HERPES-SIMPLEX-VIRUS-1-ALPHA REGULATORY PROTEIN ICP0 INTERACTS WITH AND STABILIZES THE CELL-CYCLE REGULATOR CYCLIN D3

The herpes simplex virus 1 (HSV-1) infected-cell protein O (ICPO) has the characteristics of a promiscuous transactivator of genes introduce d into cells bg infection or transfection, To identify cellular protei ns interacting with ICPO, we used a domain or exon II of ICPO that is known to be crucial for regulatory function of the protein as bait in the yeast two-hybrid screen, Our results were as follows. (i) A cDNA i n a positive yeast colony was found to encode cyclin D3, a cell cycle regulator of G(1) phase. iii) A purified chimeric protein consisting o f glutathione S-transferase (GST) fused to cyclin D3 specifically form ed complexes with ICPO contained in HSV-1-infected cell lysate, (iii) To enhance the expression of cyclin D3, the gene was inserted into the viral genome and overexpressed in infected cells. The overexpressed c yclin D3 colocalized with ICPO in nuclear structures characteristic of ND1O and which earlier have been reported to contain ICPO. (iv) The a ccumulation of cyclin D3 protein in Vero cells infected with an alpha O deletion mutant was reduced relative to that of cells infected with wild-type virus or a recombinant virus in which the deleted alpha O se quences were restored, iv) Lysates of Spodoptera frugiperda Sf9 cells doubly infected with baculoviruses genetically engineered to express c yclin D3 and cyclin-dependent kinase 4 (CDK4) phosphorylated GST fused to retinoblastoma protein (GST-pRb) but did not phosphorylate the GST -alpha(20-241) or GST-alpha O543-768 fusion protein or immunoprecipita ted ICPO proteins. Moreover, the chimeric GST-ICPOexon II protein show n to bind cyclin D3 had no effect on the activity of the kinase on GST -pRb when added to mixtures of lysates of Sf9 cells which coexpressed cyclin D3 and CDK4. These results indicate that ICPO interacts with, c olocalizes crith, and stabilizes the cyclin D3 cell cycle regulator an d does not affect its interaction with the cyclin-dependent kinase.