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BGLF4 is a serine/threonine protein kinase encoded by Epstein-Barr virus. One of the physiological substrates of BGLF4 is viral transactivator BZLF1. In the present study, it was demonstrated that alanine substitution of the serine residue at position 209 (S209A) in BZLF1 eliminated phosphorylation of the protein by BGLF4 in vitro. The S209A mutation in BZLF1, as well as a K102I mutation in BGLF4, which inactivated catalytic activity of the viral kinase, also inhibited formation of a stable BGLF4-BZLF1 complex and downregulation of BZLF1 autotransactivation activity mediated by BGLF4. These results indicate that formation of a stable complex of BGLF4-BZLF1 enables downregulation of BZLF1 autoregulation activity and it appears that BGLF4 phosphorylation of BZLF1 may be involved in these processes.

Us3 is a serine/threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). Here, we report the identification of a physiological Us3 phosphorylation site on serine at position 147 (Ser-147) which regulates its protein kinase activity in vitro. Moreover, mutation of this site influences Us3 function, including correct localization of the enzyme and induction of the usual morphological changes in HSV-1-infected cells. These conclusions are based on the following observations: (i) in in vitro kinase assays, a domain of Us3 containing Ser-147 was specifically phosphorylated by Us3 and protein kinase A, while a mutant domain in which Ser-147 was replaced with alanine was not; (ii) in vitro, alanine replacement of Ser-147 (S147A) in Us3 resulted in significant impairment of the kinase activity of the purified molecule expressed in a baculovirus system; (iii) phosphorylation of Ser-147 in Us3 tagged with the monomeric fluorescent protein (FP) VenusA206K (VenusA206K-Us3) from Vero cells infected with a recombinant HSV-1 encoding VenusA206K-Us3 was specifically detected using an antibody that recognizes phosphorylated serine or threonine residues with arginine at the -3 and -2 positions; and (iv) the S147A mutation influenced some but not all Us3 functions, including the ability of the protein to localize itself properly and to induce wild-type cytopathic effects in infected cells. Our results suggest that some of the regulatory activities of Us3 in infected cells are controlled by phosphorylation at Ser-147.

Herpes simplex virus 1 (HSV-1) UL13 is a viral protein kinase that regulates optimal viral replication in cell cultures. Identification of substrates of protein kinases is a crucial step to elucidate the mechanism by which they function. Using our developed system to analyze the specific protein kinase activity of UL13, we have shown that UL13 protein kinase directly phosphorylates the viral proteins ICP22 and UL49 previously reported to be putative substrates. We also identified UL41 as a previously unreported and novel substrate of UL13. These data will serve as a basis to clarify the mechanism by which UL13 influences viral replication.

Epstein-Barr virus (EBV) BGLF4 is a viral protein kinase that is expressed in the lytic phase of infection and is packaged in virions. We report here that BGLF4 is a tegument protein that dissociates from the virion in a phosphorylation-dependent process. We also present evidence that BGLF4 interacts with and phosphorylates BZLF1, a key viral regulator of lytic infection. These conclusions are based on the following observations. (i) In in vitro tegument release assays, a significant fraction of BGLF4 was released from virions in the presence of physiological NaCl concentrations. (ii) Addition of physiological concentrations of ATP and MgCl(2) to virions enhanced BGLF4 release, but phosphatase treatment of virions significantly reduced BGLF4 release. (iii) A recombinant protein containing a domain of BZLF1 was specifically phosphorylated by purified recombinant BGLF4 in vitro, and BGLF4 altered BZLF1 posttranslational modification in vivo. (iv) BZLF1 was specifically coimmunoprecipitated with BGLF4 in 12-O-tetradecanoylphorbol-13-acetate-treated B95-8 cells and in COS-1 cells transiently expressing both of these viral proteins. (v) BGLF4 and BZLF1 were colocalized in intranuclear globular structures, resembling the viral replication compartment, in Akata cells treated with anti-human immunoglobulin G. Our results suggest that BGLF4 functions not only in lytically infected cells by phosphorylating viral and cellular targets but also immediately after viral penetration like other herpesvirus tegument proteins.