Arindam Chakrabarti

RNase L Triggers Autophagy in Response to Viral Infections

ABSTRACT Autophagy is a programmed homeostatic response to diverse types of cellular stress that disposes of long-lived proteins, organelles, and invading microbes within double-membraned structures called autophagosomes. The 2′,5′-oligoadenylate/RNase L system is a virus-activated host RNase pathway that disposes of or processes viral and cellular single-stranded RNAs. Here we report that activation of RNase L during viral infections induces autophagy. Accordingly, infections with encephalomyocarditis virus or vesicular stomatitis virus led to higher levels of autophagy in wild-type mouse embryonic fibroblasts (MEF) than in RNase L-null MEF. Similarly, direct activation of RNase L with a 2′,5′-oligoadenylate resulted in p62(SQSTM1) degradation, LC3BI/LC3BII conversion, and appearance of autophagosomes. To determine the effect of RNase L-mediated autophagy on viral replication, we compared viral yields in wild-type and RNase L-null MEF in the absence or presence of either chemical inhibitors of autophagy (bafilomycin A1 or 3-methyladenine) or small interfering RNA (siRNA) against ATG5 or beclin-1. At a low multiplicity of infection, induction of autophagy by RNase L during the initial cycle of virus growth contributed to the suppression of virus replication. However, in subsequent rounds of infection, autophagy promoted viral replication, reducing the antiviral effect of RNase L. Our results indicate a novel function of RNase L as an inducer of autophagy that affects viral yields.

Cytosolic Double-Stranded RNA Activates the NLRP3 Inflammasome via MAVS-Induced Membrane Permeabilization and K+ Efflux

The nucleotide-binding oligomerization domain–like receptor pyrin domain–containing 3 (Nlrp3) inflammasome plays an important role in inflammation by controlling the maturation and secretion of the cytokines IL-1β and IL-18 in response to multiple stimuli including pore-forming toxins, particulate matter, and ATP. Although the pathways activated by the latter stimuli lead to a decrease in intracellular K+ concentration, which is required for inflammasome activation, the mechanism by which microbial RNA activates Nlrp3, remains poorly understood. In this study, we found that cytosolic poly(I:C), but not total RNA from healthy macrophages, macrophages undergoing pyroptosis, or mitochondrial RNA, induces caspase-1 activation and IL-1β release through the Nlrp3 inflammasome. Experiments with macrophages deficient in Tlr3, Myd88, or Trif, indicate that poly(I:C) induces Nlrp3 activation independently of TLR signaling. Further analyses revealed that the cytosolic sensors Rig-I and melanoma differentiation–associated gene 5 act redundantly via the common adaptor mitochondrial antiviral signaling (Mavs) to induce Nlrp3 activation in response to poly(I:C), but not ATP or nigericin. Mechanistically, Mavs triggered membrane permeabilization and K+ efflux independently of the inflammasome which were required for poly(I:C)-induced Nlrp3 activation. We conclude that poly (I:C) activates the inflammasome through an Mavs-dependent surveillance pathway that converges into a common K+ lowering step in the cytosol that is essential for the induction of Nlrp3 activation.

Protein Kinase R-dependent Regulation of Interleukin-10 in Response to Double-stranded RNA

The double-stranded RNA-activated protein kinase R (PKR) is an important component of antiviral defense. PKR participates in different signaling pathways in response to various stimuli to regulate translation via phosphorylation of the eukaryotic initiation factor 2α, and transcription via activating NF-κB and IRF-1, to induce pro-inflammatory cytokines. Here we show PKR regulates interleukin-10 induction in response to double-stranded RNA, bacterial lipopolysaccaride, and Sendai virus infection. Using chemical inhibitors, dominant negative constructs, and genetic knockouts, we demonstrate that the PKR-mediated interleukin-10 induction engages JNK and NF-κB. Together, our data demonstrate the role of PKR in regulating an anti-inflammatory cytokine. The findings have significance in antiviral as well as broader innate immune responses.

RNase L Targets Distinct Sites in Influenza A Virus RNAs

ABSTRACT Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8ΔNS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled. IMPORTANCE RNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease.

