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Negative Regulation of Mitotic Promoting Factor by the Checkpoint Kinase Chk1 in Simian Virus 40 Lytic Infection

Eiji Okubo, John M. Lehman, and Thomas D. Friedrich^*

Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York 12208

Received 24 May 2002/ Accepted 4 October 2002

Lytic infection of African green monkey kidney (CV-1) cells by simian virus 40 (SV40) is characterized by stimulation of DNA synthesis leading to bypass of mitosis and replication of cellular and viral DNA beyond a 4C DNA content. To define mechanisms underlying the absence of mitosis, the expression levels of upstream regulatory molecules of mitosis-promoting factor (MPF) were compared in parallel synchronized cultures of SV40-infected and uninfected CV-1 cells. The DNA replication/damage checkpoint kinase Chk1 was phosphorylated in both uninfected and SV40-infected cultures arrested at G₁/S by mimosine, consistent with checkpoint activation. Following release of uninfected cultures from G₁/S, Chk1 phosphorylation was lost even though Chk1 protein levels were retained. In contrast, G₁/S-released SV40-infected cultures exhibited dephosphorylation of Chk1 in S phase, followed by an increase in Chk1 phosphorylation coinciding with entry of infected cells into >G₂. Inhibitors of Chk1, UCN-01 and caffeine, induced mitosis and abnormal nuclear condensation and increased the protein kinase activity of MPF in SV40-infected CV-1 cells. These results demonstrate that SV40 lytic infection triggers components of a DNA damage checkpoint pathway. In addition, chemical inhibition of Chk1 activity suggests that Chk1 contributes to the absence of mitosis during SV40 lytic infection.

Present address: Michael Heidelberger Division, Department of Pathology, New York University School of Medicine, New York, N.Y.