Phosphorylation Site Mutations Affect Herpes Simplex Virus Type 1 ICP0 Function

Abstract

The herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein infected-cell protein 0 (ICP0) is a strong and global transactivator of both viral and cellular genes. In a previous study, we reported that ICP0 is highly phosphorylated and contains at least seven distinct phosphorylation signals as determined by phosphotryptic peptide mapping (D. J. Davido et al., J. Virol. 76:1077-1088, 2002). Since phosphorylation affects the activities of many viral regulatory proteins, we sought to determine whether the phosphorylation of ICP0 affects its functions. To address this question, it was first necessary to identify the regions of ICP0 that are phosphorylated. For this purpose, ICP0 was partially purified, and phosphorylation sites were mapped by microcapillary high-pressure liquid chromatography tandem mass spectrometry. Three phosphorylated regions containing 11 putative phosphorylation sites, all within or adjacent to domains important for the transactivating activity of ICP0, were identified. The 11 sites were mutated to alanine as clusters in each of the three regions by site-directed mutagenesis, generating plasmids expressing mutant forms of ICP0: Phos 1 (four mutated sites), Phos 2 (three mutated sites), and Phos 3 (four mutated sites). One-dimensional phosphotryptic peptide analysis confirmed that the phosphorylation state of each Phos mutant form of ICP0 is altered relative to that of wild-type ICP0. In functional assays, the ICP0 phosphorylation site mutations affected the subcellular and subnuclear localization of ICP0, its ability to alter the staining pattern of the nuclear domain 10 (ND10)-associated protein PML, and/or its transactivating activity in Vero cells. Only mutations in Phos 1, however, impaired the ability of ICP0 to complement the replication of an ICP0 null mutant in Vero cells. This study thus suggests that phosphorylation is an important regulator of ICP0 function.Phosphorylation is a universal posttranslational modification that alters the activities of many viral regulatory proteins. Among the best examples of this effect is large T antigen of simian virus 40, a multifunctional 708-amino-acid nuclear phospho-and oncoprotein required for the replication of simian virus 40 (reviewed in references 61 and 76). Many phosphorylation sites have been identified on T antigen, and several lie adjacent to its nuclear localization signal and origin-binding domain, modulating simian virus 40 DNA replication (reviewed in reference 61). Specifically, mutation of these sites alters T antigen's origin-binding activity, hexamer-hexamer interaction, nuclear localization, and/or DNA replication activity, affecting both its biochemical and biological functions (11, 14, 41, 50-52, 54, 78).In herpes simplex virus type 1 (HSV-1) infection, the first genes to be expressed are the immediate-early (IE) genes (39). These genes encode infected-cell protein (ICPs) 0, 4, 22, 27, and 47, which collectively exhibit diverse regulatory and immunomodulatory functions. At least four of the five IE regul...

Herpes Simplex Virus 1 ICP0 Phosphorylation Site Mutants Are Attenuated for Viral Replication and Impaired for Explant-Induced Reactivation

“…quired for productive infection and explant-induced reactivation in mice. Of the Phos mutants examined, Phos 1 was the only mutant to show replication deficiencies in cell culture (6,16) and in vivo, as demonstrated in this report. Notably, Phos 2 had no obvious viral growth defects in cell culture (6,16); however, decreased levels of viral replication and explant-induced reactivation observed with Phos 2 only became apparent when tested in the mouse ocular model, highlighting the importance of characterizing our mutants in vivo.…”

“…Of the Phos mutants examined, Phos 1 was the only mutant to show replication deficiencies in cell culture (6,16) and in vivo, as demonstrated in this report. Notably, Phos 2 had no obvious viral growth defects in cell culture (6,16); however, decreased levels of viral replication and explant-induced reactivation observed with Phos 2 only became apparent when tested in the mouse ocular model, highlighting the importance of characterizing our mutants in vivo. While we tested only one viral dose in this study, it is also possible that the replication, latency, and/or reactivation phenotypes of Phos 1, 2, and 3 may be greater or become evident when infecting mice with lower input viral doses (Ͻ2 ϫ 10 5 PFU/eye).…”

“…1) altered several properties of the protein, and at least one ICP0 phosphorylation mutant (Phos 1) was impaired for viral replication in cell culture (6,16). To determine whether the relative replication efficiencies of the Phos mutant viruses were altered in vivo, we used the mouse ocular model of HSV latency and reactivation.…”

“…The activities of cdks appear to modulate the biological properties of ICP0, including its transactivating activity (15,17). To directly determine the role phosphorylation plays in ICP0 functions, we mapped three phosphorylated regions in ICP0 and mutated phospho-acceptor sites in each of these regions, creating the mutants Phos 1, 2, and 3 (16). In transient expression assays, these phosphorylation site mutations affected several properties of ICP0, including its subcellular localization, E3 ubiquitin ligase activity, ability to dissociate proteins from ND10, and transactivation activity (6,16).…”

“…To directly determine the role phosphorylation plays in ICP0 functions, we mapped three phosphorylated regions in ICP0 and mutated phospho-acceptor sites in each of these regions, creating the mutants Phos 1, 2, and 3 (16). In transient expression assays, these phosphorylation site mutations affected several properties of ICP0, including its subcellular localization, E3 ubiquitin ligase activity, ability to dissociate proteins from ND10, and transactivation activity (6,16). When these mutations were introduced into the HSV-1 genome, only Phos 1 was impaired for HSV-1 replication in cell culture (6).…”