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Context: Toll-like receptor 4 (TLR4) activation contributes to obesity-associated insulin resistance in skeletal muscles (SM). TLR4 signaling involves two pathways: the myeloid differentiation primary response gene 88 (MyD88) leading to inflammatory cytokines production and the toll/interleukin-1 receptor domain-containing adapter-inducing interferon (IFN) I (TRIF)-dependent pathways leading to type 1 interferon (IFNI) and interferon stimulated genes (ISG) expression. The E3 ubiquitin ligase RNF41 allows the preferential activation of the TRIF-IFNI pathway; however, its role in insulin response has not been reported., Methods: We measured RNF41 level and IFNI pathway activation (ISG expression) in SM biopsies of obese insulin sensitive (OIS) and obese insulin resistant (OIR) women. Then we isolated and differentiated in myotubes, primary human SM cell progenitors from OIS and OIR SM biopsies. We modulated RNF41 and ISG expression in these myotubes and investigated their effects on insulin response., Results: RNF41 expression is down-regulated in vivo in OIR SM and myotubes compared to OIS SM and myotubes. TLR4 activation with palmitate induces TRIF-IFNI pathway and ISG in OIS myotubes but not in OIR myotubes. Inhibition of RNF41 expression with siRNF41 in OIS myotubes treated with palmitate attenuates insulin response, IFNI pathway activation and ISG induction, mimicking OIR phenotype. Further, overexpression of RNF41 in OIR myotubes increases insulin response and ISG expression. Exposure to IFNI or to its inducer polyinosinic-polycytidylic acid, restores ISG expression and insulin sensitivity in OIR myotubes and OIS myotubes transfected with siRNF41., Conclusion: Our results identify RNF41 as essential to IFNI pathway activation in order to maintain muscle insulin sensitivity during human obesity., (Copyright © 2018. Published by Elsevier Inc.)

Obesity is associated with chronic low-grade inflammation and oxidative stress that blunt insulin response in its target tissues, leading to insulin resistance (IR). IR is a characteristic feature of type 2 diabetes. Skeletal muscle is responsible for 75% of total insulin-dependent glucose uptake; consequently, skeletal muscle IR is considered to be the primary defect of systemic IR development. Interestingly, some obese people stay insulin-sensitive and metabolically healthy. With the aim of understanding this difference and identifying the mechanisms responsible for insulin sensitivity maintenance/IR development during obesity, we explored the role of the latent endoribonuclease (RNase L) in skeletal muscle cells. RNase L is a regulator of innate immunity, of double-stranded RNA sensors and of toll-like receptor (TLR) 4 signaling. It is regulated during inflammation by interferons and its activity is dependent on its binding to 2-5A, an oligoadenylate synthesized by oligoadenylate synthetases (OAS). Increased expression of RNase L or downregulation of its inhibitor (RLI) improved insulin response in mouse myogenic C2C12 cells and in primary human myotubes from normal-weight subjects treated with palmitate, a saturated free fatty acid (FFA) known to induce inflammation and oxidative stress via TLR4 activation. While RNase L and RLI levels remained unchanged, OAS level was decreased in primary myotubes from insulin-resistant obese subjects (OB-IR) compared with myotubes from insulin-sensitive obese subjects (OB-IS). TLR3 and mitochondrial manganese superoxide dismutase (MnSOD) were also underexpressed in OB-IR myotubes. Activation of RNase L by 2-5A transfection allowed to restore insulin response, OAS, MnSOD and TLR3 expression in OB-IR myotubes. Due to low expression of OAS, OB-IR myotubes present a defect in RNase L activation and TLR3 regulation. Consequently, MnSOD level is low and insulin sensitivity is reduced. These results support that RNase L activity limits FFA/obesity-induced impairment of insulin response in muscle cells via TLR3 and MnSOD expression.

