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.
Highlights
Successful completion of the life cycle of a virus depends on the function of proteins encoded by the virus and on cellular cofactors
We and others have recently demonstrated that cyclophilin A (CyPA) is an essential cofactor for hepatitis C virus (HCV) infection and serves as the direct target of a new class of clinical anti-HCV compounds, cyclosporine A (CsA) and its derivatives, that are devoid of immunosuppressive function
We report the identification of a key regulator of HCV’s dependence on CyPA and susceptibility to CsA using a novel genetic screening approach that can potentially be applied to additional cellular cofactors and other viruses. The effectiveness of this approach, termed cofactor-independent mutant (CoFIM) screening, was further supported by results obtained with a parallel CsA-based selection using additional genotypes of HCV
Summary
Successful completion of the life cycle of a virus depends on the function of proteins encoded by the virus and on cellular cofactors. Proteins with apparent functional relevance to the particular virus are characterized in detail after the discovery, but the mechanisms of action for many other cofactors remain undefined. An important step toward the illustration of the mechanism is to identify the viral agent through which a cofactor functions. In rare cases, when small chemical compounds are available whose target is the cellular cofactor, screening for compound-resistant mutant virus can provide valuable information about the viral target, but this approach is not useful for the majority of cofactors. Because our approach does not rely on prior knowledge of the function of the cofactor or the availability of chemical inhibitors, it may be broadly applicable to cellular cofactors with unknown mechanisms of action
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