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

PLoS Pathogens

Feng Yang¹  Jason M. Robotham¹  Henry Grise¹  Stephen Frausto¹  Anita Nag¹  Hengli Tang¹  Ewa Bienkiewicz²  Timothy M. Logan³  Vanesa Madan⁴  Ralf Bartenschlager⁴  Margarita Zayas⁴  Andrew E. Greenstein⁵  Margaret Robinson⁵ 

[1] Department of Biological Science, Florida State University, Tallahassee, Florida, United States of America;Department of Biomedical Sciences, Florida State University, Tallahassee, Florida, United States of America;Department of Chemistry, Florida State University, Tallahassee, Florida, United States of America;Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany;Gilead Sciences, Foster City, California, United States of America

关键词: Proline;    Microbial mutation;    Cofactors (biochemistry);    Viral replication;    Sequence motif analysis;    Hepatitis C virus;    Antimicrobial resistance;    Point mutation;   

DOI  :  10.1371/journal.ppat.1001118

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY