The Cyclin-Dependent Kinase Inhibitor Roscovitine Inhibits the Transactivating Activity and Alters the Posttranslational Modification of Herpes Simplex Virus Type 1 ICP0

Abstract

The cyclin-dependent kinase (cdk) inhibitor Roscovitine (Rosco) reduces transcription of herpes simplex virus early genes significantly, even in the presence of wild-type levels of immediate-early (IE) viral proteins, suggesting that the transactivating functions of IE proteins may require the activities of one or more Roscosensitive cdk (L. M. Schang, A. Rosenberg, and P. A. Schaffer, J. Virol. 73:2161-2172, 1999). Based on this observation, we sought to determine whether Rosco alters the transactivating activity and posttranslational modification state of the IE protein, infected cell protein 0 (ICP0), in KOS6␤-infected Vero cells. KOS6␤ is a KOS-derived recombinant virus containing an ICP0-inducible ICP6 promoter::lacZ cassette. To monitor ICP0's transactivating activity, KOS6␤-infected cells were released from a cycloheximide (CHX)-mediated protein synthesis block into medium with or without Rosco, and ␤-galactosidase activity was measured. Rosco inhibited the ability of ICP0 to transactivate the ICP6 promoter by 50-fold. This inhibition was shown not to be a consequence of inhibition of ICP6 basal promoter activity or aberrant nuclear localization of ICP0. Rosco also altered the electrophoretic mobility of a portion of ICP0 molecules derived from KOS-infected cells following reversal of a CHX block. Notably, however, Rosco had only a minimal effect on the phosphorylation state of ICP0. We conclude that ICP0's transactivating activity requires Rosco-sensitive cdks and hypothesize that these cdks regulate the functions of cellular enzymes which modify ICP0, and are, consequently, required for its transactivating activity. Thus, we propose that Rosco regulates ICP0's posttranslational state by mechanisms other than, or in addition to, phosphorylation.Productive infection of actively dividing epithelial cells and fibroblasts by herpes simplex virus type 1 (HSV-1) is characterized by expression of all or nearly all viral genes, leading to the production of new infectious virus and cell death (reviewed in reference 62). The activities of HSV immediate-early (IE) regulatory proteins are required for the transcriptional activation of early and late genes and for the repression of IE gene transcription during productive infection (34). In nondividing neurons, however, the activities of IE regulatory proteins are repressed such that the productive phase of viral gene expression does not occur or occurs transiently, leading to latent infection. After stress, neurons again become permissive for viral gene expression leading to the production of new infectious virus (reactivation). The fact that nondividing neurons can support latent as well as productive infection (reactivation) implicates the differential expression and/or activation of cellular proteins in determining whether neuronal infection will be latent or productive. In support of this concept, Schang and coworkers have shown that inhibitors of cyclin-dependent kinases (cdks) block productive infection and reactivation from neuronal latency, implicating cdks a...

Herpes Simplex Virus Type 1 ICP0 Phosphorylation Mutants Impair the E3 Ubiquitin Ligase Activity of ICP0 in a Cell Type-Dependent Manner

“…While it has long been established that ICP0 undergoes phosphorylation during infection (2,3,15,52), little is known as to how these phosphorylation events specifically regulate the biological functions of ICP0 (3,4,16,17,43). In this study we initially sought to determine whether ICP0 phosphorylation modulated its E3 ubiquitin ligase activity and its associated ability to induce the dispersal and degradation of PML and Sp100 from ND10.…”

Herpes Simplex Virus 1 ICP0 Phosphorylation Site Mutants Are Attenuated for Viral Replication and Impaired for Explant-Induced Reactivation

“…It has been demonstrated that the phosphorylation state of ICP0 is altered during the course of the viral replication cycle (1,2,15,53,68), suggesting that differential phosphorylation of ICP0 might activate or inhibit one or more specific functions of this key viral regulatory protein. Consequently, the identity of specific phosphorylation sites in regions I and II of ICP0, the potential kinases that phosphorylate these sites, and their temporal regulation and roles in ICP0's functions will likely elucidate new mechanisms in the switch between the lytic and latent infections of HSV-1.…”

“…Cellular cyclin-dependent kinases (cdks) and the viral kinase UL13 have been reported to phosphorylate ICP0 or to be required for maximal levels of ICP0 phosphorylation in the infected cells (3,15,53). The activities of cdks appear to modulate the biological properties of ICP0, including its transactivating activity (15,17). To directly determine the role phosphorylation plays in ICP0 functions, we mapped three phosphorylated regions in ICP0 and mutated phospho-acceptor sites in each of these regions, creating the mutants Phos 1, 2, and 3 (16).…”

Roscovitine Inhibits Activation of Promoters in Herpes Simplex Virus Type 1 Genomes Independently of Promoter-Specific Factors

“…Rosco also inhibits chromatin remodeling on the adenovirus type 12 E2 promoter by the viral transcription factor E1A 12S (23). Rosco inhibits posttranslational modifications of two HSV-1 IE transcriptional regulators, ICP0 and ICP4 (1,18) and the transcription-regulatory activity of ICP0 (18,19). However, it also inhibits the accumulation of IE, E, and L HSV-1 transcripts in less than 2 h and even in the presence of cycloheximide (CHX) (73)(74)(75).…”

“…However, there are no DNA sequences unique to the promoters of all E genes, and many non-HSV-1 promoters recombined into the viral genome are regulated as HSV-1 E promoters (i.e., they require IE proteins) (78,79). Consequently, it is currently accepted that ICP0 and ICP4 activate transcription through indirect mechanisms that operate independently of promoter-specific sequences or transcription factors, which are the subject of intensive research (1,7,18,19,21,22,34,54,60,80). ICP4 also inhibits the expression of IE genes, by binding with high affinity to sequences near their transcription start sites (35).…”