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.The herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is required for productive viral 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). 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with the export adaptor proteins Aly/REF (2,3,23) and UAP56 (L. A. Johnson, H. Swesey, and R. M. Sandri-Goldin, unpublished results), which form part of the TREX complex that binds to the 5Ј end of mRNA through an interaction with CBP80 (26, 32, 41). Aly/REF does not appear to bind viral RNA directly (3), and it is not essential for HSV-1 RNA export based upon small interfering RNA (siRNA) knockdown studies (20), but it contributes to the efficiency of viral RNA export (3, 23). ICP27 also interacts with the SR splicing proteins SRp20 and 9G8 (11,36), which have been shown to shuttle between the nucleus and the cytoplasm (1). SRp20 and 9G8 have also been shown to facilitate the export of some cellular RNAs (16,17,27) by binding RNA and interacting with TAP/ NXF1 (14,16,18). The knockdown of SRp20 or 9G8 adversely affects HSV-1 replication and specifically results in a nuclear accumulation of new...

Arginine Methylation of the RGG Box Does Not Appear To Regulate ICP27 Import during Herpes Simplex Virus Infection

“…Therefore, defects in viral replication seen in RGG box substitution mutants can be attributed to effects of hypomethylation on export (30) and regulation of functional interactions with cellular proteins (31). Hypomethylation of ICP27 does not affect its ability to bind RNA (8), and, as we showed here, there is no demonstrable effect on ICP27 import. This work was supported by National Institute of Allergy and Infectious Diseases (NIAID) grants AI61397 and AI21215.…”

“…Furthermore, the interaction of ICP27 with two cellular proteins, SRPK1, an SR-splicing protein-specific kinase, and Aly/ REF, an RNA export adaptor protein, was diminished under conditions of hypomethylation that resulted from infection with the substitution mutants or from adding the methylation inhibitor adenosine dialdehyde (AdOx) to HSV-1 KOS-infected cells (31). Although arginine methylation has been shown to affect the RNA and DNA binding activities of several proteins (4,14), we recently reported that hypomethylation of ICP27 does not affect its ability to bind GC-rich sequences in vitro; neither does it appear to affect RNA export during HSV-1 infection (8). However, we found that arginine methylation does regulate ICP27 export to the cytoplasm.…”

Structure of the C-Terminal Domain of the Multifunctional ICP27 Protein from Herpes Simplex Virus 1

“…Secondary structure predictions indicate that the N-terminal region up to ϳ240 residues is disordered. Circular dichroism (CD) analysis and nuclear magnetic resonance (NMR) studies on the N-terminal half (residues 1 to 160) of the protein have in fact confirmed that this region is predominantly randomcoils with no rigid three-dimensional structure (22). Another NMR study revealed that a short stretch of ICP27 (residues 103 to 110) was sufficient for binding to the RNA recognition motif of Aly/REF and was linear in the structure (23).…”

Herpes simplex virus ICP27 regulates alternative pre-mRNA polyadenylation and splicing in a sequence-dependent manner

“…Recent crystal structure studies demonstrated that the structure of ICP27 does not have KH domains and that its C-terminal region does not fold into a potentially RNA-binding hnRNPK-like structure (4, 5). ICP27's RGG motif has been shown to directly bind RNA (37,38) and appears to play a significant role in alternative polyadenylation and a lesser role in splicing inhibition. However, our in vitro transfection experiments and previous reports (18,20) showing that the RGG motif is not required for ICP27-mediated splicing inhibition suggest that there may be other RNA binding sites in ICP27 or that unknown adaptor proteins are involved in recognizing the C-rich sequences near the 5′ splice site.…”