Regulation of PKR by HCV IRES RNA: importance of domain II and NS5A.
Protein kinase R (PKR) is an essential component of the innate immune response. In the presence of double-stranded RNA (dsRNA), PKR is autophosphorylated, which enables it to phosphorylate its substrate, eukaryotic initiation factor 2alpha, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5'-untranslated region of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and noncanonical features of domain II cumulate to a total of approximately 33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV nonstructural protein 5A (NS5A) to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.
Toroney
Rebecca
R
Department of Chemistry, Pennsylvania State University, 104 Chemistry Building, University Park, PA 16802, USA.
Nallagatla
Subba Rao
SR
Boyer
Joshua A
JA
Cameron
Craig E
CE
Bevilacqua
Philip C
PC
D001483:Base Sequence; D016174:Hepacivirus / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D019892:eIF-2 Kinase; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
Virology. 1992 May;188(1):47-56
1373554
Cold Spring Harb Symp Quant Biol. 2001;66:389-96
12762041
RNA. 2003 Jul;9(7):858-70
12810919
Biochem Biophys Res Commun. 2001 Jun 15;284(3):798-807
11396973
Trends Biochem Sci. 2007 Feb;32(2):57-62
17196820
J Biol Chem. 2005 Oct 28;280(43):36417-28
16126720
J Virol. 1999 Feb;73(2):1165-74
9882318
Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4650-5
11904369
Nucleic Acids Res. 1992 Oct 11;20(19):5041-5
1329037
Nucleic Acids Res. 1994 Oct 25;22(21):4364-74
7971266
J Biol Chem. 1979 Oct 25;254(20):10180-3
489592
Nucleic Acids Res. 1987 Nov 25;15(22):9109-28
2446263
Trends Biochem Sci. 2008 Jun;33(6):274-83
18468443
Virology. 1997 Apr 14;230(2):217-27
9143277
J Virol. 1993 Jun;67(6):3338-44
8388503
RNA. 1995 Mar;1(1):7-20
7489491
J Virol. 2000 Jul;74(14):6242-50
10864633
RNA. 1996 Sep;2(9):937-51
8809020
Mol Cell Biol. 1989 Apr;9(4):1576-86
2725516
EMBO J. 2000 Mar 1;19(5):997-1009
10698941
J Mol Graph. 1996 Feb;14(1):33-8, 27-8
8744570
Nucleic Acids Res. 1981 Oct 10;9(19):5125-40
7031604
Clin Microbiol Rev. 2001 Oct;14(4):778-809, table of contents
11585785
Biochemistry. 1998 May 5;37(18):6303-16
9572845
EMBO J. 2005 Jan 12;24(1):138-48
15565168
Nucleic Acids Res. 1991 Jan 25;19(2):243-8
1673026
J Virol. 1988 Mar;62(3):902-11
2828685
EMBO J. 2001 Dec 17;20(24):7240-9
11743000
Microbiol Mol Biol Rev. 2006 Dec;70(4):1032-60
17158706
Nat Chem Biol. 2009 Dec;5(12):896-903
19801993
Immunity. 2000 Jul;13(1):129-41
10933401
Biochem Biophys Res Commun. 2002 Jan 11;290(1):105-12
11779140
Biochemistry. 1999 Nov 9;38(45):14784-92
10555960
Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14162-7
11114159
RNA. 2007 May;13(5):781-92
17449731
Science. 2007 Nov 30;318(5855):1455-8
18048689
J Med Virol. 2001 Jan;63(1):8-16
11130893
Mol Cell Biol. 1992 Nov;12(11):5238-48
1357546
J Virol. 1999 Aug;73(8):6506-16
10400746
Biotechniques. 2005 Mar;38(3):368, 370, 372
15786803
Oncogene. 1999 Nov 1;18(45):6112-20
10557102
J Mol Biol. 2009 Jul 10;390(2):319-38
19445956
Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8307-12
15150409
Acta Crystallogr D Biol Crystallogr. 2008 Apr;64(Pt 4):436-43
18391410
EMBO J. 1998 Dec 15;17(24):7505-13
9857205
Cell. 2004 Oct 29;119(3):369-80
15507208
Antivir Ther. 2004 Apr;9(2):275-86
15134190
Angew Chem Int Ed Engl. 2007;46(1-2):226-9
17131443
Nat Struct Biol. 2003 Dec;10(12):1033-8
14578934
RNA. 2005 May;11(5):592-7
15840813
Methods Enzymol. 1989;180:51-62
2482430
RNA. 1995 Jul;1(5):526-37
7489514
Virology. 1995 Jan 10;206(1):511-9
7530396
Nucleic Acids Res. 1987 Nov 11;15(21):8783-98
3684574
Chem Biol. 2009 Feb 27;16(2):173-80
19246008
J Virol. 2001 Jun;75(11):5090-8
11333890
J Virol. 1992 Mar;66(3):1476-83
1310759
Antiviral Res. 2009 Sep;83(3):228-37
19467267
Nucleic Acids Res. 1993 Sep 25;21(19):4483-90
7901835
Gut. 2004 Sep;53(9):1345-51
15306598
J Interferon Cytokine Res. 2005 May;25(5):261-70
15871663
J Mol Biol. 1999 Sep 24;292(3):513-29
10497018
Methods Mol Biol. 2008;419:53-67
18369975
J Interferon Cytokine Res. 2005 Mar;25(3):152-64
15767789
Virology. 2002 Jun 5;297(2):195-202
12083818
Cell Host Microbe. 2009 Dec 17;6(6):513-22
20006840
RNA. 2006 Jan;12(1):83-93
16373494
RNA. 2004 Dec;10(12):1934-45
15547138
Biochemistry. 1996 Aug 6;35(31):9983-94
8756460
J Virol. 2001 Apr;75(8):3719-30
11264361
Protein Expr Purif. 2004 Sep;37(1):144-53
15294292
Structure. 2005 Nov;13(11):1695-706
16271893
RNA. 1996 Sep;2(9):867-78
8809014
Science. 2001 Mar 9;291(5510):1959-62
11239155
Genes Dev. 1992 Dec;6(12B):2478-90
1364113