Replication of the human cytomegalovirus (HCMV) genome takes place in the nuclei of infected cells and is mediated by a viral-encoded DNA polymerase complex, formed by the catalytic subunit pUL54 and the processivity factor ppUL44, which tethers the catalytic subunit to DNA. ppUL44 dimerizes in the cytoplasm of infected cells before being translocated to the nucleus and has been proposed to act as a scaffold promoting the assembly of several HCMV replication fork proteins, such as pUL54 and the DNA uracil glycosylase pUL114. Although both pUL44 and pUL54 contain importin \u3b1/\u3b2 recognized nuclear localization signals (NLSs) they are also capable of being imported into the nucleus as a complex. Whereas ppUL44 nuclear import is a CK2-phosphorylation enhanced process, pUL54\u2019s is not. Here we show that ppUL44 nuclear import is likely to be also negatively regulated by protein kinase C-mediated phosphorylation of residue T427. As shown by quantitative confocal laser scanning microscopic analysis of live COS-7 cells expressing several ppUL44 GFP and DsRed2 fusion proteins, Phorbol 12-myristate 13-acetate (PMA)-induced PKC activation resulted in reduction of the nuclear accumulation of GFP-UL44 but not of the non phosphorylable A427 derivative mutant. In the absence of PMA, the phosphomimetic D427 derivative mutant exhibited reduced nuclear import when compared to the wild-type and the A427 mutant. Since ppUL114 nuclear accumulation seems to be dependent on ppUL44, we suggest that the phosphorylation state of ppUL44 could regulate the nuclear import rate of the HCMV DNA polymerase holoenzyme, and other viral proteins such as ppUL114
