Both Cyclophilin Inhibitors and Direct-Acting Antivirals Prevent PKR Activation in HCV-Infected Cells

Abstract

We and others demonstrated that the contact between NS5A and the host factor CypA is critical for HCV replication. CypI, by disrupting NS5A-CypA complexes, block HCV replication both in vitro and in patients. Since NS5A also binds to PKR, a central component of the IFN response, we investigated the possibility of a relationship between CypA, NS5A and PKR in the IFN response to HCV. HCV-infected cells treated with CypI, DAAs or IFN were analyzed for the expression and activation of various components of the innate response. We found that CypI (cyclosporine A, alisporivir, NIM811 and sanglifehrins), drastically prevented the activation/phosphorylation, but not the expression of IFN-induced PKR in HCV-infected cells. CypI had no effect on the expression or phosphorylation of other components of the innate response such as eiF2, NF-kB, IRF3, IRF9, STAT1 and STAT2, suggesting a specific effect on PKR. No significant activation of IFN-induced PKR was observed in the absence of HCV. Importantly, we found that several classes of DAAs such as NS3/4A protease, NS5B polymerase and NS5A inhibitors also prevented PKR activation. Furthermore, we found that PKR activation by the dsRNA mimic poly I:C cannot be prevented by CypI or DAAs. Our findings suggest that CypI do not have a unique effect on PKR activation, but rather the suppression of HCV replication by any anti-HCV inhibitor, abrogates PKR activation induced by IFN. Moreover, they suggest that the accumulation of dsRNA intermediates allows HCV to exploit the activation of PKR to counteract the IFN response.

Keywords: CypI; DAAs; HCV; IFN response; NS5A; PKR..

Fig. (1)

CypI prevent IFN-induced PKR activation in HCV-infected cells. (A) Schematic diagram of genomic JFH-1 replicon used is depicted. JFH-1-infected Huh7.5.1 cells were treated with or without the CypI alisporivir (ALV) or NIM811, and subsequently treated with IFNα or β for 24 or 48 h. Cells were then lysed and analyzed for their content in viral and host proteins by Western blotting. (B) Schematic diagram of subgenomic JFH-1 replicon used is depicted. Same as (A) except that JFH-1 subgenomic replicon cells were used and treated with IFNα for 24 h. (C) Same as (A) except that JFH-1-infected Huh7.5.1 cells were treated with a panel of CypI as well as with or without IFNα for 24 h. Results are representative of 3-5 independent experiments.

Fig. (2)

The PKR activation block is not unique to CypI, DAAs also prevent the IFN-induced PKR activation in HCV-infected cells. Same as (Fig. 1c) except that JFH-1-infected Huh7.5.1 cells were treated with or without CypI (cyclosporine A and alisporivir), DAAs (the HCV NS5A inhibitor daclatasvir and the HCV protease inhibitor telaprevir) and an HIV-1 inhibitor (reverse transcriptase inhibitor emtricitabine). Results are representative of 4 independent experiments.

Fig. (3)

Suppression of HCV replication and/or dsRNA accumulation correlate with the preclusion of PKR activation. (A) Noninfected or JFH-1-infected Huh7.5.1 cells were treated with or without the CypI alisporivir, the HCV NS5A inhibitor daclatasvir or the HCV NS5B polymerase inhibitor sofosbuvir and subsequently treated with IFNα for 24 h. Cells were then lysed and analyzed for their content in viral and host proteins by Western blotting. (B) Non-infected cells were treated with the dsRNA mimic poly I:C (50 µg/mL) at the time of the cell plating, treated with or without the CypI alisporivir and IFNα 24 and 48 h post-cell plating, respectively. Cells were lysed 72 h postcell plating and analyzed for their content in viral and host proteins by Western blotting. Results are representative of 3 independent experiments.

Fig. (4)

Model for the effect of CypI and DAAs on the HCV-dependent activation of IFN-induced PKR. (Left) HCV replication in hepatoma cells leads to an accumulation of dsRNA intermediates. During this established infection, PKR is slightly expressed and activated. In response to IFN, infected cells overexpress the inactive form of PKR, which is subsequently activated/phosphorylated upon the recognition of and binding to viral dsRNA. (Right) The addition of anti-HCV agents either CypI or DAAs stops viral dsRNA accumulation resulting in the prevention of the activation of IFN-induced PKR.