title: Hepatitis C virus controls interferon production through PKR activation.
abstract: Hepatitis C virus is a poor inducer of interferon (IFN), although its structured viral RNA can bind the RNA helicase RIG-I, and activate the IFN-induction pathway. Low IFN induction has been attributed to HCV NS3/4A protease-mediated cleavage of the mitochondria-adapter MAVS. Here, we have investigated the early events of IFN induction upon HCV infection, using the cell-cultured HCV JFH1 strain and the new HCV-permissive hepatoma-derived Huh7.25.CD81 cell subclone. These cells depend on ectopic expression of the RIG-I ubiquitinating enzyme TRIM25 to induce IFN through the RIG-I/MAVS pathway. We observed induction of IFN during the first 12 hrs of HCV infection, after which a decline occurred which was more abrupt at the protein than at the RNA level, revealing a novel HCV-mediated control of IFN induction at the level of translation. The cellular protein kinase PKR is an important regulator of translation, through the phosphorylation of its substrate the eIF2alpha initiation factor. A comparison of the expression of luciferase placed under the control of an eIF2alpha-dependent (IRES(EMCV)) or independent (IRES(HCV)) RNA showed a specific HCV-mediated inhibition of eIF2alpha-dependent translation. We demonstrated that HCV infection triggers the phosphorylation of both PKR and eIF2alpha at 12 and 15 hrs post-infection. PKR silencing, as well as treatment with PKR pharmacological inhibitors, restored IFN induction in JFH1-infected cells, at least until 18 hrs post-infection, at which time a decrease in IFN expression could be attributed to NS3/4A-mediated MAVS cleavage. Importantly, both PKR silencing and PKR inhibitors led to inhibition of HCV yields in cells that express functional RIG-I/MAVS. In conclusion, here we provide the first evidence that HCV uses PKR to restrain its ability to induce IFN through the RIG-I/MAVS pathway. This opens up new possibilities to assay PKR chemical inhibitors for their potential to boost innate immunity in HCV infection.
authors.affiliation: Institut Pasteur, Unité Hépacivirus et Immunité Innée, Paris, France.
authors.forename: Noëlla
authors.initials: N
authors.lastname: Arnaud
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Dabo
authors.forename: Patrick
authors.initials: P
authors.lastname: Maillard
authors.forename: Agata
authors.initials: A
authors.lastname: Budkowska
authors.forename: Katerina I
authors.initials: KI
authors.lastname: Kalliampakou
authors.forename: Penelope
authors.initials: P
authors.lastname: Mavromara
authors.forename: Dominique
authors.initials: D
authors.lastname: Garcin
authors.forename: Jacques
authors.initials: J
authors.lastname: Hugon
authors.forename: Anne
authors.initials: A
authors.lastname: Gatignol
authors.forename: Daisuke
authors.initials: D
authors.lastname: Akazawa
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
authors.forename: Eliane F
authors.initials: EF
authors.lastname: Meurs
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D016174:Hepacivirus / Q000187:drug effects / Q000276:immunology*; D006526:Hepatitis C / Q000276:immunology / Q000821:virology; D006801:Humans; D007372:Interferons / Q000096:biosynthesis*; D007700:Kinetics; D018448:Models, Immunological; D010766:Phosphorylation / Q000187:drug effects; D014176:Protein Biosynthesis; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015852:Eukaryotic Initiation Factor-2; C505371:MAVS protein, human; D047428:Protein Kinase Inhibitors; D014157:Transcription Factors; D000070584:Tripartite Motif Proteins; D007372:Interferons; C084523:TRIM25 protein, human; D044767:Ubiquitin-Protein Ligases; D019892:eIF-2 Kinase