Cell-Type-Specific Effects of RNase L on Viral Induction of Beta Interferon

ABSTRACT The interferon (IFN)-inducible antiviral state is mediated in part by the 2′,5′-oligoadenylate (2-5A) synthetase (OAS)/RNase L system. 2-5A, produced from ATP by OAS proteins in response to viral double-stranded RNA, binds to and activates RNase L. RNase L restricts viral infections by degrading viral and cellular RNA, inducing autophagy and apoptosis, and producing RNA degradation products that amplify production of type I interferons (IFNs) through RIG-I-like receptors. However, the effects of the OAS/RNase L pathway on IFN induction in different cell types that vary in basal levels of these proteins have not been previously reported. Here we report higher basal expression of both RNase L and OAS in mouse macrophages in comparison to mouse embryonic fibroblasts (MEFs). In MEFs, RNase L gene knockout decreased induction of IFN-β by encephalomyocarditis virus infection or poly(rI):poly(rC) (pIC) transfection. In contrast, in macrophages, RNase L deletion increased (rather than decreased) induction of IFN-β by virus or pIC. RNA damage from RNase L in virus-infected macrophages is likely responsible for reducing IFN-β production. Similarly, direct activation of RNase L by transfection with 2-5A induced IFN-β in MEFs but not in macrophages. Also, viral infection or pIC transfection caused RNase L-dependent apoptosis of macrophages but not of MEFs. Our results suggest that cell-type-specific differences in basal levels of OAS and RNase L are determinants of IFN-β induction that could affect tissue protection and survival during viral infections. IMPORTANCE Type I interferons (IFNs) such as IFN-β are essential antiviral cytokines that are often required for animal survival following infections by highly pathogenic viruses. Therefore, host factors that regulate type I IFN production are critically important for animal and human health. Previously we reported that the OAS/RNase L pathway amplifies antiviral innate immunity by enhancing IFN-β production in mouse embryonic fibroblasts and in virus-infected mice. Here we report that high basal levels of OAS/RNase L in macrophages reduce, rather than increase, virus induction of IFN-β. RNA damage and apoptosis caused by RNase L were the likely reasons for the decreased IFN-β production in virus-infected macrophages. Our studies suggest that during viral infections, the OAS/RNase L pathway can either enhance or suppress IFN production, depending on the cell type. IFN regulation by RNase L is suggested to contribute to tissue protection and survival during viral infections. Type I interferons (IFNs) such as IFN-β are essential antiviral cytokines that are often required for animal survival following infections by highly pathogenic viruses. Therefore, host factors that regulate type I IFN production are critically important for animal and human health. Previously we reported that the OAS/RNase L pathway amplifies antiviral innate immunity by enhancing IFN-β production in mouse embryonic fibroblasts and in virus-infected mice. Here we report that high basal levels of OAS/RNase L in macrophages reduce, rather than increase, virus induction of IFN-β. RNA damage and apoptosis caused by RNase L were the likely reasons for the decreased IFN-β production in virus-infected macrophages. Our studies suggest that during viral infections, the OAS/RNase L pathway can either enhance or suppress IFN production, depending on the cell type. IFN regulation by RNase L is suggested to contribute to tissue protection and survival during viral infections.

RNase-L deficiency exacerbates experimental colitis and colitis-associated cancer.

Background:The endoribonuclease RNase-L is a type-I interferon (IFN)-regulated component of the innate immune response that functions in antiviral, antibacterial, and antiproliferative activities. RNase-L produces RNA agonists of RIG-I–like receptors, sensors of cytosolic pathogen-associated RNAs that induce cytokines including IFN-&bgr;. IFN-&bgr; and RIG-I–like receptors signaling mediate protective responses against experimental colitis and colitis-associated cancer and contribute to gastrointestinal homeostasis. Therefore, we investigated a role for RNase-L in murine colitis and colitis-associated cancer and its association with RIG-I–like receptors signaling in response to bacterial RNA. Methods:Colitis was induced in wild type–deficient and RNase-L–deficient mice (RNase-L−/−) by administration of dextran sulfate sodium (DSS). Colitis-associated cancer was induced by DSS and azoxymethane (AOM). Histological analysis and immunohistochemistry were performed on colon tissue to analyze immune cell infiltration and tissue damage after induction of colitis. Expression of cytokines was measured by quantitative real-time–PCR and ELISA. Results:DSS-treated RNase-L−/− mice exhibited a significantly higher clinical score, delayed leukocyte infiltration, reduced expression of IFN-&bgr;, tumor necrosis factor &agr;, interleukin-1&bgr;, and interleukin-18 at early times post-DSS exposure, and increased mortality as compared with wild-type mice. DSS/AOM-treated RNase-L−/− mice displayed an increased tumor burden. Bacterial RNA triggered IFN-&bgr; production in an RNase-L–dependent manner and provided a potential mechanism by which RNase-L contributes to the gastrointestinal immune response to microbiota and protects against experimental colitis and colitis-associated cancer. Conclusions:RNase-L promotes the innate immune response to intestinal damage and ameliorates murine colitis and colitis-associated cancer. The RNase-L–dependent production of IFN-&bgr; stimulated by bacterial RNA may be a mechanism to protect against gastrointestinal inflammatory disease.

Abstract 2863: RNase-L modulates the immune response to intestinal damage and ameliorates murine colitis and colitis-associated cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The endoribonuclease RNase-L is a Type-I interferon (IFN)-regulated component of the innate immune response that functions in antiviral and antibacterial activities. Here we identify a protective role for RNase-L in the inflammatory response to gastrointestinal (GI) injury and commensal bacteria. In a dextran sulphate sodium (DSS)-induced model of ulcerative colitis RNase-L-/- mice exhibited a more severe clinical score and increased mortality compared to wild type animals. The enhanced sensitivity to GI damage was accompanied by delayed leukocyte infiltration and reduced expression of the proinflammatory cytokines IFNβ, TNFα, IL-1β and IL-18 at early times post-DSS exposure. This impaired innate immune response corresponded with increased inflammation and a decreased capacity to recover from GI injury at later times. Consistent with the established role of inflammation as a risk factor for colorectal cancer, in a model of colitis-associated cancer DSS treated RNase-L-/- mice displayed an increased carcinogen-induced tumor burden and mortality. A potential mechanism by which RNase-L functions in the response to GI damage and commensal bacteria is via the production of RNA agonists of RIG-I-like receptors (RLRs), sensors of cytosolic pathogen-associated RNAs that initiate signaling to induce proinflammatory cytokines important to GI homeostasis. Indeed, we found that bacterial RNA triggers IFNβ induction in macrophages in an RNase-L-dependent manner. Together, these results support a model in which the protective role for RNase-L in experimental colitis and colitis-associated cancer occurs through RNase-L-dependent RLR signaling and IFNβ induction. Citation Format: Tiha M. Long, Arindam Chakrabarti, Heather Ezelle, Sarah Brennan-Laun, Jean-Pierre Raufman, Irina Polyakova, Robert Silverman, Bret Hassel. RNase-L modulates the immune response to intestinal damage and ameliorates murine colitis and colitis-associated cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2863. doi:10.1158/1538-7445.AM2013-2863