Adipose tissue structure is altered during obesity, leading to deregulation of whole-body metabolism. Its function depends on its structure, in particular adipocytes number and differentiation stage. To better understand the mechanisms regulating adipogenesis, we have investigated the role of an endoribonuclease, endoribonuclease L (RNase L), using wild-type and RNase L-knockout mouse embryonic fibroblasts (RNase L(-/-)-MEFs). Here, we identify C/EBP homologous protein 10 (CHOP10), a dominant negative member of the CCAAT/enhancer-binding protein family, as a specific RNase L target. We show that RNase L is associated with CHOP10 mRNA and regulates its stability. CHOP10 expression is conserved in RNase L(-/-)-MEFs, maintaining preadipocyte state while impairing their terminal differentiation. RNase L(-/-)-MEFs have decreased lipids storage capacity, insulin sensitivity and glucose uptake. Expression of ectopic RNase L in RNase L(-/-)-MEFs triggers CHOP10 mRNA instability, allowing increased lipids storage, insulin response and glucose uptake. Similarly, downregulation of CHOP10 mRNA with CHOP10 siRNA in RNase L(-/-)-MEFs improves their differentiation in adipocyte. In vivo, aged RNase L(-)/(-) mice present an expanded adipose tissue, which, however, is unable to correctly store lipids, illustrated by ectopic lipids storage in the liver and in the kidney. These findings highlight RNase L as an essential regulator of adipogenesis via the regulation of CHOP10 mRNA.

We have coupled a spectrophotometer with a scanning near-field optical microscope to obtain, with a single scan, simultaneously scanning near-field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.

Skeletal muscle maintenance and repair involve several finely coordinated steps in which pluripotent stem cells are activated, proliferate, exit the cell cycle and differentiate. This process is accompanied by activation of hundreds of muscle-specific genes and repression of genes associated with cell proliferation or pluripotency. Mechanisms controlling myogenesis are precisely coordinated and regulated in time to allow the sequence of activation/inactivation of genes expression. Muscular differentiation is the result of the interplay between several processes such as transcriptional induction, transcriptional repression and mRNA stability. mRNA stability is now recognized as an essential mechanism of control of gene expression. For instance, we previously showed that the endoribonuclease L (RNase L) and its inhibitor (RLI) regulates MyoD mRNA stability and consequently muscle differentiation.We now performed global gene expression analysis by SAGE to identify genes that were down-regulated upon activation of RNase L in C2C12 myogenic cells, a model of satellite cells. We found that RNase L regulates mRNA stability of factors implicated in the control of pluripotency and cell differentiation. Moreover, inappropriate RNase L expression in C2C12 cells led to inhibition of myogenesis and differentiation into adipocytes even when cells were grown in conditions permissive for muscle differentiation. Conversely, over-expression of RLI allowed muscle differentiation of myogenic C2C12 cells even in non permissive conditions.These findings reveal the central role of RNase L and RLI in controlling gene expression and cell fate during myogenesis. Our data should provide valuable insights into the mechanisms that control muscle stem cell differentiation and into the mechanism of metaplasia observed in aging or muscular dystrophy where adipose infiltration of muscle occurs.

The 2-5A/RNase L pathway is one of the first cellular defences against viruses. RNase L is an unusual endoribonuclease which activity is strictly regulated by its binding to a small oligonucleotide, 2-5A. 2-5A itself is very unusual, consisting of a series of 5'- triphosphorylated oligoadenylates with 2'-5' bonds. But RNase L activity is not limited to viral RNA cleavage. RNase L plays a central role in innate immunity, apoptosis, cell growth and differentiation by regulating cellular RNA stability and expression. Default in its activity leads to increased susceptibility to virus infections and to tumor development. RNase L gene has been identified as HPC1 (Hereditary Prostate Cancer 1) gene. Study of RNase L variant R462Q in etiology of prostate cancer has led to the identification of the novel human retrovirus closely related to xenotropic murine leukemia viruses (MuLVs) and named XMRV.

Interferons (IFNs) inhibit the growth of many different cell types by altering the expression of specific genes. IFNs activities are partly mediated by the 2'-5' oligoadenylates-RNase L RNA decay pathway. RNase L is an endoribonuclease requiring activation by 2'-5' oligoadenylates to cleave single-stranded RNA. Here, we present evidence that degradation of mitochondrial mRNA by RNase L leads to cytochrome c release and caspase 3 activation during IFNalpha-induced apoptosis. We identify and characterize the mitochondrial translation initiation factor (IF2mt) as a new partner of RNase L. Moreover, we show that specific inhibition of mitochondrial translation with chloramphenicol inhibits the IFNalpha-induced degradation of mitochondrial mRNA by RNase L. Finally, we demonstrate that overexpression of IF2mt in human H9 cells stabilizes mitochondrial mRNA, inhibits apoptosis induced by IFNalpha and partially reverses IFNalpha-cell growth inhibition. On the basis of our results, we propose a model describing how RNase L regulates mitochondrial mRNA stability through its interaction with IF2mt.

