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.
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.
Corbin-Lickfett
Kara A
KA
Department of Microbiology and Molecular Genetics, School of Medicine, Medical Sciences I, University of California at Irvine, Irvine, CA 92697-4025, USA.
Souki
Stuart K
SK
Cocco
Melanie J
MJ
Sandri-Goldin
Rozanne M
RM
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
C057120:ICP27 protein, human herpesvirus 1; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D001120:Arginine; D008239:Lysine
Nucleic Acids Res. 2009 Nov;37(21):7290-301
19783816
J Virol. 2009 Oct;83(20):10336-46
19656898
J Virol. 2008 Apr;82(7):3538-45
18216091
J Biomol NMR. 1994 Nov;4(6):845-58
7812156
Curr Protoc Cytom. 2001 May;Chapter 7:Unit 7.12
18770724
PLoS One. 2008 Jan 30;3(1):e1491
18231578
EMBO J. 2001 Oct 15;20(20):5769-78
11598019
J Virol. 1996 Nov;70(11):7445-53
8892862
J Virol. 2005 Apr;79(7):3949-61
15767397
Virology. 2010 Jun 5;401(2):155-64
20227104
J Virol. 2005 Apr;79(7):4120-31
15767413
J Virol. 1997 Mar;71(3):2031-40
9032335
RNA. 2003 Oct;9(10):1198-207
13130134
Nucleic Acids Res. 2003 Jun 1;31(11):2944-51
12771220
J Virol. 2009 Sep;83(17):8970-5
19553338
Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9666-70
15210956
Cell. 2001 Nov 16;107(4):489-99
11719189
J Virol. 2009 Jul;83(13):6335-46
19369354
J Virol. 2010 Mar;84(5):2212-22
20015986
Genes Dev. 1998 Jan 1;12(1):55-66
9420331
EMBO J. 2006 Nov 1;25(21):5126-37
17036044
EMBO J. 2003 Apr 1;22(7):1608-19
12660167
Genes Cells. 2004 Oct;9(10):959-65
15461666
Virology. 1992 Jan;186(1):74-86
1309283
J Virol. 2009 Feb;83(3):1184-92
19019956
Nucleic Acids Res. 2010 Jun;38(10):3351-61
20129943
Front Biosci. 2008 May 01;13:5241-56
18508584
J Biomol NMR. 1995 Nov;6(3):277-93
8520220
Mol Cell. 2001 Apr;7(4):899-905
11336712
Methods Enzymol. 1994;239:363-92
7830591
J Virol. 2010 May;84(9):4124-35
20164236
Nature. 2002 May 16;417(6886):304-8
11979277
Genes Dev. 2005 Jul 1;19(13):1512-7
15998806
Biochemistry. 1994 May 17;33(19):5984-6003
7514039
Mol Cell. 2003 Mar;11(3):837-43
12667464
Chem Biol Drug Des. 2009 Aug;74(2):101-20
19566697
Curr Opin Cell Biol. 2005 Jun;17(3):269-73
15901496
J Virol. 2002 Dec;76(24):12877-89
12438613
J Virol. 2006 Apr;80(7):3567-81
16537625
J Virol. 2010 Mar;84(5):2200-11
20015991
J Virol. 2009 Jun;83(11):5309-20
19321610
Genes Dev. 1998 Mar 15;12(6):868-79
9512520