Cyclophilin A Interacts with Domain II of Hepatitis C Virus NS5A and Stimulates RNA Binding in an Isomerase-Dependent Manner

Abstract

NS5A plays a critical, yet poorly defined, role in hepatitis C virus genome replication. The protein consists of three domains, each of which is able to bind independently to the 3 untranslated region (UTR) of the viral positive strand genomic RNA. The peptidyl-prolyl isomerase cyclophilin A (CypA) binds to domain II, catalyzing cis-trans isomerization. CypA inhibitors such as cyclosporine (CsA) have been shown to inhibit hepatitis C virus (HCV) replication. We show here that CypA stimulated domain II RNA binding activity, and this stimulation was abrogated by CsA. An isomerase mutant of CypA (H126Q) failed to bind to domain II and did not stimulate RNA binding. Finally, we demonstrate that the RNA binding of two domain II mutants, the D316E and D316E/Y317N mutants, previously shown to exhibit CypA independence for RNA replication, was unaffected by CypA. This study provides an insight into the molecular mechanism of CypA activity during HCV replication and further validates the use of CypA inhibitors in HCV therapy.

A Synthetic Codon-Optimized Hepatitis C Virus Nonstructural 5A DNA Vaccine Primes Polyfunctional CD8+ T Cell Responses in Wild-Type and NS5A-Transgenic Mice

“…Therefore, NS5A may be considered as a therapeutic vaccine candidate for chronic HCV infection. Moreover, even though NS5A may interfere with cellular signaling pathways including molecules and cells involved in innate and adaptive immunity (20,34,35), long-term intrahepatic NS5A protein expression does not induce any liver disease (20,36).…”

“…Domain III is required for proper assembly of viral particles (31) but is not needed for RNA replication (32). NS5A has been described to interact both with cellular proteins like Raf-1, p53, PI3K, and Grb2, which are important for host cell signaling (20,33), but also to interfere with components of innate immunity by inhibiting PKR and cyclophilin A (33)(34)(35). Hence, NS5A may affect IFN signaling pathways and production of proinflammatory cytokines.…”

Correlation between NS5A Dimerization and Hepatitis C Virus Replication

“…Unfortunately, a connection between the NS5A RNA binding activity and a specific aspect of RNA replication or virion assembly remains to be established (30). A recent study showed that both Domains I and II of NS5A exhibit RNA binding activities (20). However, the ability to bind specifically to uridine-and guanosine-rich RNA resides in domain I and the adjacent linker region (33).…”

“…As a control, we used GSTCypA as bait to capture full-length NS5A-His because this interaction has been shown to be direct (15)(16)(17)(18)(19)(20)(21)(22). As expected, both GST-CypA and GST-Domain I, but not GST, capture fulllength NS5A-His (Fig.…”

“…NS5A is a nonstructural protein that has no defined role in the virus life cycle but is absolutely required for RNA replication (8 -10) and particle assembly (11)(12)(13)(14). It has also been shown to interact with a large number of host proteins including CypA (cyclophilin A) (15)(16)(17)(18)(19)(20)(21)(22). NS5A is peripherally anchored to membranes by an N-terminal amphipathic helix (23)(24)(25)(26)(27).…”