title: DDX6 (Rck/p54) is required for efficient hepatitis C virus replication but not for internal ribosome entry site-directed translation.
abstract: 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.
authors.affiliation: 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.
authors.forename: Rohit K
authors.initials: RK
authors.lastname: Jangra
authors.forename: MinKyung
authors.initials: M
authors.lastname: Yi
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Lemon
mesh_terms: 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
chemical_list: C531907:MIRN122 microRNA, human; D035683:MicroRNAs; D011518:Proto-Oncogene Proteins; D012367:RNA, Viral; C094683:DDX6 protein, human; D053487:DEAD-box RNA Helicases
references.citation: Biochem Soc Trans. 2008 Dec;36(Pt 6):1220-3
references.pmid: 19021529
references.citation: Cell Host Microbe. 2008 Jul 17;4(1):77-85
references.pmid: 18621012
references.citation: Nucleic Acids Res. 2006;34(15):4168-80
references.pmid: 16936318
references.citation: Nucleic Acids Res. 2006 Jun 12;34(10):3082-94
references.pmid: 16769775
references.citation: Proc Natl Acad Sci U S A. 2006 Feb 14;103(7):2310-5
references.pmid: 16461899
references.citation: Nucleic Acids Res. 2007;35(14):4715-27
references.pmid: 17604308
references.citation: J Virol. 2004 May;78(10):5288-98
references.pmid: 15113910
references.citation: Nat Rev Mol Cell Biol. 2007 Jan;8(1):9-22
references.pmid: 17183357
references.citation: Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12884-9
references.pmid: 17616579
references.citation: J Virol Methods. 2006 Apr;133(1):62-9
references.pmid: 16300833
references.citation: Cell. 2005 Sep 23;122(6):875-86
references.pmid: 16179257
references.citation: Mol Biol Cell. 2009 May;20(9):2464-72
references.pmid: 19297524
references.citation: Gastroenterology. 2000 Jan;118(1):152-62
references.pmid: 10611164
references.citation: Curr Opin Pharmacol. 2009 Oct;9(5):537-44
references.pmid: 19762279
references.citation: J Virol. 2005 Nov;79(21):13473-82
references.pmid: 16227268
references.citation: Gastroenterology. 2009 Oct;137(4):1448-58
references.pmid: 19632233
references.citation: Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13517-22
references.pmid: 19628699
references.citation: J Virol. 2007 Jan;81(2):629-38
references.pmid: 17079282
references.citation: Mol Cell. 2005 May 13;18(4):425-34
references.pmid: 15893726
references.citation: PLoS Biol. 2006 Jul;4(7):e210
references.pmid: 16756390
references.citation: RNA. 2000 Apr;6(4):563-70
references.pmid: 10786847
references.citation: Science. 1999 Jul 2;285(5424):110-3
references.pmid: 10390360
references.citation: J Virol. 2007 Aug;81(16):8374-83
references.pmid: 17537845
references.citation: J Virol. 2004 Aug;78(15):7904-15
references.pmid: 15254163
references.citation: Science. 2005 Sep 2;309(5740):1577-81
references.pmid: 16141076
references.citation: J Virol. 2009 Sep;83(17):8379-95
references.pmid: 19515772
references.citation: J Gen Virol. 2010 Jan;91(Pt 1):122-32
references.pmid: 19793905
references.citation: Mol Cell. 2005 Jul 1;19(1):111-22
references.pmid: 15989969
references.citation: J Hepatol. 2006 Oct;45(4):529-38
references.pmid: 16879891
references.citation: J Virol. 1999 Dec;73(12):9718-25
references.pmid: 10559281
references.citation: J Virol. 2006 Feb;80(3):1181-90
references.pmid: 16414995
references.citation: J Viral Hepat. 2003 Jul;10(4):241-8
references.pmid: 12823589
references.citation: Proc Natl Acad Sci U S A. 2007 Apr 24;104(17):7253-8
references.pmid: 17438296
references.citation: J Virol. 2007 Dec;81(24):13922-6
references.pmid: 17855521
references.citation: Virology. 1999 May 10;257(2):330-40
references.pmid: 10329544
references.citation: Nucleic Acids Res. 1992 Apr 25;20(8):1967-72
references.pmid: 1579499
references.citation: J Virol. 2004 Apr;78(7):3480-8
references.pmid: 15016871
references.citation: Science. 2010 Jan 8;327(5962):198-201
references.pmid: 19965718
references.citation: J Virol. 2002 Mar;76(6):2997-3006
references.pmid: 11861865
references.citation: J Virol. 2008 Mar;82(5):2182-95
references.pmid: 18094154
references.citation: Genes Cells. 2003 Aug;8(8):671-6
references.pmid: 12875652
references.citation: Genes Dev. 1998 Jan 1;12(1):67-83
references.pmid: 9420332
references.citation: Hepatology. 2002 May;35(5):1237-46
references.pmid: 11981774
references.citation: Gene. 2006 Feb 15;367:17-37
references.pmid: 16337753
references.citation: J Virol. 2004 Feb;78(3):1513-24
references.pmid: 14722306
references.citation: EMBO J. 2008 Dec 17;27(24):3300-10
references.pmid: 19020517
references.citation: Carcinogenesis. 2001 Dec;22(12):1965-70
references.pmid: 11751426
references.citation: J Virol. 2007 Apr;81(8):4378-80
references.pmid: 17301158
references.citation: J Virol. 2000 Dec;74(23):11347-58
references.pmid: 11070035