Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.
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.
Kaul
Artur
A
Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany.
Stauffer
Sarah
S
Berger
Carola
C
Pertel
Thomas
T
Schmitt
Jennifer
J
Kallis
Stephanie
S
Zayas
Margarita
M
Lopez
Margarita Zayas
MZ
Lohmann
Volker
V
Luban
Jeremy
J
Bartenschlager
Ralf
R
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*
D020815:Polyproteins; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D021984:Cyclophilin A
Agents Actions. 1994 Dec;43(3-4):179-86
7725970
Protein Sci. 1992 Sep;1(9):1092-9
1338979
Lancet. 2003 Dec 20;362(9401):2095-100
14697814
J Virol. 2007 May;81(10):5270-83
17344294
J Biol Chem. 2005 Oct 28;280(43):36417-28
16126720
FEBS Lett. 2004 Jan 16;557(1-3):99-103
14741348
J Virol. 2009 Jul;83(13):6554-65
19386705
J Virol. 2007 May;81(9):4591-603
17301154
Cell. 1996 Dec 27;87(7):1285-94
8980234
Gastroenterology. 2005 Sep;129(3):1031-41
16143140
J Biol Chem. 2009 Jun 19;284(25):16998-17005
19380579
J Biol Chem. 2002 Mar 29;277(13):11149-55
11801599
Adv Virus Res. 2004;63:71-180
15530561
J Virol. 2003 Mar;77(5):3007-19
12584326
J Virol. 2008 Jun;82(11):5269-78
18385230
Nucleic Acids Res. 2007 Jan;35(Database issue):D363-6
17142229
J Virol. 1994 Aug;68(8):5045-55
8035505
Hepatology. 2007 Oct;46(4):1026-33
17600342
Science. 1999 Jul 2;285(5424):110-3
10390360
J Virol. 2005 Mar;79(5):3187-94
15709040
Hepatology. 2003 Nov;38(5):1282-8
14578868
J Virol. 2005 Jan;79(1):380-92
15596831
Biochem Pharmacol. 2009 Apr 1;77(7):1173-80
19174155
J Virol. 2007 Dec;81(23):13168-79
17881454
J Virol. 2007 Aug;81(16):8374-83
17537845
PLoS Pathog. 2008 Mar 28;4(3):e1000035
18369481
J Virol. 1995 Dec;69(12):7519-28
7494258
Mol Cell. 2005 Jul 1;19(1):111-22
15989969
Antimicrob Agents Chemother. 2006 Sep;50(9):2976-82
16940091
J Virol. 2002 Dec;76(24):13001-14
12438626
Biochemistry. 1991 Mar 5;30(9):2306-10
2001362
J Biol Chem. 2006 Apr 14;281(15):9833-6
16407182
J Gen Virol. 2007 Aug;88(Pt 8):2204-2213
17622624
J Biol Chem. 2005 Aug 19;280(33):29604-11
15980068
J Virol. 2002 Jun;76(12):5974-84
12021330
Proc Natl Acad Sci U S A. 2006 May 9;103(19):7408-13
16651538
Nat Cell Biol. 2007 Sep;9(9):1089-97
17721513
Nat Rev Microbiol. 2007 Jun;5(6):453-63
17487147
Science. 2005 Jul 22;309(5734):623-6
15947137
FEBS Lett. 2003 Jan 30;535(1-3):34-8
12560074
Hepatology. 2007 Nov;46(5):1365-74
17668876
Science. 1984 Nov 2;226(4674):544-7
6238408
Genome Biol. 2005;6(7):226
15998457
J Virol. 1994 Dec;68(12):8147-57
7966606
PLoS Pathog. 2008 Mar 21;4(3):e1000032
18369478
Hepatology. 2008 Mar;47(3):817-26
18302285
Biochem Biophys Res Commun. 2004 Jan 2;313(1):42-7
14672695
Cell. 1991 Aug 23;66(4):807-15
1715244
J Biol Chem. 2008 Oct 17;283(42):28546-62
18644781
Proc Natl Acad Sci U S A. 2003 May 13;100(10):6104-8
12719519
J Biol Chem. 2007 Dec 21;282(51):37158-69
17942391
J Biol Chem. 2009 May 15;284(20):13589-13601
19297321
Hepatology. 2006 Apr;43(4):761-70
16557546
J Virol. 2006 Jun;80(11):5308-20
16699011
Antimicrob Agents Chemother. 2008 Apr;52(4):1302-17
18212100