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J. Virol., Oct 1997, 7328-7336, Vol 71, No. 10
Y Kawaguchi, C Van Sant and B Roizman
The herpes simplex virus 1 (HSV-1) infected-cell protein 0 (ICP0) has the
characteristics of a promiscuous transactivator of genes introduced into
cells by infection or transfection. To identify cellular proteins
interacting with ICP0, we used a domain of exon II of ICP0 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 G1
phase. (ii) A purified chimeric protein consisting of glutathione
S-transferase (GST) fused to cyclin D3 specifically formed complexes with
ICP0 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 ICP0 in nuclear structures characteristic of ND10 and which earlier
have been reported to contain ICP0. (iv) The accumulation of cyclin D3
protein in Vero cells infected with an alpha0 deletion mutant was reduced
relative to that of cells infected with wild-type virus or a recombinant
virus in which the deleted alpha0 sequences were restored. (v) 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- alpha0(20-241) or GST-alpha0(543-768) fusion
protein or immunoprecipitated ICP0 proteins. Moreover, the chimeric
GST-ICP0(exon 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
ICP0 interacts with, colocalizes with, and stabilizes the cyclin D3 cell
cycle regulator and does not affect its interaction with the cyclin-
dependent kinase.
Copyright © 1997, American Society for Microbiology
Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3
The Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, Illinois 60637, USA.
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