title: 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.
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, United States of America.
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.forename: Jason M
authors.initials: JM
authors.lastname: Robotham
authors.forename: Henry
authors.initials: H
authors.lastname: Grise
authors.forename: Stephen
authors.initials: S
authors.lastname: Frausto
authors.forename: Vanesa
authors.initials: V
authors.lastname: Madan
authors.forename: Margarita
authors.initials: M
authors.lastname: Zayas
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
authors.forename: Margaret
authors.initials: M
authors.lastname: Robinson
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Greenstein
authors.forename: Anita
authors.initials: A
authors.lastname: Nag
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Logan
authors.forename: Ewa
authors.initials: E
authors.lastname: Bienkiewicz
authors.forename: Hengli
authors.initials: H
authors.lastname: Tang
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D002942:Circular Dichroism; D021984:Cyclophilin A / Q000235:genetics / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology*; D024882:Drug Resistance, Viral; D004797:Enzyme-Linked Immunosorbent Assay; D015967:Gene Expression Regulation, Viral; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D006526:Hepatitis C / Q000188:drug therapy* / Q000235:genetics / Q000821:virology; D006801:Humans; D007124:Immunoenzyme Techniques; D007166:Immunosuppressive Agents / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D012367:RNA, Viral / Q000235:genetics; D012093:Replicon / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D021984:Cyclophilin A; D016572:Cyclosporine; D007166:Immunosuppressive Agents; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase