2Ј,5Ј-Oligoadenylate-dependent RNase L functions in the interferon-inducible, RNA decay pathway known as the 2-5A system. To determine the physiological roles of the 2-5A system, mice were generated with a targeted disruption of the RNase L gene. The antiviral effect of interferon α was impaired in RNase L -/-mice providing the first evidence that the 2-5A system functions as an antiviral pathway in animals. In addition, remarkably enlarged thymuses in the RNase L -/-mice resulted from a suppression of apoptosis. There was a 2-fold decrease in apoptosis in vivo in the thymuses and spleens of RNase L -/-mice. Furthermore, apoptosis was substantially suppressed in RNase L -/-thymocytes and fibroblasts treated with different apoptotic agents. These results suggest that both interferon action and apoptosis can be controlled at the level of RNA stability by RNase L. Another implication is that the 2-5A system is likely to contribute to the antiviral activity of interferon by inducing apoptosis of infected cells.

Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH 2 -terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i.e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L-and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections. Double-stranded RNA (dsRNA) produced during viral infections triggers stress response pathways that lead to elimination of infected cells by apoptosis. Two complementary but independent cellular dsRNA-detecting systems have been implicated in the translational inhibition in response to viral infection: the 2-5A system and the dsRNA-activated protein kinase (PKR) (for a recent review, see reference 55). The 2-5A system is composed of a family of dsRNA-dependent enzymes known as 2Ј-5Ј oligoadenylate synthetases (OAS) (5) and the dormant cytosolic RNase L (64) (for recent reviews on the 2-5A system and RNase L, see references 45 and 52, respectively). Upon dsRNA binding, OAS produce unusual second messengers, short 2Ј-5Ј-linked oligoadenylates (2-5A) (32), which, in turn, specifically bind to and activate RNase L (64). Activated RNase L cleaves diverse RNA substrates...

Antiviral proteins encoded by the interferon (IFN)-stimulated genes provide a front-line defense against viral infections. In particular, PKR, RNase L, and Mx are considered to be the principal proteins through which IFNs mount an antiviral state. To determine whether alternative antiviral pathways exist, RNase L-/- mice and PKR-/- mice were crossed onto an Mx1(-/-) background to generate a strain of triply deficient (TD) mice. After infections with encephalomyocarditis virus, the TD mice died 3-4 days earlier than infected, wild-type mice. However, there was an IFN dose-dependent increase in survival times after encephalomyocarditis virus infections for both the TD and wild-type mice. Mice that were deficient for PKR or RNase L showed intermediate survival times between those of the TD and wild-type mice. Surprisingly, cultured embryonic fibroblasts lacking RNase L, PKR, or both proteins were still able to mount a substantial residual antiviral response against encephalomyocarditis virus or vesicular stomatitis virus after IFN-alpha treatments. These results confirm the antiviral functions of RNase L and PKR in vivo but also provide unequivocal evidence for the existence of novel, innate immune pathways against viruses.

