Requirements for the nuclear-cytoplasmic translocation of infected-cell protein 0 of herpes simplex virus 1

Earlier studies have shown that wild-type infected-cell protein 0 (ICP0), a key herpes simplex virus regulatory protein, translocates from the nucleus to the cytoplasm of human embryonic lung (HEL) fibroblasts within several hours after infection (Y. Kawaguchi, R. Bruni, and B. Roizman, J. Virol. 71 :1019-1024, 1997). Translocation of ICP0 was also observed in cells infecte d with the d120 mutant, in which both copies of the gene encoding ICP4, the major regulatory protein, had been deleted (V. Calvan, R. Brandimarti, J. Munger, and B. Roizman, J. Virol. 74:1931-1938, 2000). Furthermore, a mutan t (R7914) carrying the D199A substitution in ICP0 does not bind or stabiliz e cyclin D3 and is retained in the nucleus (C. Van Sant, P. Lopez, S. J. Ad vani, and B. Roizman, J. Virol. 75:1888-1898, 2001). Studies designed to el ucidate the requirements for the translocation of ICP0 between cellular com partments revealed the following. (i) Translocation of ICP0 to the cytoplas m in productive infection maps to the D199 amino acid, inasmuch as wild-typ e ICP0 delivered in trans to cells infected with an ICP0 null mutant was tr anslocated to the cytoplasm whereas the D199A-substituted mutant ICP0 was n ot. (ii) Translocation of wild-type ICP0 requires a function expressed late in infection, inasmuch as phosphonoacetate blocked the translocation of IC P0 in wild-type virus-infected cells but not in d120 mutant-infected cells. Moreover, whereas in d120 mutant-infected cells ICP0 was translocated rapi dly from the cytoplasm to the nucleus at approximately 5 h after infection, the translocation of ICP0 in wild-type virus-infected cells extended from 5 to at least 9 h after infection. (iii) In wild-type virus-infected cells, the MG132 proteasomal inhibitor blocked the translocation of ICP0 to the c ytoplasm early in infection, but when added late in infection, it caused IC P0 to be relocated back to the nucleus from the cytoplasm. (iv) MG132 block ed the translocation of ICP0 in d120 mutant-infected cells early in infecti on but had no effect on the ICP0 aggregated in vesicle-like structures late in infection. However, in d120 mutant-infected cells treated with MG132 at late times, proteasomes formed a shell-like structure around the aggregate d ICP0. These structures were not seen in wild-type virus or R7914 mutant-i nfected cells. The results indicate the following. (i) In the absence of be ta or gamma protein synthesis, ICP0 dynamically associates with proteasomes and is translocated to the cytoplasm. (ii) In cells productively infected beyond alpha gene expression, ICP0 is retained in the nucleus until after t he onset of viral DNA synthesis and the synthesis of gamma (2) proteins. (i ii) Late in infection, ICP0 is actively sequestered in the cytoplasm by a p rocess mediated by proteasomes, inasmuch as interference with proteasomal f unction causes rapid relocation of ICP0 to the nucleus.