Role of cyclin D3 in the biology of herpes simplex virus 1 ICP0

Earlier reports from this laboratory have shown that the promiscuous transa ctivator infected cell protein 0 (ICP0) binds and stabilizes cyclin D3, tha t the binding site maps to aspartic acid 199 (D199), and that replacement o f D199 With alanine abolishes binding and reduces the capacity of the mutan t virus to replicate in quiescent cells or to cause mortality in mice infec ted by a peripheral site. The objective of this report was to investigate t he role of cyclin D3 in the biology of ICP0. We report the following result s. (i) Wild-type ICP0 activates cyclin D-dependent kinase 4 (cdk4) and stab ilizes cyclin D1 although ICP0 does not interact with this cyclin. (ii) The D199A mutant virus (R7914) does not activate cdk4 or stabilize cyclin D1, and neither the wild-type nor the mutant virus activates cdk2. (iii) Early in infection of human embryonic lung (HEL) fibroblasts both wild-type and D 199A mutant ICP0s colocalize with PML, and in these cells the ND10 nuclear structures are dispersed. Whereas wild-type ICP0 is transported to the cyto plasm between 3 and 9 h. after infection, ICP0 containing the D199A substit ution remains quantitatively in the nucleus. (iv) To examine the interactio n of ICPO with cyclin D3, we used a previously described mutant carrying a wild type ICPO but expressing cyclin D3 (R7801) and in addition constructed a virus (R7916) that was identical except that it carried the D199A-substi tuted ICP0. Early in infection with R7801, ICPO colocalized with cyclin D3 in structures similar to those containing PML. At 3 h after infection, ICPO was translocated to the cytoplasm whereas cyclin D3 remained in the nucleu s. The translocation of ICPO to the cytoplasm was accelerated in cells expr essing cyclin D3 compared with that of ICPO expressed by wild-type virus. I n contrast, ICPO carrying the D199A substitution remained in the nucleus an d did not colocalize with cyclin D3. These studies suggest the following co nclusions. (i) ICP0 brings to the vicinity of ND10 cyclin D3 and, in conseq uence, an activated cdk4. The metabolic events occurring at or near that st ructure and involving cyclin D3 cause the translocation of ICP0 to the cyto plasm. (ii) In the absence of the cyclin D3 binding site in ICP0, cyclin D3 is not brought to ND10, cyclin D is not stabilized, and the function respo nsible for the translocation of ICP0 is not expressed, and in quiescent HEL fibroblasts the yields of virus are reduced.