Research Article: Hepatitis C Virus Controls Interferon Production through PKR Activation

Date Published: May 11, 2010

Publisher: Public Library of Science

Author(s): Noëlla Arnaud, Stéphanie Dabo, Patrick Maillard, Agata Budkowska, Katerina I. Kalliampakou, Penelope Mavromara, Dominique Garcin, Jacques Hugon, Anne Gatignol, Daisuke Akazawa, Takaji Wakita, Eliane F. Meurs, Sung Key Jang.

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 eIF2α initiation factor. A comparison of the expression of luciferase placed under the control of an eIF2α-dependent (IRESEMCV) or independent (IRESHCV) RNA showed a specific HCV-mediated inhibition of eIF2α-dependent translation. We demonstrated that HCV infection triggers the phosphorylation of both PKR and eIF2α 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.

Partial Text: In response to invasion with bacterial or viral pathogens, cells are able to mount an immediate immune response through their ability to use specialized cellular molecules, referred to as pattern recognition receptors or PRRs, to detect unusual DNA, ssRNA or dsRNA structures. Among these PRRs, are the CARD-containing DexD/H RNA helicases RIG-I and MDA5, which are activated upon binding either to both 5′-triphosphate ssRNA and short double-stranded RNA structures (RIG-I) or to long dsRNA and higher-ordered RNA structures (MDA5) [1]. Once activated, these RNA helicases interact with the mitochondria-bound MAVS adapter protein [2]. In the case of RIG-I, the interaction with MAVS requires ubiquitination of the CARD domain of RIG-I by the TRIM25 ubiquitin ligase [3]. Subsequently, MAVS is able to recruit the IKK complex and the TBK1/IKKε kinases that are responsible for the activation of the NF-κB and IRF3/IRF7 transcription factors, respectively. This leads to induction of the interferons and pro-inflammatory cytokines that are involved in the innate immune response [4].

Here we report the use of HCV permissive Huh7.25.CD81 cells to analyse the effect of HCV on the RIG-I/MAVS-mediated IFN induction pathway during the early hours after infection, before the HCV NS3/4A protease can cleave MAVS and abrogate this pathway. We first observed that the IFN induction pathway was deficient in these cells, but that it could be conveniently restored upon ectopic expression of the E3 ubiquitin ligase TRIM25. This protein has been shown to bind the threonine 55 residue of RIG-I through its C terminal SPRY domain, and uses the E3 ligase activity present in its N terminal RING domain to promote a Lys63-type ubiquitination of RIG-I in its CARD domain at lysine 172. This modification is important if RIG-I is to engage in a correct interaction with its downstream adapter MAVS [3]. The nature of the TRIM25 defect in Huh7.25.CD81 cells and the relation to activation of RIG-I is now under characterization. It is interesting to note here that this defect emphasizes the importance of the RIG-I/TRIM25 pathway in the control of HCV expression since both Huh7.25.CD81 and Huh7.5 cells, which were originally selected for their ability to replicate HCV subgenomic replicons, present a defect in this pathway at the level of RIG-I for Huh7.5 cells and of TRIM25 for Huh7.25.CD81 cells. Importantly, Huh7.25.CD81 cells can be considered as a more physiological model than Huh7.5 cells to study the interaction between the IFN induction pathway and HCV infection, since Huh7.5 cells have a point mutation at the threonine 55 residue of RIG-I [5] and therefore require ectopic expression of RIG-I to restore their IFN induction pathway. In contrast, ectopic expression of TRIM25 restores the IFN induction pathway downstream of RIG-I and therefore does not perturb the initial interaction between RIG-I and the incoming HCV dsRNA structures.