Type of Document	Dissertation
Author	Yang, Feng
URN	etd-07012010-161119
Title	The Role of Cyclophilin A in Hepatitis C Virus Life Cycle
Degree	Doctor of Philosophy
Department	Biological Science, Department of
Advisory Committee	Advisor Name Title Hengli Tang Committee Chair Fanxiu Zhu Committee Member Hank W. Bass Committee Member Kenneth H. Roux Committee Member Branko Stefanovic University Representative
Keywords	Hepatitis C viurs Cyclophilin Cyclosporin A NS5A NS5B
Date of Defense	2010-06-25
Availability	unrestricted
Abstract Hepatitis C virus (HCV) infection leads to acute and chronic liver disease for thousands of people every year. With the absence of an effective treatment method, new classes of anti-HCV therapies are needed. Cyclosporine (CsA) and its derivatives potently suppress HCV replication. Recently, CsA resistant HCV replicons have been identified in vitro. We examined the dependence of the wild-type and CsA resistant HCV replicons on various cyclophilins for replication. A strong correlation between CsA resistance and reduced dependency on cyclophilin A (CyPA) for replication was identified. Silencing CyPB or CyPC expression had no significant effect on replication, whereas various forms of small interfering RNA (siRNA) directed at CyPA inhibited HCV replication of wild-type but not CsA-resistant replicons. The efficiency of a particular siRNA in suppressing CyPA expression was correlated with its potency in inhibiting HCV replication, and expression of a siRNA-resistant CyPA cDNA rescued replication. In addition, an anti-CyPA antibody blocked replication of the wild-type but not the resistant replicon in an in vitro replication assay. Depletion of CyPA alone in the CsA-resistant replicon cells eliminated CsA resistance, indicating that CyPA is the chief mediator of the observed CsA resistance. The dependency on CyPA for replication was observed for both genotype (GT) 1a and 1b replicons as well as a GT 2a infectious virus. An interaction between CyPA and HCV RNA as well as the viral polymerase that is sensitive to CsA treatment in wild-type but not in resistant replicons was detected. These findings reveal the molecular mechanism of CsA resistance and identify CyPA as a critical cellular cofactor for HCV replication and infection. In addition, we selected mutant viruses that were able to infect CyPA-knock down cells. 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 by nuclear magnetic resonance. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype. Four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. 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. This approach could be applied broadly to other cofactors in other viruses as large numbers of cellular cofactors important for viral infection have been discovered since the advent of genome-wide small interfering RNA screening.
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