Purification of a bacterially expressed herpes simplex virus type 1 origin binding protein for use in posttranslational processing studies.

Abstract The origin binding protein (OBP) encoded by the UL9 open reading frame of herpes simplex virus type 1 (HSV-1) plays an essential role in productive infection by promoting the initiation of viral DNA synthesis. In this study, OBP was inducibly expressed in Escherichia coli and purified to homogeneity using a two-step chromatographic separation procedure. The properties of this recombinant OBP (rOBP) were found to be indistinguishable from those of the virus-encoded protein. Since rOBP was synthesized in bacterial cells, it lacked the posttranslational processing which normally occurs in OBP produced in HSV-1-infected mammalian cells and could therefore be exploited in experiments which addressed the effects of protein modification on OBP function. As an initial study, the impact of phosphorylation on enzymatic activity was examined using rOBP which had been treated with a panel of purified cellular kinases. rOBP was found to act as a substrate for nearly all of the kinases tested in 32 P-labeled phosphate transfer assays. However, only phosphorylation by protein kinase A (PKA, or cAMP-dependent protein kinase) was shown to significantly alter the enzymatic properties of rOBP, as it increased by five- to eightfold the ATPase activity associated with this protein. Activation of this critical viral DNA replication enzyme by a cAMP-dependent kinase such as PKA may be of some relevance in the natural course of HSV-1 infections, since reactivation of latent virus is thought to involve both signal transduction events and the induction of viral DNA synthesis. Thus, the expression and purification strategy outlined in this work provides an economical source of unmodified HSV-1 OBP that should prove useful in future in vitro studies.