The antiviral and antiproliferative effects of interferons are mediated in part by the 2'-5' oligoadenylate-RNase L RNA decay pathway. RNase L is an endoribonuclease that requires 2'-5' oligoadenylates to cleave single-stranded RNA. In this report we present evidence demonstrating a role for RNase L in translation. We identify and characterize the human translation termination factor eRF3/GSPT1 as an interacting partner of RNase L. We show that interaction of eRF3 with RNase L leads to both increased translation readthrough efficiency at premature termination codons and increased +1 frameshift efficiency at the antizyme +1 frameshift site. On the basis of our results, we present a model describing how RNase L is involved in regulating gene expression by modulating the translation termination process.

Interferon alpha (IFNalpha) belongs to a cytokine family that exhibits antiviral properties, immuno-modulating effects, and antiproliferative activity on normal and neoplasic cells in vitro and in vivo. IFNalpha exerts antitumor action by inducing direct cytotoxicity against tumor cells. This toxicity is at least partly due to induction of apoptosis. Although the molecular basis of the inhibition of cell growth by IFNalpha is only partially understood, there is a direct correlation between the sensitivity of cells to the antiproliferative action of IFNalpha and the down-regulation of their mitochondrial mRNAs. Here, we studied the role of the 2-5A/RNase L system and its inhibitor RLI in this regulation of the mitochondrial mRNAs by IFNalpha. We found that a fraction of cellular RNase L and RLI is localized in the mitochondria. Thus, we down-regulated RNase L activity in human H9 cells by stably transfecting (i) RNase L antisense cDNA or (ii) RLI sense cDNA constructions. In contrast to control cells, no post-transcriptional down-regulation of mitochondrial mRNAs and no cell growth inhibition were observed after IFNalpha treatment in these transfectants. These results demonstrate that IFNalpha exerts its antiproliferative effect on H9 cells at least in part via the degradation of mitochondrial mRNAs by RNase L.

The 2',5'-oligoadenylate (2-5A)/RNase L pathway is one of several enzymatic pathways induced by interferons (IFN). RNase L is a latent endoribonuclease that is activated on its binding by 2-5A and inhibited by the ribonuclease L inhibitor (RLI). We have shown previously by coimmunoprecipitation that RNase L may be associated with a 90-kDa RNA binding protein (RNABP), identified with a monoclonal antibody (mAb) raised against an RNase L complex purified under native conditions on 2-5A-sepharose. Here we confirm, by gel-filtration and pull-down analysis, the association of RNase L and RNABP, and we demonstrate that this association is significantly increased in the presence of 2-5A. Moreover, we found that RNABP protein levels decrease during terminal differentiation in various cell lines but do not vary during vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) infection or following IFN-alpha/beta treatment. In this latter case, although total cellular RNABP levels do not vary, the amount of RNABP found in the cytoplasm increases in comparison to that found in the nucleus, indicating a cytoplasmic localization of RNABP after IFN-alpha/beta treatment. Finally, we demonstrate the interaction between RNase L and RNABP in intact cells. Microinjection of an mAb against RNABP into HeLa cells inhibits RNase L antiviral activity and partially inhibits the IFN-alpha/beta-induced antiviral activity.

The 2'-5' oligoadenylate (2-5A)/RNase L pathway is one of the enzymatic pathways induced by interferon. RNase L is a latent endoribonuclease which is activated by 2-5A and inhibited by a specific protein known as RLI (RNase L inhibitor). This system has an important role in regulating viral infection. Additionally, variations in RNase L activity have been observed during cell growth and differentiation but the significance of the 2-5A/RNase L/RLI pathway in these latter processes is not known. To determine the roles of RNase L and RLI in muscle differentiation, C2 mouse myoblasts were transfected with sense and antisense RLI cDNA constructs. Importantly, the overexpression of RLI in C2 cells was associated with diminished RNase L activity, an increased level of MyoD mRNA, and accelerated kinetics of muscle differentiation. Inversely, transfection of the RLI antisense construct was associated with increased RNase L activity, a diminished level of MyoD mRNA, and delayed differentiation. In agreement with these data, MyoD mRNA levels were also decreased in C2 cells transfected with an inducible RNase L construct. The effect of RNase L activity on MyoD mRNA levels was relatively specific because expression of several other mRNAs was not altered in C2 transfectants. Therefore, RNase L is directly involved in myoblast differentiation, probably through its role in regulating MyoD stability. This is the first identification of a potential mRNA target for RNase L.