Alleles at the Flv locus determine disease outcome after a flavivirus infection in mice. Although comparable numbers of congenic resistant and susceptible mouse embryo fibroblasts (MEFs) are infected by the flavivirus West Nile virus (WNV), resistant MEFs produce ϳ100-to 150-fold lower titers than susceptible ones and flavivirus titers in the brains of resistant and susceptible animals can differ by >10,000-fold. The Flv locus was previously identified as the 2-5 oligoadenylate synthetase 1b (Oas1b) gene. Oas gene expression is upregulated by interferon (IFN), and after activation by double-stranded RNA, some mouse synthetases produce 2-5A, which activates latent RNase L to degrade viral and cellular RNAs. To determine whether the lower levels of intracellular flavivirus genomic RNA from resistant mice detected in cells at all times after infection were mediated by RNase L, RNase L activity levels in congenic resistant and susceptible cells were compared. Similar moderate levels of RNase L activation by transfected 2-5A were observed in both types of uninfected cells. After WNV infection, the mRNAs of IFN-␤ and three Oas genes were up-regulated to similar levels in both types of cells. However, significant levels of RNase L activity were not detected until 72 h after WNV infection and the patterns of viral RNA cleavage products generated were similar in both types of cells. When RNase L activity was down-regulated in resistant cells via stable expression of a dominant negative RNase L mutant, ϳ5-to 10-times-higher yields of WNV were produced. Similarly, about ϳ5-to 10-times-higher virus yields were produced by susceptible C57BL/6 RNase L Variation in susceptibility to flavivirus-induced disease among mice was first observed in the 1920s and subsequently shown to be controlled by a single locus (56,72). This virus-specific resistance is dominant. Studies with the C3H.PRI-Flv r and C3H/HeJ congenic pair of mouse strains (26) showed that although resistant mice support the replication of flaviviruses, virus titers in their tissues are significantly lower and the spread of infection is slower compared to that in susceptible mice (4,24,32,65). Cell cultures prepared from various tissues obtained from resistant mice also produce lower yields of flaviviruses than do comparable cell cultures from susceptible mice (13; reviewed in reference 8). Coinheritance of the Flv alleles with those of the Ric locus on chromosome 5 identified the chromosomal location of the Flv locus (36). The Flv locus was then mapped on mouse chromosome 5 by linkage analysis first with known flanking genes (61) and then with microsatellite markers (67). The Oas1b gene was identified as the Flv locus by a positional cloning strategy (51). Mashimo et al. (45) confirmed the identification of this gene. The transcript of the Oas1b allele in susceptible mice contains a premature stop codon and encodes a truncated protein. The mechanism(s) through which the products of the different Oas1b alleles confer differential susceptibility to flavivirus-induced ...

Apoptosis of viral infected cells appears to be one defense strategy to limit viral infection. Interferon can also confer viral resistance by the induction of the 2-5A system comprised of 2'-5' oligoadenylate synthetase (OAS), and RNase L. Since rRNA is degraded upon activation of RNase L and during apoptosis and since both of these processes serve antiviral functions, we examined the role RNase L may play in cell death. Inhibition of RNase L activity, by transfection with a dominant negative mutant, blocked staurosporine-induced apoptosis of NIH3T3 cells and SV40-transformed BALB/c cells. In addition, K562 cell lines expressing inactive RNase L were more resistant to apoptosis induced by decreased glutathione levels. Hydrogen peroxide-induced death of NIH3T3 cells did not occur by apoptosis and was not dependent upon active RNAse L. Apoptosis regulatory proteins of the Bcl-2 family did not exhibit altered expression levels in the absence of RNase L activity. RNase L is required for certain pathways of cell death and may help mediate viral-induced apoptosis.

