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1.
J Virol ; 84(13): 6367-76, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20410270

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.


Subject(s)
GC Rich Sequence , Herpesvirus 1, Human/physiology , Immediate-Early Proteins/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , Virus Replication , Amino Acid Substitution/genetics , Animals , Arginine/genetics , Chlorocebus aethiops , HeLa Cells , Humans , Immediate-Early Proteins/chemistry , Immediate-Early Proteins/genetics , Lysine/genetics , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Tertiary , RNA Transport , Vero Cells
2.
J Virol ; 84(5): 2212-22, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20015986

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.


Subject(s)
Herpesvirus 1, Human , Immediate-Early Proteins , Nuclear Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Alanine/genetics , Alanine/metabolism , Amino Acid Substitution , Animals , Glutamic Acid/genetics , Glutamic Acid/metabolism , HeLa Cells , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/metabolism , Humans , Immediate-Early Proteins/genetics , Immediate-Early Proteins/metabolism , Mutagenesis, Site-Directed , Nuclear Proteins/genetics , Nucleocytoplasmic Transport Proteins/genetics , Phosphorylation , RNA, Viral/genetics , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics
3.
J Virol ; 84(5): 2200-11, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20015991

ABSTRACT

Herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is posttranslationally modified by phosphorylation during viral infection. ICP27 has been shown to be phosphorylated on three serine residues, specifically serine residues 16 and 18, which are within casein kinase 2 (CK2) sites, and serine residue 114, which is within a protein kinase A (PKA) site. Phosphorylation is an important regulatory mechanism that is reversible and controls many signaling pathways, protein-protein interactions, and protein subcellular localization. To determine the role of phosphorylation in modulating the activities of ICP27, we constructed phosphorylation site mutations at each of the three serine residues. Single, double, and triple viral mutants were created in which alanine or glutamic acid was substituted for serines 16, 18, and 114. ICP27 phosphorylation site mutants were defective in viral replication and viral gene expression. Notably, ICP4-containing replication compartment formation was severely compromised, with the appearance of small ring-like structures that persisted even at late times after infection. Neither the colocalization of ICP27 with RNA polymerase II nor the formation of Hsc70 nuclear foci was observed during infection with the phosphorylation site mutants, both of which occur during wild-type HSV-1 infection. These data indicate that several key events in which ICP27 plays a role are curtailed during infection with ICP27 phosphorylation site mutants.


Subject(s)
Gene Expression Regulation, Viral , Herpesvirus 1, Human/physiology , Immediate-Early Proteins/genetics , Virus Replication/genetics , Animals , Cell Line , HSC70 Heat-Shock Proteins/genetics , HSC70 Heat-Shock Proteins/metabolism , Herpesvirus 1, Human/genetics , Humans , Immediate-Early Proteins/metabolism , Mutagenesis, Site-Directed , Phosphorylation , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Serine/genetics , Serine/metabolism
4.
Nucleic Acids Res ; 37(21): 7290-301, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19783816

ABSTRACT

Herpes simplex virus 1 (HSV-1) protein ICP27, an important regulator for viral gene expression, directly recognizes and exports viral RNA through an N-terminal RGG box RNA binding motif, which is necessary and sufficient for RNA binding. An ICP27 N-terminal peptide, including the RGG box RNA binding motif, was expressed and its binding specificity was analyzed using EMSA and SELEX. DNA oligonucleotides corresponding to HSV-1 glycoprotein C (gC) mRNA, identified in a yeast three-hybrid analysis, were screened for binding to the ICP27 N-terminal peptide in EMSA experiments. The ICP27 N-terminus was able to bind most gC substrates. Notably, the ICP27 RGG box was unable to bind G-quartet structures recognized by the RGG domains of other proteins. SELEX analysis identified GC-rich RNA sequences as a common feature of recognition. NMR analysis of SELEX and gC sequences revealed that sequences able to bind to ICP27 did not form secondary structures and conversely, sequences that were not able to bind to ICP27 gave spectra consistent with base-pairing. Therefore, the ICP27 RGG box is unique in its recognition of nucleic acid sequences compared to other RGG box proteins; it prefers flexible, GC-rich substrates that do not form stable secondary structures.


Subject(s)
GC Rich Sequence , Immediate-Early Proteins/metabolism , RNA, Viral/chemistry , RNA-Binding Proteins/metabolism , Base Sequence , Binding Sites , DNA, Viral/chemistry , Electrophoretic Mobility Shift Assay , G-Quadruplexes , Immediate-Early Proteins/chemistry , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Tertiary , RNA, Viral/metabolism , RNA-Binding Proteins/chemistry , SELEX Aptamer Technique , Viral Envelope Proteins/genetics
5.
Virology ; 315(1): 234-44, 2003 Oct 10.
Article in English | MEDLINE | ID: mdl-14592775

ABSTRACT

A complex of the Adenovirus (Ad) early region 1b 55-kDa protein (E1b-55kDa) and the early region 4 ORF6 34-kDa protein (E4-34kDa) promotes viral late RNA accumulation in the cytoplasm while inhibiting the transport of most newly synthesized cellular mRNA. The E4 ORF3 11-kDa protein (E4-11kDa) functionally compensates for at least some of the activities of this complex. We find that the same large central region of E4-34kDa that is required for proteasome-mediated degradation of p53 (J. Virol. 75, (2001) 699-709) is also required to promote viral late gene expression in a complementation assay. E4-34kDa does not promote late gene expression in complementation assays performed in the presence of proteasome inhibitors. A proteasome inhibitor also dramatically reduced late gene expression by a virus that lacks the E4-11kDa gene and therefore relies on E4-34kDa for late gene expression. Our results suggest that E4-34kDa activity in promoting late gene expression depends on the proteasome.


Subject(s)
Acetylcysteine/analogs & derivatives , Adenovirus E4 Proteins/metabolism , Adenoviruses, Human/physiology , Gene Expression Regulation, Viral , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex , Acetylcysteine/pharmacology , Adenovirus E4 Proteins/genetics , Adenoviruses, Human/genetics , Animals , Cell Line , Chlorocebus aethiops , Genetic Complementation Test , HeLa Cells , Humans , Mutation , Virus Replication
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