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Roscovitine, a Specific Inhibitor of Cellular Cyclin-Dependent Kinases, Inhibits Herpes Simplex Virus DNA Synthesis in the Presence of Viral Early Proteins

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076, USA.
Journal of Virology (Impact Factor: 4.65). 04/2000; 74(5):2107-20. DOI: 10.1128/JVI.74.5.2107-2120.2000
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ABSTRACT We have previously shown that two inhibitors specific for cellular cyclin-dependent kinases (cdks), Roscovitine (Rosco) and Olomoucine (Olo), block the replication of herpes simplex virus (HSV). Based on these results, we demonstrated that HSV replication requires cellular cdks that are sensitive to these drugs (L. M. Schang, J. Phillips, and P. A. Schaffer. J. Virol. 72:5626-5637, 1998). We further established that at least two distinct steps in the viral replication cycle require cdks: transcription of immediate-early (IE) genes and transcription of early (E) genes (L. M. Schang, A. Rosenberg, and P. A. Schaffer, J. Virol. 73:2161-2172, 1999). Since Rosco inhibits HSV replication efficiently even when added to infected cells at 6 h postinfection, we postulated that cdks may also be required for viral functions that occur after E gene expression. In the study presented herein, we tested this hypothesis directly by measuring the efficiency of viral replication, viral DNA synthesis, and expression of several viral genes during infections in which Rosco was added after E proteins had already been synthesized. Rosco inhibited HSV replication, and specifically viral DNA synthesis, when the drug was added at the time of release from a 12-h phosphonoacetic acid (PAA)-induced block in viral DNA synthesis. Inhibition of DNA synthesis was not a consequence of inhibition of expression of IE or E genes in that Rosco had no effect on steady-state levels of two E transcripts under the same conditions in which it inhibited viral DNA synthesis. Moreover, viral DNA synthesis was inhibited by Rosco even in the absence of protein synthesis. In a second series of experiments, the replication of four HSV mutants harboring temperature-sensitive mutations in genes essential for viral DNA replication was inhibited when Rosco was added at the time of shift-down from the nonpermissive to the permissive temperature. Viral DNA synthesis was inhibited by Rosco under these conditions, whereas expression of viral E genes was not affected. We conclude that cellular Rosco-sensitive cdks are required for replication of viral DNA in the presence of viral E proteins. This requirement may indicate that HSV DNA synthesis is functionally linked to transcription, which requires cdks, or that both viral transcription and DNA replication, independently, require viral or cellular factors activated by Rosco-sensitive cdks.

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    • "However, this appears to contradict the known biology of the virus, as HSV-1 requires the cell nucleus for its replication. Moreover, HSV-1 replicates independently of the cell cycle, and the lytic cycle does not depend on cell arrest at the mitotic phase [75]. "
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    • "The purine analogue, roscovitine (2- (1-Ethyl-2-hydroxyethylamino)-6-ben- zylamino-9-isopropylpurine) is a potent, but reversible specific competitive inhibitor of Cdks 1, 2, and 5 (Worland et al., 1993; De Azevedo et al., 1996; Meijer et al., 1997; Alessi et al., 1998; Gray et al., 1998; Hajduch et al., 1999; Senderowicz and Sausville, 2000; Wang et al., 2001), and there is some evidence that roscovitine can also inhibit Cdks7–9 that regulate transcription (Hajduch et al., 1999; Wang et al., 2001). In human cell lines examined, roscovitine arrests cells in the G 1 and the G 2 /M phases of the cell cycle (Meijer et al., 1997; Pippin et al., 1997; Alessi et al., 1998), inhibits DNA synthesis (Yakisich et al., 1999; Schang et al., 2000), causes nucleolar fragmentation (David-Pfeuty, 1999), induces apoptosis (Mgbonyebi et al., 1999; Atienza et al., 2000), and inhibits apoptosis (Maas et al., 1998; Choi et al., 1999; Hiromura et al., 1999; Padmanabhan et al., 1999; Giardina and Beart, 2002). "
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