The Isomerase Active Site of Cyclophilin A Is Critical for Hepatitis C Virus Replication

Abstract

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies. Hepatitis C virus (HCV)2 is the main contributing agent of acute and chronic liver diseases worldwide (1). Primary infection is often asymptomatic or associated with mild symptoms. However, persistently infected individuals develop high risks for chronic liver diseases such as hepatocellular carcinoma and liver cirrhosis (1). The combination of IFN␣ and ribavirin that serves as current therapy for chronically HCV-infected patients not only has a low success rate (about 50%) (2) but is often associated with serious side effects (2). There is thus an urgent need for the development of novel anti-HCV treatments (2).The immunosuppressive drug cyclosporine A (CsA) was reported to be clinically effective against HCV (3). Controlled trials showed that a combination of CsA with IFN␣ is more effective than IFN␣ alone, especially in patients with a high viral load (4, 5). Moreover, recent in vitro studies provided evidence that CsA prevents both HCV RNA replication and HCV protein production in an IFN␣-independent manner (6 -10). CsA exerts this anti-HCV activity independently of its immunosuppressive activity because the nonimmunosuppressive Cyp inhibitors such as Debio 025, NIM811, and SCY-635 also block HCV RNA and...

Inhibition of cyclophilin A suppresses H2O2‐enhanced replication of HCMV through the p38 MAPK signaling pathway

“…Similarly, the host cellular protein CyPA is also known to be involved in the replication of these viruses 16, 34, 35. It has been reported that HCMV infection induces the generation of ROS minutes after entry into the host cell 4.…”

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

“…For HIV-1, the binding of CypA to the viral Capsid protein seems required (66), whereas the infection of E. coli by the filamentous phage fd is regulated by the Cyclophilin18-catalyzed cis/trans-isomer-ization of its Pro 213 (67,68). Regarding HCV and its mandatory host factor CypA, this important question remains to be answered (69). However, because mutations in the active site of CypA abolishing its PPIase activity also interfere with its binding property (69,70), it is challenging to distinguish between these two possible mechanisms, which are moreover not mutually exclusive.…”

“…Regarding HCV and its mandatory host factor CypA, this important question remains to be answered (69). However, because mutations in the active site of CypA abolishing its PPIase activity also interfere with its binding property (69,70), it is challenging to distinguish between these two possible mechanisms, which are moreover not mutually exclusive. We show that CypA-catalyzed cis/trans (c 7 T) isomerization of Pro 314 is significantly faster when the structural motif is absent (Fig.…”

Targeting Mitochondrial Function for the Treatment of Acute Spinal Cord Injury

“…At the present time, NIM811 and two other cyclophilin inhibitors are in clinical trials as co-treatments for hepatitis C virus (HCV). These compounds are thought to partially inhibit HCV replication by binding to cyclophilin A and possibly other cyclophilins, of which there are at least 16 expressed in mammalian cells [115][116][117]. Additional studies suggest that co-treatment with cyclophilin inhibitors and protease or polymerase inhibitors leads to a reduction in HCV replication and limits development of resistance to monotherapy alone [117][118][119][120].…”