Phosphorylation of the herpes simplex virus type 1 origin binding protein.
The herpes simplex virus type 1 (HSV-1) origin binding protein (OBP), the product of the UL9 gene, is one of seven HSV-encoded proteins required for viral DNA replication. OBP performs multiple functions characteristic of a DNA replication initiator protein, including origin-specific DNA binding and ATPase and helicase activities, as well as the ability to interact with viral and cellular proteins involved in DNA replication. Replication initiator proteins in other systems, including those of other DNA viruses, are known to be regulated by phosphorylation; however, the role of phosphorylation in OBP function has been difficult to assess due to the low level of OBP expression in HSV-infected cells. Using a metabolic labeling and immunoprecipitation approach, we obtained evidence that OBP is phosphorylated during HSV-1 infection. Kinetic analysis of metabolically labeled cells indicated that the levels of OBP expression and phosphorylation increased at approximately 4 h postinfection. Notably, when expressed from a transfected plasmid, a recombinant baculovirus, or a recombinant adenovirus (AdOBP), OBP was phosphorylated minimally, if at all. In contrast, superinfection of AdOBP-infected cells with an OBP-null mutant virus increased the level of OBP phosphorylation approximately threefold, suggesting that HSV-encoded viral or HSV-induced cellular factors enhance the level of OBP phosphorylation. Using HSV mutants inhibited at sequential stages of the viral life cycle, we demonstrated that this increase in OBP phosphorylation is dependent on early protein synthesis and is independent of viral DNA replication. Based on gel mobility shift assays, phosphorylation does not appear to affect the ability of OBP to bind to the HSV origins.
Isler
J A
JA
Department of Microbiology and Cell and Molecular Biology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Schaffer
P A
PA
D000818:Animals; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006561:Herpes Simplex / Q000821:virology*; D018259:Herpesvirus 1, Human / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D018741:Replication Origin / Q000502:physiology; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000096:biosynthesis; D014764:Viral Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C044768:glycoprotein E, herpes simplex virus type 1; C064565:UL9 protein, Human herpesvirus 1; D010744:Phosphoric Monoester Hydrolases