Purpose: Recent studies have revealed abnormalities in the ribonuclease L pathway in peripheral blood mononuclear cells of patients with the chronic fatigue syndrome. We conducted a blinded study to detect possible differences in the distribution of 2-5A binding proteins in the cells of patients with chronic fatigue syndrome and controls., Patients and Methods: We studied 57 patients with chronic fatigue syndrome and 53 control subjects (28 healthy subjects and 25 patients with depression or fibromyalgia). A radioactive probe was used to label 2-5A binding proteins in unfractionated peripheral blood mononuclear cell extracts and to compare their distribution in the three groups., Results: A 37 kDa 2-5A binding polypeptide was found in 50 (88%) of the 57 patients with chronic fatigue syndrome compared with 15 (28%) of the 53 controls (P < 0.01). When present, the amount of 37 kDa protein was very low in the control groups. When expressed as the ratio of the 37 kDa protein to the 80 kDa protein, 41 (72%) of the 57 patients with chronic fatigue syndrome had a ratio > 0.05, compared with 3 (11%) of the 28 healthy subjects and none of the patients with fibromyalgia or depression., Conclusion: The presence of a 37 kDa 2-5A binding protein in extracts of peripheral blood mononuclear cells may distinguish patients with chronic fatigue syndrome from healthy subjects and those suffering from other diseases.

The interferon-regulated 2-5A/RNase L pathway plays a major role in the antiviral and antiproliferative activities of these cytokines. Several viruses, however, have evolved strategies to escape the antiviral activity of the 2-5A/RNase L pathway. In this context, we have cloned a cDNA coding for the RNase L inhibitor (RLI), a protein that specifically inhibits RNase L and whose regulated expression in picornavirus-infected cells down regulates the activity of the 2-5A/RNase L pathway. We show here that RLI increases during the course of human immunodeficiency virus type 1 (HIV-1) infection, which may be related to the downregulation of RNase L activity that has been described to occur in HIV-infected cells. In order to establish a possible causal relationship between these observations, we have stably transfected H9 cells with RLI sense or antisense cDNA-expressing vectors. The overexpression of RLI causes a decrease in RNase L activity and a twofold enhancement of HIV production. This increase in HIV replication correlates with an increase in HIV RNA and proteins. In contrast, reduction of RLI levels in RLI antisense cDNA-expressing clones reverses the inhibition of RNase L activity associated with HIV multiplication and leads to a threefold decrease in the viral load. This anti-HIV activity correlated with a decrease in HIV RNA and proteins. These findings demonstrate that the level of RLI, via its modulation of RNase L activity, can severely impair HIV replication and suggest the involvement of RLI in the inhibition of the 2-5A/RNase L system observed during HIV infection.

The (2-5A)-RNase L pathway is an important component of interferon (IFN) action. Its central role in the antiviral effect of IFN against Picornaviridae has been clearly demonstrated. We have characterized and cloned a new component of this pathway, the RNase L inhibitor (RLI). RLI is a cellular protein whose mRNA is not regulated by IFN but is induced by viruses, such as encephalomyocarditis virus (EMCV). RLI inhibits RNase L during the time course of EMCV infection, and overexpression of RLI in HeLa cells partially reverses the antiviral action of IFN against EMCV. The replicative complexes of several viruses consist of double-stranded RNA structures. These dsRNAs could activate gene transcription as demonstrated for IFNs and could be responsible for RLI induction. We describe the increased expression of RLI mRNA and RLI protein induced by synthetic dsRNAs, such as poly(I):poly(C). This induction gives rise to an inhibition of the 2-5A-binding activity of RNase L. The inhibition of RNase L activity is transcient, probably due to the rapid turnover of RLI protein.

The interferon-(IFN)-inducible 2',5'-oligoadenylate (2-5A)/endoribonuclease L (RNase L) pathway plays a major role in the antiviral and antiproliferative effects of IFN. The 2-5A/RNase L pathway appears to be regulated by the cell-growth status or viral infection. Viruses, and picornaviruses in particular, have evolved strategies to escape the 2-5A/RNase L-pathway-associated antiviral activity. We have recently cloned a cDNA coding for RLI, a RNase-L-specific protein inhibitor. Its regulated expression by viral infection could provide a new strategy to modulate the 2-5A/RNase L pathway. Since RNase L had been shown to be down regulated upon encephalomyocarditis (EMCV) infection, we stably transfected HeLa cells with a RLI antisense cDNA expressing vector. Four independent clones named VAS1, VAS2, VAS3 and VAS4 and one clone transfected with the empty vector (VV) as control, were analyzed. The level of RLI was decreased by 20% for VAS1, 25% for VAS2, 75% for VAS3 and 50% for VAS4. The inactivation of RNase L observed during EMCV infection was decreased in these clones as compared to control HeLa cells. Here again the results vary between the four clones. The maximum inhibition of RNase L (90%) was observed in control cells and in VAS1 while 48% inhibition was observed in VAS4 and 25% in VAS3. The reversal in RNase L inhibition thus reflects closely the resulting RLI level, in keeping with a major role of RLI in EMCV-induced down regulation of 2-5A-binding activity of RNase L. Moreover, cells expressing a low level of RLI (VAS3 and VAS 4) are partially resistant to EMCV infection.

