The HSV-1 ICP27 RGG box specifically binds flexible, GC-rich sequences but not G-quartet structures

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.

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

“…Recently, we reported that several GC-rich sequences from the glycoprotein C (gC) gene, which were derived as 30-mers from a 300-nucleotide clone that was positive for ICP27 binding in a yeast three-hybrid screen (5), bound the N-terminal portion of ICP27 from amino acids 1 to 160 in EMSAs (5,6). This portion of ICP27 includes the RGG box from residues 138 to 152 (Fig.…”

“…ICP27 was shown previously to bind RNA through an RGG box motif located at amino acids 138 to 152 within the 512-amino-acid protein (28,34). Using electrophoretic mobility shift assays (EMSAs), we showed that the N-terminal portion of ICP27 from amino acids 1 to 160 bound specifically to viral oligonucleotides that are GC rich and that are flexible and relatively unstructured (5). Here we report the importance of three arginine residues within the RGG box for ICP27 binding to GC-rich sequences in vitro and for viral RNA export during infection.…”

“…ICP27 interacts with a number of cellular proteins, and it binds RNA (35). One of the functions that ICP27 performs is to escort viral mRNAs from the nucleus to the cytoplasm for translation (2,3,5,10,13,21,34). ICP27 binds viral RNAs (5, 34) and interacts directly with the cellular mRNA export receptor TAP/NXF1 (2, 21), which is required for the export of HSV-1 mRNAs (20,21).…”

“…One of the functions that ICP27 performs is to escort viral mRNAs from the nucleus to the cytoplasm for translation (2,3,5,10,13,21,34). ICP27 binds viral RNAs (5,34) and interacts directly with the cellular mRNA export receptor TAP/NXF1 (2,21), which is required for the export of HSV-1 mRNAs (20,21). ICP27 also interacts with the export adaptor proteins Aly/REF (2,3,23) and UAP56 (L. A. Johnson, H. Swesey, and R. M. Sandri-Goldin, unpublished results), which form part of the TREX complex that binds to the 5Ј end of mRNA through an interaction with CBP80 (26,32,41).…”

“…All NMR protein samples were prepared in the same buffer (50 mM Na 2 HPO 4 , 50 mM NaH 2 PO 4 , 100 mM NaCl, 200 mM K 2 SO 4 [pH 8], and 10% D 2 O) at a final concentration of 0.3 mM. The gC oligonucleotides used in this study (gC 1-30, gC 11-40, gC 31-60, gC 71-100, and gC 221-250) were described previously (5). The gC oligonucleotides (Operon) were resuspended in a solution containing 50 mM Tris (pH 8), 150 mM KCl, and 1 mM EDTA (pH 8) at a final concentration of 10 mM.…”