title: Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.
abstract: Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.
authors.affiliation: Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Artur
authors.initials: A
authors.lastname: Kaul
authors.forename: Sarah
authors.initials: S
authors.lastname: Stauffer
authors.forename: Carola
authors.initials: C
authors.lastname: Berger
authors.forename: Thomas
authors.initials: T
authors.lastname: Pertel
authors.forename: Jennifer
authors.initials: J
authors.lastname: Schmitt
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kallis
authors.forename: Margarita
authors.initials: M
authors.lastname: Zayas
authors.forename: Margarita Zayas
authors.initials: MZ
authors.lastname: Lopez
authors.forename: Volker
authors.initials: V
authors.lastname: Lohmann
authors.forename: Jeremy
authors.initials: J
authors.lastname: Luban
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
mesh_terms: D045744:Cell Line, Tumor; D021984:Cyclophilin A / Q000502:physiology*; D020868:Gene Silencing; D016174:Hepacivirus / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D009154:Mutation; D020815:Polyproteins / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D020815:Polyproteins; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D021984:Cyclophilin A
references.citation: Agents Actions. 1994 Dec;43(3-4):179-86
references.pmid: 7725970
references.citation: Protein Sci. 1992 Sep;1(9):1092-9
references.pmid: 1338979
references.citation: Lancet. 2003 Dec 20;362(9401):2095-100
references.pmid: 14697814
references.citation: J Virol. 2007 May;81(10):5270-83
references.pmid: 17344294
references.citation: J Biol Chem. 2005 Oct 28;280(43):36417-28
references.pmid: 16126720
references.citation: FEBS Lett. 2004 Jan 16;557(1-3):99-103
references.pmid: 14741348
references.citation: J Virol. 2009 Jul;83(13):6554-65
references.pmid: 19386705
references.citation: J Virol. 2007 May;81(9):4591-603
references.pmid: 17301154
references.citation: Cell. 1996 Dec 27;87(7):1285-94
references.pmid: 8980234
references.citation: Gastroenterology. 2005 Sep;129(3):1031-41
references.pmid: 16143140
references.citation: J Biol Chem. 2009 Jun 19;284(25):16998-17005
references.pmid: 19380579
references.citation: J Biol Chem. 2002 Mar 29;277(13):11149-55
references.pmid: 11801599
references.citation: Adv Virus Res. 2004;63:71-180
references.pmid: 15530561
references.citation: J Virol. 2003 Mar;77(5):3007-19
references.pmid: 12584326
references.citation: J Virol. 2008 Jun;82(11):5269-78
references.pmid: 18385230
references.citation: Nucleic Acids Res. 2007 Jan;35(Database issue):D363-6
references.pmid: 17142229
references.citation: J Virol. 1994 Aug;68(8):5045-55
references.pmid: 8035505
references.citation: Hepatology. 2007 Oct;46(4):1026-33
references.pmid: 17600342
references.citation: Science. 1999 Jul 2;285(5424):110-3
references.pmid: 10390360
references.citation: J Virol. 2005 Mar;79(5):3187-94
references.pmid: 15709040
references.citation: Hepatology. 2003 Nov;38(5):1282-8
references.pmid: 14578868
references.citation: J Virol. 2005 Jan;79(1):380-92
references.pmid: 15596831
references.citation: Biochem Pharmacol. 2009 Apr 1;77(7):1173-80
references.pmid: 19174155
references.citation: J Virol. 2007 Dec;81(23):13168-79
references.pmid: 17881454
references.citation: J Virol. 2007 Aug;81(16):8374-83
references.pmid: 17537845
references.citation: PLoS Pathog. 2008 Mar 28;4(3):e1000035
references.pmid: 18369481
references.citation: J Virol. 1995 Dec;69(12):7519-28
references.pmid: 7494258
references.citation: Mol Cell. 2005 Jul 1;19(1):111-22
references.pmid: 15989969
references.citation: Antimicrob Agents Chemother. 2006 Sep;50(9):2976-82
references.pmid: 16940091
references.citation: J Virol. 2002 Dec;76(24):13001-14
references.pmid: 12438626
references.citation: Biochemistry. 1991 Mar 5;30(9):2306-10
references.pmid: 2001362
references.citation: J Biol Chem. 2006 Apr 14;281(15):9833-6
references.pmid: 16407182
references.citation: J Gen Virol. 2007 Aug;88(Pt 8):2204-2213
references.pmid: 17622624
references.citation: J Biol Chem. 2005 Aug 19;280(33):29604-11
references.pmid: 15980068
references.citation: J Virol. 2002 Jun;76(12):5974-84
references.pmid: 12021330
references.citation: Proc Natl Acad Sci U S A. 2006 May 9;103(19):7408-13
references.pmid: 16651538
references.citation: Nat Cell Biol. 2007 Sep;9(9):1089-97
references.pmid: 17721513
references.citation: Nat Rev Microbiol. 2007 Jun;5(6):453-63
references.pmid: 17487147
references.citation: Science. 2005 Jul 22;309(5734):623-6
references.pmid: 15947137
references.citation: FEBS Lett. 2003 Jan 30;535(1-3):34-8
references.pmid: 12560074
references.citation: Hepatology. 2007 Nov;46(5):1365-74
references.pmid: 17668876
references.citation: Science. 1984 Nov 2;226(4674):544-7
references.pmid: 6238408
references.citation: Genome Biol. 2005;6(7):226
references.pmid: 15998457
references.citation: J Virol. 1994 Dec;68(12):8147-57
references.pmid: 7966606
references.citation: PLoS Pathog. 2008 Mar 21;4(3):e1000032
references.pmid: 18369478
references.citation: Hepatology. 2008 Mar;47(3):817-26
references.pmid: 18302285
references.citation: Biochem Biophys Res Commun. 2004 Jan 2;313(1):42-7
references.pmid: 14672695
references.citation: Cell. 1991 Aug 23;66(4):807-15
references.pmid: 1715244
references.citation: J Biol Chem. 2008 Oct 17;283(42):28546-62
references.pmid: 18644781
references.citation: Proc Natl Acad Sci U S A. 2003 May 13;100(10):6104-8
references.pmid: 12719519
references.citation: J Biol Chem. 2007 Dec 21;282(51):37158-69
references.pmid: 17942391
references.citation: J Biol Chem. 2009 May 15;284(20):13589-13601
references.pmid: 19297321
references.citation: Hepatology. 2006 Apr;43(4):761-70
references.pmid: 16557546
references.citation: J Virol. 2006 Jun;80(11):5308-20
references.pmid: 16699011
references.citation: Antimicrob Agents Chemother. 2008 Apr;52(4):1302-17
references.pmid: 18212100