The interferon (IFN)-inducible 2 0 -5 0 -oligoadenylate synthetase (OAS)/RNase L pathway blocks infections by some types of viruses through cleavage of viral and cellular single-stranded RNA. Viruses induce type I IFNs that initiate signaling to the OAS genes. OAS proteins are pathogen recognition receptors for the viral pathogenassociated molecular pattern, double-stranded RNA. Double-stranded RNA activates OAS to produce p x 5 0 A(2 0 p5 0 A) n ; x ¼ 1-3; n > 2 (2-5A) from ATP. Upon binding 2-5A, RNase L is converted from an inactive monomer to a potently active dimeric endoribonuclease for single-stranded RNA. RNase L contains, from N-to Cterminus, a series of 9 ankyrin repeats, a linker, several protein kinase-like motifs, and a ribonuclease domain homologous to Ire1 (involved in the unfolded protein response). In the past few years, it has become increasingly apparent that RNase L and OAS contribute to innate immunity in many ways. For example, small RNA cleavage products produced by RNase L during viral infections can signal to the retinoic acid-inducible-I like receptors to amplify and perpetuate signaling to the IFN-b gene. In addition, RNase L is now implicated in protecting the central nervous system against viral-induced demyelination. A role in tumor suppression was inferred by mapping of the RNase L gene to the hereditary prostate cancer 1 (HPC1) gene, which in turn led to discovery of the xenotropic murine leukemia-related virus. A broader role in innate immunity is suggested by involvement of RNase L in cytokine induction and endosomal pathways that suppress bacterial infections. These newly described findings about RNase L could eventually provide the basis for developing broad-spectrum antimicrobial drugs.

SUMMARY The NLRP3 inflammasome assembles in response to danger signals, triggering self-cleavage of procaspase-1 and production of the proinflammatory cytokine IL-1β. Although virus infection activates the NLRP3 inflammasome, the underlying events remain incompletely understood. We report that virus activation of the NLRP3 inflammasome involves the 2′,5′-oligoadenylate (2-5A) synthetase (OAS)/RNase L system, a component of the interferon-induced antiviral response that senses double stranded RNA and activates endoribonuclease RNase L to cleave viral and cellular RNAs. The absence of RNase L reduces IL-1β production in influenza A virus-infected mice. RNA cleavage products generated by RNase L enhance IL-1β production but require the presence of 2′,3′-cyclic phosphorylated termini characteristic of RNase L activity. Additionally, these cleavage products stimulate NLRP3 complex formation with the DExD/H-box helicase, DHX33, and mitochondrial adapter protein, MAVS, which are each required for effective NLRP3 inflammasome activation. Thus, RNA cleavage events catalyzed by RNase L are required for optimal inflammasome activation during viral infections.

The 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 inintracellular K+concentration,which is required for inflammasome activation,the mechanism by whichmicrobial RNA activates Nlrp3, remains poorly understood. Here 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 orTrif, indicate that poly(I:C)induces Nlrp3 activation independently of TLR signaling. Further analyses revealed that the cytosolic sensorsRig-I andMda5act redundantly viathe common adaptor Mavsto induceNlrp3 activation in response topoly(I:C), but not ATP or Nigericin. Mechanistically, Mavstriggeredmembrane permeabilizationand K+ efflux independently of the inflammasome which were required for poly(I:C)-induced Nlrp3 activation. We conclude thatpoly (I:C)activates the inflammasome through an Mavs-dependent surveillance pathway that convergesinto a common K+ lowering step in the cytosol that is essential for the induction of Nlrp3 activation.

Influenza

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 A utophagy (or "self-eating") is an essential and ancient cellular process for degrading and recycling components of longlived proteins, organelles, and microbial invaders (7,22). These materials are sequestered in the cytoplasm within double-membrane vesicles termed autophagosomes that fuse with lysosomes, causing the contents to be degraded. While autophagy is often induced in response to viral infections, the impact of autophagy on pathogenesis and virus replication can be unpredictable. For instance, intracerebral injection of Sindbis virus in mice induced autophagy, causing degradation of viral proteins while promoting viral clearance and protecting against virus-mediated neuronal cell death (32). However, while animal survival was enhanced by autophagy there was no effect on virus replication. There is also a complex, reciprocal relationship between autophagy and the mammalian immune system (22). The fact that many viruses have evolved strategies for countering and/or subverting autophagy supports a role for autophagy in the host antiviral response (31, 43). As an example of cross talk between autophagy and the innate immune response, autophagy mediates cellular recognition of some single-strand RNA (ssRNA) viruses, leading to alpha interferon (IFN-␣) induction in plasmacytoid dendritic cells (20). In addition, downstream events in IFN-regulated pathways regulate the autophagocytic process itself. For instance, PKR is an IFNinducible serine/threonine protein kinase that inhibits translation by phosphorylating eIF2␣ and that promotes autophagocytic degradation of HSV-1 (37, 38). Effects of PKR on both translation and autophagy are blocked by the HSV-1 neurovirulence gene product ICP34.5, which redirects protein phosphatase 1␣ to dephosphorylate eIF2␣. Here we implicate a second IFN-regulated pathway, the 2=,5=-oligoadenylate (OAS)/RNase L system (3, 34), in the control of autophagy.OASs are IFN-inducible pathogen recognition receptors (PRR) that produce 2=,5=-linked oligonucleotides of the formula p x 5=A(2=p5=A) n (x ϭ 1 to 3; n Ն 2) (2-5A) from ATP in response to viral double-stranded RNA (dsRNA) (15). The ...