The Differential Requirement for Cyclin-Dependent Kinase Activities Distinguishes Two Functions of Herpes Simplex Virus Type 1 ICP0

Abstract

Herpes simplex virus type 1 (HSV-1) ICP0 directs the degradation of cellular proteins associated with nuclear structures called ND10, a function thought to be closely associated with its broad transactivating activity. Roscovitine (Rosco), an inhibitor of cyclin-dependent kinases (cdks), inhibits the replication of HSV-1, HSV-2, human cytomegalovirus, varicella-zoster virus, and human immunodeficiency virus type 1 by inhibiting specific steps or activities of viral regulatory proteins, indicating the broad and pleiotropic effects that cdks have on the replication of these viruses. We previously demonstrated that Rosco inhibits the transactivating activity of ICP0. In the present study, we asked whether Rosco also affects the ability of ICP0 to direct the degradation of ND10-associated proteins. For this purpose, WI-38 cells treated with cycloheximide (CHX) were mock infected or infected with wild-type HSV-1 or an ICP0؊ mutant (7134). After release from the CHX block, the infections were allowed to proceed for 2 h in the presence or absence of Rosco at a concentration known to inhibit ICP0's transactivating activity. The cells were then examined for the presence of ICP0 and selected ND10-associated antigens (promyelocytic leukemia antigen [PML], sp100, hDaxx, and NDP55) by immunofluorescence. Staining for the ND10-associated antigens was detected in <20% of KOS-infected cells in the presence or absence of Rosco, demonstrating that Rosco-sensitive kinases are not required for ICP0's ability to direct the dispersal or degradation of these antigens. In contrast, >90% of 7134-and mock-infected cells stained positive for all ND10-associated antigens in the presence or absence of Rosco. Similar results were obtained with a non-ND10-associated antigen, DNA-PK cs , a known target of ICP0-directed degradation. The results of the PML and DNA-PK cs immunofluorescence studies correlated with a decrease in the levels of these proteins as determined by Western blotting. Thus, the differential requirement for Rosco-sensitive cdk activities distinguishes ICP0's ability to direct the dispersal or degradation of cellular proteins from its transactivating activity.The molecular mechanisms responsible for the activation of herpes simplex virus (HSV) gene expression during productive infection and reactivation from latency are not well understood. Available evidence indicates, however, that the functions of viral regulatory proteins that activate viral-gene expression are themselves activated by cell cycle regulatory proteins. For example, two cellular proteins, HCF (36) and TAF250 (84), whose expression and activities are cell cycle associated, activate the functions of two viral regulatory proteins, VP16 (36) and ICP4 (12), respectively, and cyclin-dependent kinases (cdks), which drive the cell cycle, are required for the synthesis and activities of HSV immediate-early (IE) regulatory proteins (75-77). Notably, inhibitors of cdks have been shown to inhibit the replication of HSV type 1 (HSV-1) (74), HSV-2 (74), human cytomegaloviru...

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

“…1354, 2002). Because several PCIs inhibit viral replication in vitro, they have also been proposed as potential antiviral drugs (8,13,17,19,20,24,25,38,52,53,55,56,65,69,73,84). However, not all biological activities of these drugs have been characterized, and purineand nonpurine-type PCIs may even act via different mechanisms.…”

“…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).…”

“…Considering that Rosco has been repeatedly proposed as a potential antiviral agent (8,13,17,19,24,25,38,52,55,56,65,69,73,84) and is already in human clinical trials to assess its activity against proliferative diseases (10, 25, 40; Benson et al, Abstr. 92nd Annu.…”

“…For example, Pharmacological Cyclin-dependent kinase (CDK) inhibitors (PCIs) inhibit the replication of humanpathogenic viruses such as human immunodeficiency virus type 1 (70), human cytomegalovirus (9), varicella-zoster virus (54, 82a), Epstein-Barr virus (38), and herpes simplex virus types 1 (HSV-1) (73) and 2 (HSV-2) (70). Since PCIs are proving surprisingly safe for humans in clinical trials of their effects against cancer (25,26), they have been repeatedly proposed as potential antiviral drugs (8,13,17,19,20,24,25,38,52,53,55,56,65,69,73,84).…”

“…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).…”