title: Three arginine residues within the RGG box are crucial for ICP27 binding to herpes simplex virus 1 GC-rich sequences and for efficient viral RNA export.
abstract: ICP27 is a multifunctional protein that is required for herpes simplex virus 1 mRNA export. ICP27 interacts with the mRNA export receptor TAP/NXF1 and binds RNA through an RGG box motif. Unlike other RGG box proteins, ICP27 does not bind G-quartet structures but instead binds GC-rich sequences that are flexible in structure. To determine the contribution of arginines within the RGG box, we performed in vitro binding assays with N-terminal proteins encoding amino acids 1 to 160 of wild-type ICP27 or arginine-to-lysine substitution mutants. The R138,148,150K triple mutant bound weakly to sequences that were bound by the wild-type protein and single and double mutants. Furthermore, during infection with the R138,148,150K mutant, poly(A)(+) RNA and newly transcribed RNA accumulated in the nucleus, indicating that viral RNA export was impaired. To determine if structural changes had occurred, nuclear magnetic resonance (NMR) analysis was performed on N-terminal proteins consisting of amino acids 1 to 160 from wild-type ICP27 and the R138,148,150K mutant. This region of ICP27 was found to be highly flexible, and there were no apparent differences in the spectra seen with wild-type ICP27 and the R138,148,150K mutant. Furthermore, NMR analysis with the wild-type protein bound to GC-rich sequences did not show any discernible folding. We conclude that arginines at positions 138, 148, and 150 within the RGG box of ICP27 are required for binding to GC-rich sequences and that the N-terminal portion of ICP27 is highly flexible in structure, which may account for its preference for binding flexible sequences.
authors.affiliation: Department of Microbiology and Molecular Genetics, School of Medicine, Medical Sciences I, University of California at Irvine, Irvine, CA 92697-4025, USA.
authors.forename: Kara A
authors.initials: KA
authors.lastname: Corbin-Lickfett
authors.forename: Stuart K
authors.initials: SK
authors.lastname: Souki
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Cocco
authors.forename: Rozanne M
authors.initials: RM
authors.lastname: Sandri-Goldin
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D002522:Chlorocebus aethiops; D020862:GC Rich Sequence; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034443:RNA Transport; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D014709:Vero Cells; D014779:Virus Replication
chemical_list: C057120:ICP27 protein, human herpesvirus 1; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D001120:Arginine; D008239:Lysine
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