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 introduced into cells bg infection or transfection, To identify cellular proteins 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 in a positive yeast colony was found to encode cyclin D3, a cell cycle regulator of G(1) phase. iii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to cyclin D3 specifically formed 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 cyclin D3 colocalized with ICPO in nuclear structures characteristic of ND1O and which earlier have been reported to contain ICPO. (iv) The accumulation 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 sequences were restored, iv) Lysates of Spodoptera frugiperda Sf9 cells doubly infected with baculoviruses genetically engineered to express cyclin 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 immunoprecipitated ICPO proteins. Moreover, the chimeric GST-ICPOexon II protein shown 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, colocalizes crith, and stabilizes the cyclin D3 cell cycle regulator and does not affect its interaction with the cyclin-dependent kinase.