Cutting Edge: Induced Loss of Rasgrp1 in Peripheral CD4+ T Cells of Conditional Rasgrp1-Deficient Mice Reveals an Essential Role for Rasgrp1 in TCR/CD28-Induced Ras-MAPK Signaling.

Ras guanine nucleotide-releasing protein 1 (Rasgrp1) is a Ras guanine nucleotide exchange factor that participates in the activation of the Ras-ERK signaling pathway in developing T cells and is required for efficient thymic T cell positive selection. However, the role of Rasgrp1 in mature peripheral T cells has not been definitively addressed, in part because peripheral T cells from constitutive Rasgrp1-deficient mice show an abnormal activated phenotype. In this study, we generated an inducible Rasgrp1-deficient mouse model to allow acute disruption of Rasgrp1 in peripheral CD4+ T cells in the context of normal T cell development. TCR/CD28-mediated activation of Ras-ERK signaling was blocked in Rasgrp1-deficient peripheral CD4+ T cells. Furthermore, Rasgrp1-deficient CD4+ T cells were unable to synthesize IL-2 and the high-affinity IL-2R and were unable to proliferate in response to TCR/CD28 stimulation. These findings highlight an essential function for Rasgrp1 for TCR/CD28-induced Ras-ERK activation in peripheral CD4+ T cells.

Viral Hemorrhagic Septicemia Virus Activates Integrated Stress Response Pathway and Induces Stress Granules to Regulate Virus Replication.

Virus infection activates integrated stress response (ISR) and stress granule (SG) formation and viruses counteract by interfering with SG assembly, suggesting an important role in antiviral defense. The infection of fish cells by Viral Hemorrhagic Septicemia Virus (VHSV), activates the innate immune recognition pathway and the production of type I interferon (IFN). However, the mechanisms by which VHSV interacts with ISR pathway regulating SG formation is poorly understood. Here, we demonstrate that fish cells respond to heat shock, oxidative stress and VHSV infection by forming SG that localized key SG marker, Ras GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1). We show that PKR-like endoplasmic reticulum kinase (PERK), but not (dsRNA)-dependent protein kinase (PKR), is required for VHSV-induced SG formation. Furthermore, in VHSV Ia infected cells, PERK activity is required for IFN production, antiviral signaling and viral replication. SG formation required active virus replication as individual VHSV Ia proteins or inactive virus did not induce SG. Cells lacking G3BP1 produced increased IFN, antiviral genes and viral mRNA, however viral protein synthesis and viral titers were reduced. We show a critical role of the activation of ISR pathway and SG formation highlighting a novel role of G3BP1 in regulating VHSV protein translation and replication.

ABCE1 Regulates RNase L-Induced Autophagy during Viral Infections.

Host response to a viral infection includes the production of type I interferon (IFN) and the induction of interferon-stimulated genes that have broad antiviral effects. One of the key antiviral effectors is the IFN-inducible oligoadenylate synthetase/ribonuclease L (OAS/RNase L) pathway, which is activated by double-stranded RNA to synthesize unique oligoadenylates, 2-5A, to activate RNase L. RNase L exerts an antiviral effect by cleaving diverse RNA substrates, limiting viral replication; many viruses have evolved mechanisms to counteract the OAS/RNase L pathway. Here, we show that the ATP-binding cassette E1 (ABCE1) transporter, identified as an inhibitor of RNase L, regulates RNase L activity and RNase L-induced autophagy during viral infections. ABCE1 knockdown cells show increased RNase L activity when activated by 2-5A. Compared to parental cells, the autophagy-inducing activity of RNase L in ABCE1-depleted cells is enhanced with early onset. RNase L activation in ABCE1-depleted cells inhibits cellular proliferation and sensitizes cells to apoptosis. Increased activity of caspase-3 causes premature cleavage of autophagy protein, Beclin-1, promoting a switch from autophagy to apoptosis. ABCE1 regulates autophagy during EMCV infection, and enhanced autophagy in ABCE1 knockdown cells promotes EMCV replication. We identify ABCE1 as a host protein that inhibits the OAS/RNase L pathway by regulating RNase L activity, potentially affecting antiviral effects.

RNase L Amplifies Interferon Signaling by Inducing Protein Kinase R-Mediated Antiviral Stress Granules.

Virus infection leads to activation of the interferon (IFN)-induced endoribonuclease RNase L, which results in degradation of viral and cellular RNAs. Both cellular and viral RNA cleavage products of RNase L bind pattern recognition receptors (PRRs), like retinoic acid-inducible I (Rig-I) and melanoma differentiation-associated protein 5 (MDA5), to further amplify IFN production and antiviral response. Although much is known about the mechanics of ligand binding and PRR activation, how cells coordinate RNA sensing with signaling response and interferon production remains unclear. We show that RNA cleavage products of RNase L activity induce the formation of antiviral stress granules (avSGs) by regulating activation of double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) and recruit the antiviral proteins Rig-I, PKR, OAS, and RNase L to avSGs. Biochemical analysis of purified avSGs showed interaction of a key stress granule protein, G3BP1, with only PKR and Rig-I and not with OAS or RNase L. AvSG assembly during RNase L activation is required for IRF3-mediated IFN production, but not IFN signaling or proinflammatory cytokine induction. Consequently, cells lacking avSG formation or RNase L signaling produced less IFN and showed higher susceptibility during Sendai virus infection, demonstrating the importance of avSGs in RNase L-mediated host defense. We propose a role during viral infection for RNase L-cleaved RNAs in inducing avSGs containing antiviral proteins to provide a platform for efficient interaction of RNA ligands with pattern recognition receptors to enhance IFN production to mount an effective antiviral response.IMPORTANCE Double-stranded RNAs produced during viral infections serve as pathogen-associated molecular patterns (PAMPs) and bind pattern recognition receptors to stimulate IFN production. RNase L is an IFN-regulated endoribonuclease that is activated in virus-infected cells and cleaves single-stranded viral and cellular RNAs. The RNase L-cleaved dsRNAs signal to Rig-like helicases to amplify IFN production. This study identifies a novel role of antiviral stress granules induced by RNase L as an antiviral signaling hub to coordinate the RNA ligands with cognate receptors to mount an effective host response during viral infections.

