Cyclosporine Inhibits a Direct Interaction between Cyclophilins and Hepatitis C NS5A

Abstract

BackgroundHepatitis C Virus (HCV) infection is a leading indication for liver transplantation. HCV infection reoccurs almost universally post transplant, decreasing both graft longevity and patient survival. The immunosuppressant, cyclosporine A (CsA) has potent anti-HCV activity towards both HCV replicons and the genotype 2a cell culture infectious virus. Previously, we isolated mutations in the 1bN replicon with less sensitivity to CsA that mapped to both NS5A and NS5B regions of the virus. Mutations in NS5A alone conferred decreased CsA susceptibility regardless of NS5B mutations.Methodology/Principal FindingsWe examined the mechanisms by which NS5A mutations contribute to CsA resistance and if they are strain dependent. Using in vitro mutagenesis, the amino acid position 321 mutation of NS5A was restored to the wild-type tyrosine residue conferring partial CsA susceptibility on the mutant replicon. The 321 mutation also alters CsA susceptibility of the JFH cell culture virus. Additionally, we demonstrated a novel CsA-sensitive interaction between NS5A and both cyclophilin A and B. Both the mutant NS5A and wild type NS5A bind cyclophilin in vitro. The NS5A: cyclophilin interaction requires both the NS5A region identified by the resistance mutants and cyclophilin catalytic residues. In cell culture, NS5A from CsA resistant mutant has an enhanced interaction with cyclophilin B. Additionally; NS5B facilitates a stronger binding of mutant NS5A to endogenous cyclophilin B than wild-type in cell culture.Conclusions/SignificanceCollectively, this data suggests direct interactions between cyclophilins and NS5A are critical to understand for optimal use of cyclophilin inhibitors in anti-HCV therapy.

Correlation between NS5A Dimerization and Hepatitis C Virus Replication

“…As a control, we used GSTCypA as bait to capture full-length NS5A-His because this interaction has been shown to be direct (15)(16)(17)(18)(19)(20)(21)(22). As expected, both GST-CypA and GST-Domain I, but not GST, capture fulllength NS5A-His (Fig.…”

“…NS5A is a nonstructural protein that has no defined role in the virus life cycle but is absolutely required for RNA replication (8 -10) and particle assembly (11)(12)(13)(14). It has also been shown to interact with a large number of host proteins including CypA (cyclophilin A) (15)(16)(17)(18)(19)(20)(21)(22). NS5A is peripherally anchored to membranes by an N-terminal amphipathic helix (23)(24)(25)(26)(27).…”

Peptidyl-Prolyl Isomerase Pin1 Is a Cellular Factor Required for Hepatitis C Virus Propagation

“…Like other PPIases, Pin1 interacts with phosphorylated proteins through the serine/threonine-proline motif. Because both CypA and CypB interact with the HCV NS5A and NS5B proteins (2,5,10,30,35), we speculated that Pin1 might upregulate HCV propagation through interaction with either NS5A or NS5B protein. Both NS5A and NS5B are phosphoproteins that contain various phosphorylated serine/threonineproline motifs; thus, both proteins could be potential substrates of PPIases.…”

“…Previous studies have demonstrated that both CypA and CypB interact with the NS5A and NS5B proteins and thus modulate HCV replication (2,5,10). We wondered whether these PPIases competed for interaction with HCV proteins.…”

Domain 3 of NS5A Protein from the Hepatitis C Virus Has Intrinsic α-Helical Propensity and Is a Substrate of Cyclophilin A

“…However, we showed in a recent study that NS5A-D2 from the JFH-1 strain (genotype 2a) interacts with, and is a substrate for, the PPIase of human cyclophilin A (35). In addition, we could correlate the CypA interaction sites with mutations in D2 that significantly influence viral RNA replication (27) and that confer resistance to cyclophilin inhibitors (14,22,37). Finally, D3 (residues 356 -447) has been shown, at least in the context of the JFH-1 strain (2a), to be involved in the production and assembly of viral particles (26,38).…”