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...
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| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
September 2010
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| In: |
PLoS pathogens
Year: 2010, Volume: 6, Issue: 9, Pages: 1-16 |
| ISSN: | 1553-7374 |
| DOI: | 10.1371/journal.ppat.1001118 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1371/journal.ppat.1001118 Verlag, lizenzpflichtig, Volltext: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1001118 
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| Author Notes: | 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 |
| Summary: | 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. |
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| Item Description: | Gesehen am 22.05.2024 |
| Physical Description: | Online Resource |
| ISSN: | 1553-7374 |
| DOI: | 10.1371/journal.ppat.1001118 |