Mutations in the hepatitis C virus polymerase that increase RNA binding can confer resistance to cyclosporine A

Abstract

Hepatitis C virus (HCV) infection leads to acute and chronic liver diseases, and new classes of anti-HCV therapeutics are needed. Cyclosporine A (CsA) inhibits HCV replication and CsA derivatives that lack the immunosuppressive function are currently in clinical trials as candidate anti-HCV drugs. Here we characterize several independently derived HCV replicons with varying levels of CsA resistance due to mutations in nonstructural protein 5B (NS5B), the HCV-encoded polymerase. Mutant HCV replicons engineered with these mutations showed resistance to CsA. The mutations reside in two distinct patches in the polymerase: the template channel and one face of a concave surface behind the template channel. Mutant NS5B made by cells expressing the HCV replicon had increased ability to bind to RNA in the presence of CsA. Purified recombinant NS5B proteins containing the mutations were better at de novo initiated RNA synthesis than the wild-type control. Furthermore, the mutant proteins were able to bind RNA with Ϸ8-fold higher affinity. Last, mutation near the template channel alleviated the lethal phenotype of a mutation in the concave patch, P540A. This intramolecular compensation for the HCV replicase function by amino acid changes in different domains was further confirmed in an infectious cell culture-derived virus system. C yclosporine A (CsA), a commonly used immunosuppressant for transplant patients, has recently emerged as a potential new anti-hepatitis C virus (HCV) therapeutic. CsA and its derivatives potently inhibit HCV replication both in cell-culture systems and in mice with transplanted human liver, 1-3 although no consensus has emerged on the in vivo benefits of using CsA over Tacrolimus (FK506), a compound that lacks anti-HCV effect in vitro, for HCV-infected liver-transplant patients. [4][5][6][7][8][9] More recent clinical trials with a CsA derivative, DEBIO-025, yield promising results in human immunodeficiency virus (HIV) and HCV-coinfected patients, but drug resistance in vivo has not been studied. 10 Studies of the antiviral effect of CsA on HCV replicons has led to the identification of cyclophilin (CyP) as an essential cofactor for HCV replication [11][12][13] ; point mutations in nonstructural protein 5B (NS5B) and NS5A are associated with CsA resistance in vitro. 14,15 In addition, NS5B interacts with both CyPA and CyPB both in vitro and in vivo. 12,13 These data establish NS5B as an indirect viral target for the CsA-mediated inhibition of HCV replication. The CyPA-NS5B interaction has also been shown to be the principal mediator of cyclosporine resistance in vitro. 12

Regulation of De Novo -Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions

“…We examined ⌬21 ( Fig. 2A) along with several mutant derivatives that are affected for oligomerization (E18R mutant [46]), de novo initiation (m26-30 and I432V mutants [10,27]), or in the GTPbinding allosteric site (P495/V499A mutant, named PV [6]) (Fig. 2B).…”

“…Therefore, we analyzed several other mutant RdRps and found that the I432V mutant was a good choice for reconstruction. The I432V substitution was originally identified in a mutant HCV replicon that was resistant to cyclosporin A and subsequently was shown to be better at de novo initiation than the wild-type (WT) ⌬21 protein, especially at higher enzyme concentrations (27) (Fig. 9A and B).…”

A Conserved Tandem Cyclophilin-Binding Site in Hepatitis C Virus Nonstructural Protein 5A Regulates Alisporivir Susceptibility

“…It would be interesting to systematically determine whether any additional NS5A interactions, especially the ones that involve domains II and III, are dependent on CyPA's PPIase activity and thus sensitive to CPIs. CyPA has also been proposed to regulate NS5A/5B cleavage kinetics (27) and/or RNA binding by HCV replicase (17,33). Although domain I is likely the major RNA-binding domain of NS5A and some controversies currently surround whether domains II and III are directly involved in NS5A's RNA-binding ability or not (16,25), it is theoretically possible for structural changes in domain II and LCS-II to influence the configuration of domain I dimers.…”

“…The importance of CyPA's PPIase activity in HCV replication has been confirmed by several independent labs using both chemical and RNA interference approaches (5, 27, 34), but the relevant substrates of the PPIases are much less defined. At least three HCV proteins (NS2, NS5A, and NS5B) have been implicated in the action of CyPA as an HCV cofactor (7,21,33), and increasing evidence supports a direct interaction between CyPA and NS5A (8,14,21,58,63). Recombinant CyPA protein interacts with both full-length NS5A from infected cell lysates and purified NS5A domains.…”

Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

“…A-782759 selects highly fit mutations at NS5B residue M414 that confer a large decrease in drug susceptibility (17,18). In contrast, a single HCV mutation that confers a more than fivefold shift in potency has not been reported despite a large body of literature on CsA (19)(20)(21). When a large (10 5 ) population of 51C-RFP-1a replicon cells was treated with each of these drugs, a dramatic difference in the frequency of resistance foci was observed (Fig.…”