RNase L Mediates Transient Control of the Interferon Response through Modulation of the Double-stranded RNA-dependent Protein Kinase PKR

Abstract

(1) and mRNA turnover regulation by the AU-rich elements in the 3Ј-untranslated regions (2). The IFN antiviral response is a highly regulated process involving the transient inhibition of viral and cellular protein synthesis. The translational inhibition is mediated predominately by the activity of PKR, the double-stranded RNAdependent protein kinase. PKR is a serine/threonine protein kinase of 65 and 68 kDa in murine and human cells, respectively, that is induced by IFN treatment of cells and phosphorylates itself and other proteins, notably eIF2␣, in response to viral double-stranded RNA (3). DsRNA is produced as the replicative intermediates of many RNA viruses and also by annealing of complementary RNA strands transcribed from some DNA viruses (3). Phosphorylated eIF2␣ sequesters the guanine nucleotide exchange factor, eIF2B, which becomes trapped as inactive complex with GDP resulting in translational arrest (4, 5).Among the principal effectors of the IFN-induced antiviral state are the 2Ј,5Ј-oligoadenylate (2-5A) synthetases that convert ATP to 2-5A, activators of RNase L, in response to viral double-stranded RNA (6, 7). Thus, 2-5A is an alarmone that alerts the cells to the presence of virus by signaling to RNase L. Both RNase L and PKR have been implicated in the action of IFN-␣ against a variety of viruses (reviewed in Refs. 8 and 9). RNase L is widely distributed in different tissues, and it has been suggested that low levels of 2-5A lead to RNase L-mediated selective degradation of viral mRNA (10), whereas higher levels may lead to broader effects such as cleavage of 18 S and 28 S ribosomal RNAs (11). During the course of experiments on the role of RNase L in the inhibition of viral protein synthesis during acute infections, we observed that an absence of RNase L led to selective stabilization of PKR mRNA, extended kinetics of eIF2␣ phosphorylation, and potent inhibition of viral protein synthesis. Our findings suggest that RNase L truncates and limits the induction of PKR, possibly contributing to the transient nature of the IFN response against viral infections. Cell Culture, Viral Infections, and IFN Treatments-RNase L ϩ/ϩ and RNase L Ϫ/Ϫ mouse embryonic fibroblast (MEF) cell lines were of mixed or C57BL/6 genetic backgrounds. The cell lines are post-crisis derivatives of primary MEFs as described previously (12). MEFs were cultured in DMEM with high glucose supplemented with 10% FBS and antibiotics (Invitrogen, Gaithersburg, MD). Bone marrow macrophages collected from the femurs of RNase L ϩ/ϩ and RNase L Ϫ/Ϫ mice, both on a background of C57BL/6, were cultured in L-cell-conditioned medium for 8 days and plated at a density of 10 6 cells per 10-cm plate. WISH cell line (HeLa markers) was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in RPMI 1640 supplemented with 10% FBS and antibiotics. EXPERIMENTAL PROCEDURES

A scientific journey through the 2-5A/RNase L system

“…Meanwhile, in complementary work, Catherine Bisbal and co-workers were successful in cloning the RNase L inhibitor (RLI), an ATP binding cassette protein also known as ABCE1 [55]. Khalid Khabar showed that RNase L could affect the stability of PKR mRNA and levels of PKR, demonstrating that the two pathways are in a type of balance [56]. Studies by the groups of Robert Suhadolnik [57,58] and Bernard LeBleu [59,60] described up-regulation of the 2-5A system and accumulation of a 37 kDa polypeptide from RNase L in PBMC of patients with chronic fatigue syndrome.…”

Post-transcriptional control of the interferon system

“…This pathway is reviewed by Silverman and Bisbal in this same issue. In recent years, it has been accepted that RNase L participates in the degradation of selected cellular mRNAs [63][64][65][66][67]. Specifically, RNase L has been shown to downregulate PKR mRNA [64].…”

“…In recent years, it has been accepted that RNase L participates in the degradation of selected cellular mRNAs [63][64][65][66][67]. Specifically, RNase L has been shown to downregulate PKR mRNA [64]. During the IFN antiviral response in normal cells, PKR mRNA expression is transient, but in RNase L-null cells, extended kinetics of PKR mRNA expression is observed, due to increased mRNA stability [64].…”

“…Specifically, RNase L has been shown to downregulate PKR mRNA [64]. During the IFN antiviral response in normal cells, PKR mRNA expression is transient, but in RNase L-null cells, extended kinetics of PKR mRNA expression is observed, due to increased mRNA stability [64]. The effect results in prolongation of the PKR-dependent phosphorylation of the subunit of eukaryotic translation initiation factor 2, eIF2α; a process that leads to inhibition of viral protein synthesis [64].…”

“…During the IFN antiviral response in normal cells, PKR mRNA expression is transient, but in RNase L-null cells, extended kinetics of PKR mRNA expression is observed, due to increased mRNA stability [64]. The effect results in prolongation of the PKR-dependent phosphorylation of the subunit of eukaryotic translation initiation factor 2, eIF2α; a process that leads to inhibition of viral protein synthesis [64]. Thus, RNAse L contributes to the transient nature of the IFN response in order to ensure a brief translational arrest imparted by PKR.…”