Cyclophilin inhibitors reduce phosphorylation of RNA-dependent protein kinase to restore expression of IFN-stimulated genes in HCV-infected cells.
OBJECTIVE
Cyclophilin inhibitors are being developed for treatment of hepatitis C virus (HCV) infection. They are believed to inhibit the HCV replication complex. We investigated whether cyclophilin inhibitors interact with interferon (IFN) signaling in cultured cells infected with HCV.


METHODS
We used immunoblot assays to compare expression of IFN-stimulated genes (ISGs) and of components of IFN signaling in HCV-infected and uninfected cells.


RESULTS
Incubation with IFN alfa induced expression of ISGs in noninfected cells and, to a lesser extent, in HCV-infected cells; addition of the cyclophilin inhibitor SCY-635 restored expression of ISG products in HCV-infected cells. SCY-635 reduced phosphorylation of double-strand RNA-dependent protein kinase (PKR) and its downstream factor eIF2α; the phosphorylated forms of these proteins are negative regulators of ISG translation. Cyclophilin A interacted physically with PKR; this interaction was disrupted by SCY-635. SCY-635 also suppressed PKR-mediated formation of stress granules. Cyclophilin inhibitors were found to inhibit PKR phosphorylation and stress granule formation in HCV-infected and uninfected cells.


CONCLUSIONS
In cultured cells, cyclophilin inhibitors reverse the attenuation of the IFN response by HCV, in addition to their effects on HCV replication complex. Cyclophilin A regulation of PKR has been proposed as a mechanism for observed effects of cyclophilin inhibitors on IFN signaling. We found that cyclophilin inhibitors reduce phosphorylation of PKR and eIF2α during HCV infection to allow for translation of ISG products. Proteins in this pathway might be developed as targets for treatment of HCV infection.
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; SCYNEXIS, Inc, Durham, North Carolina.
Takuji
T
Daito
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan. Electronic address: kwatashi@nih.go.jp.
Koichi
K
Watashi
SCYNEXIS, Inc, Durham, North Carolina.
Ann
A
Sluder
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
Hirofumi
H
Ohashi
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
Syo
S
Nakajima
SCYNEXIS, Inc, Durham, North Carolina.
Katyna
K
Borroto-Esoda
Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto University, Kyoto, Japan.
Takashi
T
Fujita
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
Takaji
T
Wakita
D000998:Antiviral Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D021984:Cyclophilin A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003524:Cyclosporins / Q000494:pharmacology*; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005786:Gene Expression Regulation; D016174:Hepacivirus / Q000187:drug effects* / Q000378:metabolism; D006526:Hepatitis C / Q000188:drug therapy* / Q000201:enzymology / Q000821:virology; D006801:Humans; D050835:Interferon Regulatory Factors / Q000235:genetics / Q000378:metabolism*; D016898:Interferon-alpha / Q000378:metabolism; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000821:virology; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D019892:eIF-2 Kinase / Q000378:metabolism*
D000998:Antiviral Agents; D003524:Cyclosporins; D004791:Enzyme Inhibitors; D015852:Eukaryotic Initiation Factor-2; D050835:Interferon Regulatory Factors; D016898:Interferon-alpha; D012333:RNA, Messenger; C583541:EIF2AK1 protein, human; D019892:eIF-2 Kinase; D021984:Cyclophilin A; C544142:SCY-635
Cyclosporin; Innate Immunity; Replication; Signal Transduction