RNase L Induces Expression of A Novel Serine/Threonine Protein Kinase, DRAK1, to Promote Apoptosis.

Apoptosis of virus-infected cells is an effective antiviral mechanism in addition to interferon induction to establish antiviral state to restrict virus spread. The interferon-inducible 2'-5' oligoadenylate synthetase/RNase L pathway results in activation of RNase L in response to double stranded RNA and cleaves diverse RNA substrates to amplify interferon induction and promote apoptosis. Here we show that RNase L induces expression of Death-associated protein kinase-Related Apoptosis-inducing protein Kinase 1 (DRAK1), a member of the death-associated protein kinase family and interferon-signaling pathway is required for induction. Overexpression of DRAK1 triggers apoptosis in the absence of RNase L activation by activating c-Jun N-terminal kinase (JNK), translocation of BCL2 Associated X (Bax) to the mitochondria accompanied by cytochrome C release and loss of mitochondrial membrane potential promoting cleavage of caspase 3 and Poly(ADP-Ribose) Polymerase 1 (PARP). Inhibitors of JNK and caspase 3 promote survival of DRAK1 overexpressing cells demonstrating an important role of JNK signaling pathway in DRAK1-mediated apoptosis. DRAK1 mutant proteins that lack kinase activity or nuclear localization fail to induce apoptosis highlighting the importance of cellular localization and kinase function in promoting cell death. Our studies identify DRAK1 as a mediator of RNase L-induced apoptosis.

RNase L Suppresses Androgen Receptor Signaling, Cell Migration and Matrix Metalloproteinase Activity in Prostate Cancer Cells.

The interferon antiviral pathways and prostate cancer genetics converge on a regulated endoribonuclease, RNase L. Positional cloning and linkage studies mapped Hereditary Prostate Cancer 1 (HPC1) to RNASEL. To date, there is no correlation of viral infections with prostate cancer, suggesting that RNase L may play additional roles in tumor suppression. Here, we demonstrate a role of RNase L as a suppressor of androgen receptor (AR) signaling, cell migration and matrix metalloproteinase activity. Using RNase L mutants, we show that its nucleolytic activity is dispensable for both AR signaling and migration. The most prevalent HPC1-associated mutations in RNase L, R462Q and E265X, enhance AR signaling and cell migration. RNase L negatively regulates cell migration and attachment on various extracellular matrices. We demonstrate that RNase L knockdown cells promote increased cell surface expression of integrin ß1 which activates Focal Adhesion Kinase-Sarcoma (FAK-Src) pathway and Ras-related C3 botulinum toxin substrate 1-guanosine triphosphatase (Rac1-GTPase) activity to increase cell migration. Activity of matrix metalloproteinase (MMP)-2 and -9 is significantly increased in cells where RNase L levels are ablated. We show that mutations in RNase L found in HPC patients may promote prostate cancer by increasing expression of AR-responsive genes and cell motility and identify novel roles of RNase L as a prostate cancer susceptibility gene.

RNase L Cleavage Products Promote Switch from Autophagy to Apoptosis by Caspase-Mediated Cleavage of Beclin-1.

Autophagy and apoptosis share regulatory molecules enabling crosstalk in pathways that affect cellular homeostasis including response to viral infections and survival of tumor cells. Ribonuclease L (RNase L) is an antiviral endonuclease that is activated in virus-infected cells and cleaves viral and cellular single-stranded RNAs to produce small double-stranded RNAs with roles in amplifying host responses. Activation of RNase L induces autophagy and apoptosis in many cell types. However, the mechanism by which RNase L mediates crosstalk between these two pathways remains unclear. Here we show that small dsRNAs produced by RNase L promote a switch from autophagy to apoptosis by caspase-mediated cleavage of Beclin-1, terminating autophagy. The caspase 3-cleaved C-terminal fragment of Beclin-1 enhances apoptosis by translocating to the mitochondria along with proapoptotic protein, Bax, and inducing release of cytochrome C to the cytosol. Cleavage of Beclin-1 determines switch to apoptosis since expression of caspase-resistant Beclin-1 inhibits apoptosis and sustains autophagy. Moreover, inhibiting RNase L-induced autophagy promotes cell death and inhibiting apoptosis prolongs autophagy in a cross-inhibitory mechanism. Our results demonstrate a novel role of RNase L generated small RNAs in cross-talk between autophagy and apoptosis that impacts the fate of cells during viral infections and cancer.