Scripps Research Logo

The Gallay Laboratory

Projects at the Gallay Laboratory

Click here for Human Immunodeficiency Virus (HIV) Project

Understanding the Role of Cyclophilins in HCV Replication and the Mechanisms of Action of Cyclophilin Inhibitors

The immunosuppressive drug cyclosporine A (CsA) was recently reported to be effective against HCV replication in vitro. More recent studies provided evidence that non-immunosuppressive CsA analogs such as NIM811, Alisporivir (Debio 025) and SCY-635 inhibit HCV RNA replication in vitro in an IFNα-independent manner. Remarkably, Alisporivir and SCY-635 have shown great promise in early clinical trials. These compounds are active against all HCV genotypes. Alisporivir is currently tested for safety and efficacy in a phase III study. Despite intense academic and pharmaceutical industry research, the anti-HCV mechanisms of action of CsA, NIM811, Alisporivir and SCY-635 remain poorly understood. A growing body of evidence suggests that cyclophilins (Cyps) are important for the HCV life cycle, suggesting that CsA, Alisporivir, NIM811 or SCY-635, by acting on intracellular Cyps, block HCV replication. CsA and CsA analogs - Alisporivir, NIM811 or SCY-635 - are now called Cyps inhibitors. Cyps act as peptidyl-prolyl isomerases, catalysing the cis-trans isomerisation of the peptide bond preceding prolyl residues. A number of knockout studies in various species, including mice and human cells, have shown that Cyps are non-essential for cellular growth and survival, validating their potential as therapeutic targets. As host-targeted antivirals (HTAs), Cyp inhibitors are likely to provide a higher barrier to selection of HCV resistance than that of direct-acting antivirals (DAA), which target viral proteins such as protease inhibitors. Supporting this notion, Alisporivir has been reported to exhibit the highest hurdle for the selection of resistance of HCV therapies tested. Our laboratory recently demonstrated that HCV highly relies on cyclophilin A (CypA) to replicate in human hepatocytes. We showed that CypA, but not CypB, CypC and CypD, is critical for HCV replication. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which control the isomerase activity of CypA, is critical for HCV replication. By selecting Cyp inhibitor-resistant HCV mutants in vitro, we mapped mutations mainly into the HCV nonstructural (NS) protein NS5A, suggesting that NS5A serves as a viral ligand for CypA. Supporting this hypothesis, we found that NS5A and CypA form a stable complex. We confirmed the NS5A-CypA interaction by pulldown experiments, ELISA and fluorescence resonance energy transfer (FRET) (Figure 5).

Figure 5: NS5A-CypA Interaction Detected by FRET

Figure 5: NS5A-CypA Interaction Detected by FRET.

Remarkably, Cyp inhibitors prevent the CypA-NS5A interaction in a dose-dependent manner. The CypA-NS5A interaction is conserved among genotypes and is interrupted by Cyp inhibitors. Moreover, CypA, devoid of its isomerase activity, fails to bind NS5A. Altogether our data suggest that CypA, via its isomerase pocket, binds directly to NS5A, and most importantly, that disrupting this interaction stops HCV replication.

research figure

Figure 6: Model for the mechanisms of action of Cyp inhibitors. Several models have been proposed to explain how Cyp inhibitors block HCV replication. Inhibiting HCV polyprotein processing: C-terminal NS5A mutations, which confer Cyp inhibitor resistance, slow down the processing of JFH-1 polyprotein precursor. In this model, CypA neutralization by Cyp inhibitors would interfere with RC formation (Model I). It remains to be demonstrated that CypA affects the processing of polyproteins of other genotypes. Preventing CypA recruitment into RC: CypA locates in a protease-resistant compartment similar to that where HCV replicates. Since CsA blocks CypA association with this compartment, one can postulate that Cyp inhibitors deplete RC of CypA, leading to abortive RC. If this model is correct (Model II), it would suggest that HCV exploits a protected compartment enriched with CypA to initiate RC formation. Interfering with NS5B polymerase activity: Because CypA binds NS5B, one could postulate that CypA enhances both NS5B affinity to viral RNA and HCV replication. Supporting this model, CsA resistance is associated with NS5B mutations, which increase NS5B binding to viral RNA in the presence of CsA. In this model (Model III), Cyp inhibitors, by preventing the contact between CypA and NS5B, would weaken NS5B binding to viral RNA, leading to abortive RC. Neutralizing NS5A activities: CypA-NS5A interactions could regulate several steps of the HCV life cycle. Since NS5A binds both NS5B and viral RNA, CypA neutralization by Cyp inhibitors could hamper HCV RNA replication (Model III). In this model, NS5A mutations in Cyp inhibitor-resistant variants would preserve the contact between NS5A and NS5B or the viral RNA. The interplay between CypA, NS5A and NS5B remains to be examined at a molecular level. Since NS5A also plays an essential role in HCV assembly, one could envision that CypA neutralization by Cyp inhibitors could also interfere with particle release (Model IV). Neutralizing NS2 activities: NS2 contributes to RNA replication by cleaving off itself from NS3. NS2 has been implicated in both modulation of host cell gene expression and apoptosis. One could envision that CypA-NS2 contacts trigger the proper folding of NS2 and enhance NS2 activities. In this model, CypA neutralization by Cyp inhibitors, would interfere with NS2 functions (i.e., polyprotein processing) (Model I).

Nevertheless, the molecular requirements for CypA in HCV replication and how NS5A-CypA interactions govern HCV replication remain to be understood. It is important to emphasize that very recent findings suggest that the NS5B polymerase as well as NS2 could represent additional indirect ligands for CypA. Therefore, at this stage one cannot exclude the possibility that Cyp inhibitors block HCV replication by interfering with several steps of the HCV replication cycle as suggested in the model below (Figure 6). A main goal in the laboratory is to understand why HCV exploits CypA to replicate in humans, to understand at a molecular level how CypA assist HCV, and to unravel the mechanisms of action of this novel class of promising anti-HCV agents - the Cyps inhibitors. These studies should not only provide new tools to fight this prime threat to humans – HCV – but also shed light on our understanding of the true cellular functions of Cyps.

___________ 

Overview
___________

Projects
___________

Lab Members
___________

Selected Publications
___________

Photos
___________

Contacts
___________