The differential requirement for cyclin-dependent kinase activities distinguishes two functions of herpes simplex virus type 1 ICP0.
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.
Davido
David J
DJ
Department of Medicine, Harvard Medical School at the Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
Von Zagorski
William F
WF
Maul
Gerd G
GG
Schaffer
Priscilla A
PA
D000818:Animals; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D006868:Hydrolysis; D017874:Immediate-Early Proteins / Q000378:metabolism*; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011687:Purines / Q000494:pharmacology; D000077546:Roscovitine; D015533:Transcriptional Activation / Q000187:drug effects; D044767:Ubiquitin-Protein Ligases; D014709:Vero Cells
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D011687:Purines; D000077546:Roscovitine; D044767:Ubiquitin-Protein Ligases; C051804:Vmw110 protein, Human herpesvirus 1; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases