DDX6 (Rck/p54) is required for efficient hepatitis C virus replication but not for internal ribosome entry site-directed translation.
DDX6 (Rck/p54) is an evolutionarily conserved member of the SF2 DEAD-box RNA helicase family that contributes to the regulation of translation and storage and the degradation of cellular mRNAs. It interacts with multiple proteins and is a component of the micro-RNA (miRNA)-induced silencing complex (miRISC). Since miRNA-122 (miR-122) is essential for efficient hepatitis C virus (HCV) replication, we investigated the requirement for DDX6 in HCV replication in cultured hepatoma cells. Small interfering RNA (siRNA)-mediated knockdown of DDX6 and rescue with an siRNA-resistant mutant demonstrated that DDX6 expression is indeed required for optimal HCV replication. However, DDX6 knockdown did not impair miR-122 biogenesis or alter HCV responsiveness to miR-122 supplementation. Overexpression of DDX6 fused to EYFP (EYFP-DDX6) enhanced replication, whereas a helicase-deficient mutant with a substitution in the conserved DEAD-box motif II (DQAD) had a dominant-negative effect, reducing HCV yields. Coimmunoprecipitation experiments revealed an intracellular complex containing DDX6, HCV core protein, and both viral and cellular RNAs, the formation of which was dependent upon the C-terminal domain of DDX6 but not DDX6 helicase activity. However, since DDX6 abundance influenced the replication of subgenomic HCV RNAs lacking core sequence, the relevance of this complex is uncertain. Importantly, DDX6 knockdown caused minimal reductions in cellular proliferation, generally stimulated cellular translation ([(35)S]Met incorporation), and did not impair translation directed by the HCV internal ribosome entry site. Thus, DDX6 helicase activity is essential for efficient HCV replication, reflecting essential roles for DDX6 in HCV genome amplification and/or maintenance of cellular homeostasis.
Jangra
Rohit K
RK
Center for Hepatitis Research, Institute for Human Infections and Immunity, and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-0610, USA.
Yi
MinKyung
M
Lemon
Stanley M
SM
D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D016174:Hepacivirus / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000378:metabolism; D014176:Protein Biosynthesis; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014779:Virus Replication
C531907:MIRN122 microRNA, human; D035683:MicroRNAs; D011518:Proto-Oncogene Proteins; D012367:RNA, Viral; C094683:DDX6 protein, human; D053487:DEAD-box RNA Helicases
Biochem Soc Trans. 2008 Dec;36(Pt 6):1220-3
19021529
Cell Host Microbe. 2008 Jul 17;4(1):77-85
18621012
Nucleic Acids Res. 2006;34(15):4168-80
16936318
Nucleic Acids Res. 2006 Jun 12;34(10):3082-94
16769775
Proc Natl Acad Sci U S A. 2006 Feb 14;103(7):2310-5
16461899
Nucleic Acids Res. 2007;35(14):4715-27
17604308
J Virol. 2004 May;78(10):5288-98
15113910
Nat Rev Mol Cell Biol. 2007 Jan;8(1):9-22
17183357
Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12884-9
17616579
J Virol Methods. 2006 Apr;133(1):62-9
16300833
Cell. 2005 Sep 23;122(6):875-86
16179257
Mol Biol Cell. 2009 May;20(9):2464-72
19297524
Gastroenterology. 2000 Jan;118(1):152-62
10611164
Curr Opin Pharmacol. 2009 Oct;9(5):537-44
19762279
J Virol. 2005 Nov;79(21):13473-82
16227268
Gastroenterology. 2009 Oct;137(4):1448-58
19632233
Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13517-22
19628699
J Virol. 2007 Jan;81(2):629-38
17079282
Mol Cell. 2005 May 13;18(4):425-34
15893726
PLoS Biol. 2006 Jul;4(7):e210
16756390
RNA. 2000 Apr;6(4):563-70
10786847
Science. 1999 Jul 2;285(5424):110-3
10390360
J Virol. 2007 Aug;81(16):8374-83
17537845
J Virol. 2004 Aug;78(15):7904-15
15254163
Science. 2005 Sep 2;309(5740):1577-81
16141076
J Virol. 2009 Sep;83(17):8379-95
19515772
J Gen Virol. 2010 Jan;91(Pt 1):122-32
19793905
Mol Cell. 2005 Jul 1;19(1):111-22
15989969
J Hepatol. 2006 Oct;45(4):529-38
16879891
J Virol. 1999 Dec;73(12):9718-25
10559281
J Virol. 2006 Feb;80(3):1181-90
16414995
J Viral Hepat. 2003 Jul;10(4):241-8
12823589
Proc Natl Acad Sci U S A. 2007 Apr 24;104(17):7253-8
17438296
J Virol. 2007 Dec;81(24):13922-6
17855521
Virology. 1999 May 10;257(2):330-40
10329544
Nucleic Acids Res. 1992 Apr 25;20(8):1967-72
1579499
J Virol. 2004 Apr;78(7):3480-8
15016871
Science. 2010 Jan 8;327(5962):198-201
19965718
J Virol. 2002 Mar;76(6):2997-3006
11861865
J Virol. 2008 Mar;82(5):2182-95
18094154
Genes Cells. 2003 Aug;8(8):671-6
12875652
Genes Dev. 1998 Jan 1;12(1):67-83
9420332
Hepatology. 2002 May;35(5):1237-46
11981774
Gene. 2006 Feb 15;367:17-37
16337753
J Virol. 2004 Feb;78(3):1513-24
14722306
EMBO J. 2008 Dec 17;27(24):3300-10
19020517
Carcinogenesis. 2001 Dec;22(12):1965-70
11751426
J Virol. 2007 Apr;81(8):4378-80
17301158
J Virol. 2000 Dec;74(23):11347-58
11070035