RNase L is an antiviral endoribonuclease that cleaves viral mRNAs after single-stranded UA and UU dinucleotides. Poliovirus (PV) mRNA is surprisingly resistant to cleavage by RNase L due to an RNA structure in the 3C Pro open reading frame (ORF). The RNA structure associated with the inhibition of RNase L is phylogenetically conserved in group C enteroviruses, including PV type 1 (PV1), PV2, PV3, coxsackie A virus 11 (CAV11), CAV13, CAV17, CAV20, CAV21, and CAV24. The RNA structure is not present in other human enteroviruses (group A, B, or D enteroviruses). Coxsackievirus B3 mRNA and hepatitis C virus mRNA were fully sensitive to cleavage by RNase L. HeLa cells expressing either wild-type RNase L or a dominant-negative mutant RNase L were used to examine the effects of RNase L on PV replication. PV replication was not inhibited by RNase L activity, but rRNA cleavage characteristic of RNase L activity was detected late during the course of PV infection, after assembly of intracellular virus. Rather than inhibiting PV replication, RNase L activity was associated with larger plaques and better cell-to-cell spread. Mutations in the RNA structure associated with the inhibition of RNase L did not affect the magnitude of PV replication in HeLa cells expressing RNase L, consistent with the absence of observed RNase L activity until after virus assembly. Thus, PV carries an RNA structure in the 3C protease ORF that potently inhibits the endonuclease activity of RNase L, but this RNA structure does not prevent RNase L activity late during the course of infection, as virus assembly nears completion.RNase L is a latent endoribonuclease in an interferon-regulated and double-stranded RNA (dsRNA)-activated antiviral pathway (reviewed in reference 47). Although RNase L is expressed in most human cells, it becomes active only after viral dsRNA accumulates and provokes the synthesis of 2Ј-5Ј oligoadenylate (2-5A) by 2Ј-5Ј oligoadenylate synthetases (2-5 OAS) (18, 64). 2-5A binds to ankyrin repeats within the N terminus of monomeric RNase L, provoking conformational changes which lead to RNase L dimerization and activation of endoribonuclease activity (20,21,57). The endoribonuclease of RNase L cleaves RNAs predominantly after single-stranded UA and UU dinucleotides (22,62). Viral mRNAs like that of hepatitis C virus (HCV) are exquisitely sensitive to cleavage by RNase L in vitro (26,27). Reduced frequencies of UA and UU dinucleotides within the open reading frames (ORFs) of HCV mRNAs are consistent with the selective pressure of RNase L. Activated RNase L also cleaves cellular RNAs, including rRNA (61). rRNA cleavage characteristic of activated RNase L is associated with the synthesis and accumulation of viral dsRNA during the course of infections (6,16,52,55).RNase L is thought to manifest antiviral activity via two independent mechanisms, namely, by cleaving viral RNA and by promoting apoptosis (65). Poliovirus (PV) infection activates apoptotic pathways; however, PV also delays apoptotic death to accommodate the time needed f...

Virus replication in higher vertebrates is restrained by IFNs that cause cells to transcribe genes encoding antiviral proteins, such as 2 -5 oligoadenylate synthetases. 2 -5 oligoadenylate synthetase is stimulated by dsRNA to produce 5 -phosphorylated, 2 -5 -linked oligoadenylates (2-5A), whose function is to activate RNase L. Although RNase L is required for a complete IFN antiviral response and mutations in the RNase L gene (RNASEL or HPC1) increase prostate cancer rates, it is unknown how 2-5A affects these biological endpoints through its receptor, RNase L. Presently, we show that 2-5A activation of RNase L produces a remarkable stimulation of transcription (>20-fold) for genes that suppress virus replication and prostate cancer. Unexpectedly, exposure of DU145 prostate cancer cells to physiologic levels of 2-5A (0.1 M) induced approximately twice as many RNA species as it downregulated. Among the 2-5A-induced genes are several IFN-stimulated genes, including IFN-inducible transcript 1͞P56, IFN-inducible transcript 2͞P54, IL-8, and IFN-stimulated gene 15. 2-5A also potently elevated RNA for macrophage inhibitory cytokine-1͞non-steroidal antiinflammatory drug-activated gene-1, a TGF-␤ superfamily member implicated as an apoptotic suppressor of prostate cancer. Transcriptional signaling to the macrophage inhibitory cytokine-1͞nonsteroidal antiinflammatory drug-activated gene-1 promoter by 2-5A was deficient in HeLa cells expressing a nucleasedead mutant of RNase L and was dependent on the mitogenactivated protein kinases c-Jun N-terminal kinase and extracellular signal-regulated kinase, both of which were activated in response to 2-5A treatments. Because 2-5A and RNase L participate in defenses against viral infections and prostate cancer, our findings have implications for basic cellular mechanisms that control major pathogenic processes.prostate ͉ macrophage inhibotory cytokine 1 ͉ HPC1 ͉ RNASEL

We have used a behavioral genetic approach to identify six X-linked loci which specify olfaction in Drosophila melanogaster. Mutations in five of these genes lead to partial anosmias affecting responses either to aldehydes (olfA, olfB, olfE and olfF) or to acetate esters (olfC). Only one of the mutants obtained in our screening (olfD) resulted in a insensitivity to several different odorants. olfA, olfE and olfC map close together in a small region of the chromosome between 7C and 7D. The alleles at the olfC locus fall into two phenotypic classes according to their responses to different acetate esters. The two groups of olfC alleles interact in-trans.