Identification of proteins phosphorylated directly by the Us3 protein kinase encoded by herpes simplex virus 1

We have developed a system to analyze the specific protein kinase activity of herpes simplex virus 1 Us3 in vitro and shown that Us3 directly phosphorylates viral proteins UL34, ICP22, and Us9 and the cellular protein Bad, previously reported to be putative substrates. Using this system, we determined the phosphorylation sites of UL34 and identified UL31 as a previously unreported, novel substrate of Us3. This system will be useful for further identification of Us3 substrates and their phosphorylation sites, clarification of the role of Us3 in viral replication, and identification of additional Us3 function(s).