ROLE OF PROTEIN-KINASE A AND THE SERINE-RICH REGION OF HERPES-SIMPLEXVIRUS TYPE-1 ICP4 IN VIRAL REPLICATION

Efficient expression of herpes simplex virus genes requires the synthe sis of functional ICP4, a nuclear phosphoprotein that contains a promi nent serine rich region between amino acids 142 and 210. Residues in t his region not only are potential sites for phosphorylation but also a re involved in the functions of ICP4. By comparing the growth of a vir us in which this region is deleted (d8-10) with wild-type virus (KOS) in PC12 cells or PC12 cells that are deficient in cyclic AMP-dependent protein kinase (PKA), two observations were made: (i) the growth of w ild-type virus was impaired by 1 to 2 orders of magnitude in the PKA d eficient cells, indicating the involvement of PKA in the growth cycle of herpes simplex virus type 1, and (ii) while the growth of d8-10 was impaired by almost 2 orders of magnitude in wild-type cells, it was n ot further impaired (as was that of wild-type virus) in PKA-deficient cells, implicating the region deleted in d8-10 as a possible target fo r cellular PKA. In trigeminal ganglia of mice, the d8-10 mutant virus grew poorly; however, it established latency in nearly 90% of ganglia tested. Studies of the phosphorylation of wild-type and d8-10 ICP? pro teins revealed that the serine-rich region is a major determinant for phosphorylation of ICP4 in vivo and that the phosphorylation state cou ld change as a function of the PKA activity. Consistent with this obse rvation, the serine-rich region of ICP4 was shown to be a target for P KA in vitro. While intact ICP4 was readily phosphorylated by ICP4 in v itro, the d8-10 mutant ICP4 was not, Moreover, a synthetic peptide rep resenting a sequence in the serine tract that is predicted to be a sub strate for PKA was phosphorylated by PKA in vitro, having a K-m within the physiological range. These data suggest that PKA plays a role in viral growth through phosphorylation of one or more sites on the ICP4 molecule.