HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A-cyclophilin A interaction to cyclophilin inhibitors

Abstract

Background & aims: 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.

Fig. 1. Specific and Direct Interaction Between CypA and Full-Length NS5A

(A) GST (lane 1), GST-CypA (lane 2) or GST-H126Q CypA (lane 3) (100 ng) was mixed with NS5A Con1-His (10 ng) for 3 h at 4° C. Glutathione beads were added to the GST-CypA/NS5A mixture for 30 min at 4° C and washed. Bound material was eluted and analyzed by Western blotting using anti-His and anti-GST antibodies. (B) Plates were coated with BSA, GST, GST-CypA and GST-H126Q CypA (10 μg/ml) for 16 h at 4° C. Recombinant NS5A-His (1 ng/ml) was added to wells for 16 h at 4° C. Captured NS5A-His was detected using mouse anti-His IgG (1 μg/ml) followed by anti-mouse-HRP IgG (1:1000 dilution). Adsorded levels of GST, GST-CypA and GST-H126Q CypA were monitored using mouse anti-GST IgG (1 μg/ml) and rabbit anti-CypA IgG (1μg/ml) followed by anti-mouse and -rabbit-HRP IgG (1:1000 dilution).

Fig. 2. CsA Disrupts the CypA-NS5A Interaction in a Dose-Dependent Manner

(A) Same as Fig. 4A, except that CsA (2.5 μM) was added to GST, GST-CypA and GST-H126Q CypA 15 min prior to the addition of NS5A Con1-His. (B) Same as A, except that increasing concentrations of CsA were used: 0 (lane 2), 0.3125 (lane 3), 0.625 (lane 4), 1.25 (lane 5) and 2.5 μM (lane 6). (C) Same as 1B. (D) Huh7 cells were co-transfected with pBIND, pact and pG5luc. Three days post-transfection, luciferase activity was measured in cell lysates. Data (triplicates) are representative of four independent experiments.

Fig. 3. CypA-NS5A Interaction Conserved Among HCV Genotypes

(A) GST-CypA (100 ng) preincubated for 15 min with increasing concentrations of CsA (from 0.3125 to 1.25 μM) was mixed with recombinant NS5A-His (10 ng) derived from genotypes 1a, 1b, 2a and 2b for 3 h at 4° C. Glutathione beads were added to the GST-CypA/NS5A mixture for 30 min at 4° C and washed. Bound material was eluted and analyzed by Western blotting using anti-His antibodies. (B) Same as 2C.

Fig. 4. The D320E NS5A Mutation Does Not Render CypA-NS5A Interactions Resistant to Cs

(A) GST-CypA (100 ng) preincubated for 15 min with increasing concentrations of CsA (from 0.3125 to 1.25 μM) was mixed with recombinant wild-type or D320E mutant NS5A-His (10 ng) for 3 h at 4 C. Glutathione beads were added to the GST-CypA/NS5A mixture for 30 min at 4° C and washed. Bound material was eluted and analyzed by Western blotting using anti-His antibodies. (B) Same as 2C.