A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach.
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...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2010-09-01
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| Series: | PLoS Pathogens |
| Online Access: | http://europepmc.org/articles/PMC2944805?pdf=render |
| _version_ | 1819262595264151552 |
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| author | Feng Yang Jason M Robotham Henry Grise Stephen Frausto Vanesa Madan Margarita Zayas Ralf Bartenschlager Margaret Robinson Andrew E Greenstein Anita Nag Timothy M Logan Ewa Bienkiewicz Hengli Tang |
| author_facet | Feng Yang Jason M Robotham Henry Grise Stephen Frausto Vanesa Madan Margarita Zayas Ralf Bartenschlager Margaret Robinson Andrew E Greenstein Anita Nag Timothy M Logan Ewa Bienkiewicz Hengli Tang |
| author_sort | Feng Yang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-23T20:00:11Z |
| format | Article |
| id | doaj.art-590f2528938b41288d334ed17974daf9 |
| institution | Directory Open Access Journal |
| issn | 1553-7366 1553-7374 |
| language | English |
| last_indexed | 2024-12-23T20:00:11Z |
| publishDate | 2010-09-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Pathogens |
| spelling | doaj.art-590f2528938b41288d334ed17974daf92022-12-21T17:33:06ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742010-09-0169e100111810.1371/journal.ppat.1001118A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach.Feng YangJason M RobothamHenry GriseStephen FraustoVanesa MadanMargarita ZayasRalf BartenschlagerMargaret RobinsonAndrew E GreensteinAnita NagTimothy M LoganEwa BienkiewiczHengli TangSince 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.http://europepmc.org/articles/PMC2944805?pdf=render |
| spellingShingle | Feng Yang Jason M Robotham Henry Grise Stephen Frausto Vanesa Madan Margarita Zayas Ralf Bartenschlager Margaret Robinson Andrew E Greenstein Anita Nag Timothy M Logan Ewa Bienkiewicz Hengli Tang A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach. PLoS Pathogens |
| title | A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach. |
| title_full | A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach. |
| title_fullStr | A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach. |
| title_full_unstemmed | A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach. |
| title_short | A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach. |
| title_sort | major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis c virus identified by a genetic approach |
| url | http://europepmc.org/articles/PMC2944805?pdf=render |
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