A Major Determinant of Cyclophilin Dependence and Cyclosporine Susceptibility of Hepatitis C Virus Identified by a Genetic Approach

Abstract

Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials.

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).…”

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

“…In this respect, NS5A is of particular interest because it is the target of efficient direct acting antivirals (e.g. daclatasvir) (7) and its interaction with the human cyclophilin A (CypA), an essential peptidylprolyl cis-trans isomerase (PPIase) (8,9), is also targeted by the most advanced host-targeted antiviral (alisporivir) (10,11).…”

“…CypA-Because the structural motif is located in the CypA binding site (8,27,51) and CypA is a mandatory host factor for HCV replication, we investigated the possibility that the structural PW turn element was involved in the binding of CypA. (Fig.…”

Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

“…Sequencing of the pooled 51C-RFP-1a replicon RNA indicated the presence of the C316Y NS5B mutation in these cells. A similar selection for resistance was conducted with the host-factor inhibitor CsA in the 51C-RFP-1a replicon cells, and the expected NS5A D320E mutation, known to confer resistance to both CsA treatment and cyclophilin A (CypA) knockdown, was observed (16). These experiments demonstrate that selections for drug resistance performed with 51C-RFP-1a cells yield populations of cells that maintain high levels of replicon and reporter gene expression and exhibit properties of genotypic and phenotypic resistance consistent with other replicon systems.…”