THE HUMAN CYTOMEGALOVIRUS UL97 PROTEIN IS A PROTEIN-KINASE THAT AUTOPHOSPHORYLATES ON SERINES AND THREONINES

The product of the human cytomegalovirus (CMV) UL97 gene, which contro ls ganciclovir phosphorylation in virus-infected cells, is homologous to known protein kinases but diverges from them at a number of positio ns that are functionally important. To investigate UL97, we raised an antibody against it and overexpressed it in baculovirus-infected insec t cells. Recombinant baculovirus expressing full-length UL97 directed the phosphorylation of ganciclovir in insect cells, which was abolishe d by a four-codon deletion that confers ganciclovir resistance to CMV. When incubated with [gamma-P-32]ATP, full-length UL97 was phosphoryla ted on serine and threonine residues. Phosphorylation was severely imp aired by a point mutation that alters lysine-355 in a motif that align s with subdomain II of protein kinases. However, phosphorylation was i mpaired much less severely by the four-codon deletion. A UL97 fusion p rotein expressed from recombinant baculovirus was purified to near hom ogeneity. It too was phosphorylated upon incubation with [gamma-P-32]A TP in vitro. This phosphorylation, which was abolished by the lysine 3 55 mutation, was optimal at high NaCl and high pH. The activity requir ed either Mn2+ or Mg2+, with a preference for Mn2+, and utilized eithe r ATP or GTP as a phosphate donor, with K(m)s of 2 and 4 mu M, respect ively. The phosphorylation rate was first order with protein concentra tion, consistent with autophosphorylation. These data strongly argue t hat UL97 is a serine/threonine protein kinase that autophosphorylates and suggest that the four-codon deletion affects its substrate specifi city.