The role of ICP27 phosphorylation on regulating the activities of ICP27 during Herpes simplex virus Type-1 infection

Herpes Simplex Virus Type-1 immediate early protein ICP27 is a 63 kDa phosphoprotein that is essential for viral replication. ICP27 is a multifunctional protein that shuttles between the nucleus and the cytoplasm. The N-terminus of ICP27 contains a leucine rich region (LRR) required for its export to the cytoplasm and a nuclear localization signal (NLS) important for import. We previously reported that ICP27 is phosphorylated at three serine residues located in the N-terminus. Casein Kinase II (CKII) phosphorylates ICP27 at serines 16 and 18, in the LRR. A Protein Kinase A (PKA) site at serine 114 in the NLS was found to be phosphorylated. To study the role of phosphorylation on the activities of ICP27, we constructed serine to alanine and serine to glutamic acid substitution mutations and subsequently, single, double, and triple recombinant viral mutants. In one-step viral growth experiments, viral yields were reduced 2 to 2.5 logs compared to wild type virus for all mutants. In addition, DNA replication and transcription with phosphorylation site mutants were greatly decreased. Immunofluorescence studies showed that the localization of ICP27 during infection with phosphorylation mutants was predominantly nuclear, and ICP27 remained associated with splicing structures even at late times post infection. Formation of transcription/replication compartment did not fully form but abnormal ring-like structures were seen as late as 16 hours post infection. Wild type ICP27 interacts with cellular RNA polymerase II (RNAP II) and recruits RNAP II to viral transcription/replication compartments. In infections with phosphorylation site mutants, RNAP II was diffusely distributed throughout the nucleus with some recruitment to ICP4-ring-like structures.;Immunofluorescence studies also showed that the phosphoserine-2 form of RNAP II was not degraded and Hsc70 nuclear foci were absent in infections with the ICP27 phosphorylation mutants. Aly/REF was not found to colocalize with mutant ICP27 or recruited to viral transcription/replication compartments. ICP27 phosphorylation site mutants did not colocalize with TAP/NXF1. Together these data demonstrate that ICP27's interactions with RNAP II, Hsc70, Aly/REF, and TAP/NXF1 are adversely affected by the phosphorylation site mutations, suggesting that phosphorylation may regulate ICP27's functional interactions during infection.