HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A-cyclophilin A interaction to cyclophilin inhibitors.
OBJECTIVE
The cyclophilin (Cyp) inhibitors - cyclosporine A (CsA), NIM811, Debio 025, and SCY 635 - block HCV replication both in vitro and in vivo, and represent a novel class of potent anti-HCV agents. We and others showed that HCV relies on cyclophilin A (CypA) to replicate. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which controls the isomerase activity of CypA, is critical for HCV replication. Recent studies showed that under Cyp inhibitor selection, mutations arose in the HCV nonstructural 5A (NS5A) protein. This led us to postulate that CypA assists HCV by acting on NS5A.


METHODS
We tested this hypothesis by developing several interaction assays including GST pull-down assays, ELISA, and mammalian two-hybrid binding assays.


RESULTS
We demonstrated that full-length NS5A and CypA form a stable complex. Remarkably, CsA prevents the CypA-NS5A interaction in a dose-dependent manner. Importantly, the CypA-NS5A interaction is conserved among genotypes and is interrupted by CsA. Surprisingly, the NS5A mutant protein, which arose in CsA-resistant HCV variants, behaves similarly to wild-type NS5A in terms of both CypA binding and CsA-mediated release from CypA. This latter finding suggests that HCV resistance to CsA does not correlate with a resistance of the CypA-NS5A interaction to Cyp inhibitors. Moreover, we found that CypA, devoid of its isomerase activity, fails to bind NS5A.


CONCLUSIONS
Altogether these data suggest that CypA, via its isomerase pocket, binds directly to NS5A, and most importantly, that disrupting this interaction stops HCV replication.
Chatterji
Udayan
U
The Scripps Research Institute, La Jolla, USA.
Lim
Precious
P
Bobardt
Michael D
MD
Wieland
Stefan
S
Cordek
Daniel G
DG
Vuagniaux
Gregoire
G
Chisari
Francis
F
Cameron
Craig E
CE
Targett-Adams
Paul
P
Parkinson
Tanya
T
Gallay
Philippe A
PA
D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000494:pharmacology; D001665:Binding Sites; D021984:Cyclophilin A / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D016572:Cyclosporine / Q000494:pharmacology*; D024882:Drug Resistance, Viral / Q000235:genetics; D005814:Genes, Viral; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase
D000998:Antiviral Agents; D021984:Cyclophilin A; D016572:Cyclosporine; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
Antimicrob Agents Chemother. 2008 Sep;52(9):3267-75
18591281
J Virol. 2008 Jun;82(11):5269-78
18385230
Mol Cell. 2005 Jul 1;19(1):111-22
15989969
Protein Sci. 1992 Sep;1(9):1092-9
1338979
Antimicrob Agents Chemother. 2006 Sep;50(9):2976-82
16940091
Arch Virol. 2009;154(10):1671-7
19779801
Antimicrob Agents Chemother. 2010 Feb;54(2):660-72
19933795
J Gastroenterol. 2003;38(6):567-72
12825133
Antimicrob Agents Chemother. 2009 Mar;53(3):967-76
19104013
PLoS Pathog. 2009 Aug;5(8):e1000546
19680534
Hepatology. 2006 Feb;43(2 Suppl 1):S207-20
16447262
Hepatology. 2008 Mar;47(3):817-26
18302285
Gastroenterology. 2005 Sep;129(3):1031-41
16143140
J Biol Chem. 2009 Jun 19;284(25):16998-17005
19380579
Cancer Sci. 2009 Oct;100(10):1943-50
19659609
Transplant Proc. 2005 Mar;37(2):1233-4
15848679
Hepatology. 2007 Oct;46(4):1026-33
17600342
Hepatology. 2003 Nov;38(5):1282-8
14578868
J Gastroenterol. 2006 Jan;41(1):17-27
16501853
Clin Liver Dis. 2009 Aug;13(3):403-17
19628157
Biochem Biophys Res Commun. 2006 May 12;343(3):879-84
16564500
Hepatology. 2009 May;49(5):1460-8
19353740
Protein Expr Purif. 2004 Sep;37(1):144-53
15294292
J Biol Chem. 2009 May 15;284(20):13589-13601
19297321
EMBO J. 1999 Dec 1;18(23):6771-85
10581250
Hepatology. 2006 Apr;43(4):761-70
16557546
J Virol. 2006 May;80(9):4510-20
16611911
Bone Marrow Transplant. 1997 Dec;20(11):993-5
9422481
J Virol. 2009 Jul;83(13):6554-65
19386705