an efficient lytic replication system without external stimuli does not exist for the virus. Expression of the EBV lytic-switch transactivator BZLF1 protein in EBV-negative epithelial tumor cell lines, however, is known to arrest the cell cycle in G₀/G₁ by induction of the tumor suppressor protein p53 and the cyclin-dependent kinase (CDK) inhibitors p21^WAF-1/CIP-1 and p27^KIP-1, followed by the accumulation of a hypophosphorylated form of the Rb protein. In order to determine the effect of the onset of lytic viral replication on cellular events in latently EBV-infected B LCLs, a tightly controlled induction system of the EBV lytic-replication program by inducible BZLF1 protein expression was established in B95-8 cells. The induction of lytic replication completely arrested cell cycle progression and cellular DNA replication. Surprisingly, the levels of p53, p21^WAF-1/CIP-1, and p27^KIP-1 were constant before and after induction of the lytic program, indicating that the cell cycle arrest induced by the lytic program is not mediated through p53 and the CDK inhibitors. Furthermore, although cellular DNA replication was blocked, elevation of cyclin E/A expression and accumulation of hyperphosphorylated forms of Rb protein were observed, a post-G₁/S phase characteristic of cells. Thus, while the EBV lytic program promoted specific cell cycle-associated activities involved in the progression from G₁ to S phase, it inhibited cellular DNA synthesis. Such cellular conditions appear to especially favor viral lytic replication.

Present address: Virology Division, National Cancer Center, Tokyo 104-0045, Japan.

Present address: Division of Immunology, Aichi Cancer Center, Nagoya 464-8681, Japan.