title: ICP27 phosphorylation site mutants display altered functional interactions with cellular export factors Aly/REF and TAP/NXF1 but are able to bind herpes simplex virus 1 RNA.
abstract: Herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is phosphorylated. Phosphorylation can affect protein localization, protein interactions, and protein function. The major sites of ICP27 that are phosphorylated are serine residues 16 and 18, within a CK2 site adjacent to a leucine-rich region required for ICP27 export, and serine 114, within a PKA site in the nuclear localization signal. Viral mutants bearing serine-to-alanine or glutamic acid substitutions at these sites are defective in viral replication and gene expression. To determine which interactions of ICP27 are impaired, we analyzed the subcellular localization of ICP27 and its colocalization with cellular RNA export factors Aly/REF and TAP/NXF1. In cells infected with phosphorylation site mutants, ICP27 was confined to the nucleus even at very late times after infection. ICP27 did not colocalize with Aly/REF or TAP/NXF1, and overexpression of TAP/NXF1 did not promote the export of ICP27 to the cytoplasm. However, in vitro binding experiments showed that mutant ICP27 was able to bind to the same RNA substrates as the wild type. Nuclear magnetic resonance (NMR) analysis of the N terminus of ICP27 from amino acids 1 to 160, compared to mutants with triple substitutions to alanine or glutamic acid, showed that the mutations affected the overall conformation of the N terminus, such that mutant ICP27 was more flexible and unfolded. These results indicate that these changes in the structure of ICP27 altered in vivo protein interactions that occur in the N terminus but did not prevent RNA binding.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of California, Irvine, California 92697-4025, USA.
authors.forename: Kara A
authors.initials: KA
authors.lastname: Corbin-Lickfett
authors.forename: Santos
authors.initials: S
authors.lastname: Rojas
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Cocco
authors.forename: Rozanne M
authors.initials: RM
authors.lastname: Sandri-Goldin
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: C105242:ALYREF protein, human; C057120:ICP27 protein, human herpesvirus 1; D017874:Immediate-Early Proteins; C122111:NXF1 protein, human; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D018698:Glutamic Acid; D000409:Alanine