The 2-5A/RNase L system is one of the pathways induced by interferon (IFN). It plays a major role in the antiviral and antiproliferative activities of IFNs. Recently, we have shown that the activity of the RNase L could be inhibited by a proteic inhibitor, the RNase L Inhibitor (RLI). Human RLI (Hu-RLI) was cloned and characterized. We describe here the isolation and characterization of the cDNA encoding the murine RLI (Mu-RLI). Hu-RLI and Mu-RLI protein have 98% amino acid identity. Mu-RLI is functionally homologous to Hu-RLI, and all the structural features and amino acid sequence motifs of Hu-RLI are conserved in Mu-RLI. Moreover, reticulocyte lysate translated Mu-RLI protein is also able to inhibit 2-5A binding on 2-5A-dependent RNAse-L. Northern blot analysis revealed that Mu-RLI cDNA hybridizes with one mRNA of 3.5 kb except for the testis where two mRNA of 3.5 and 2.1 kb, respectively, are detected, suggesting a tissue-specific regulation.

The 2-5A/RNase L system is considered as a central pathway of interferon (IFN) action and could possibly play a more general physiological role as for instance in the regulation of RNA stability in mammalian cells. We describe here the expression cloning and initial characterization of RLI (for RNase L inhibitor), a new type of endoribonuclease inhibitor. RLI cDNA codes for a 68-kDa polypeptide whose expression is not regulated by IFN. Its expression in reticulocyte extracts antagonizes the 2-5A binding ability and the nuclease activity of endogenous RNase L or the cloned 2DR polypeptide. The inhibition requires the association of RLI with the nuclease and is dependent on the ratio between the two proteins. Likewise RLI is coimmunoprecipitated with the RNase L complex by a nuclease-specific antibody. RLI does not lead to 2-5A degradation or to irreversible modification of RNase L. The overexpression of RLI in stably transfected HeLa cells inhibits the antiviral activity of IFN on encephalomyocarditis virus but not on vesicular stomatitis virus. RLI therefore appears as the first described and potentially important mediator of the 2-5A/RNase L pathway.

The subunit composition of RNase L, a key enzyme in the interferon system, has been characterized. RNase L was purified from human Daudi cells on a column of 2-5A-Sepharose and used to immunize Balb/c mice. A specific monoclonal antibody which recognizes a protein of 80 kDa has been isolated. This protein has been characterized and shown to be an RNA-binding protein with nuclease activity which is associated with, but distinct from, the 80-kDa 2-5A-binding protein known previously as RNase L. It is therefore proposed that the 2-5A-dependent RNase L is a complex of two distinct subunits: an 80-kDa RNA-binding protein (i.e. the catalytic subunit) and an 80-kDa 2-5A-binding protein (i.e. the regulatory subunit).

A rapid and convenient new procedure for detecting RNase L activity following Western blot by renaturation of the enzyme on the nitrocellulose sheets is described. This method allows the simultaneous analysis of enzymatic activity (e.g., cleavage of poly(uridylic acid)-3'-[32P]pCp) and RNase L binding to radioactivE probes (e.g., 2-5A-3'-[32P]pCp) in the same sample. Unlike previously published methods, this procedure eliminates interference from proteases or other RNases during the analysis of RNase L activity. The detection of RNase(s) L is also affected by the presence of endogenous 2-5A, 2-5A derivatives, or other possible "inhibitors" in cell extracts; this Western blot assay allows of RNase(s) L to be detected independently of intracellular 2-5A or analogs. Differences between the procedures used so far and this Western blot technique can indeed be demonstrated. It is shown with this Western blot assay that although RNase L has been described as a protein of 185-200 kDa under nondenaturating conditions, its 80-kDa (and 40-kDa) component is able to bind 2-5A and to cleave poly(uridylic acid) in a 2-5A-dependent way, independently of other subunit(s) or cofactor(s).

RNase L activated by 2-5A (a series of 2'-5'-linked adenylic oligoribonucleotides) is a key enzyme of the interferon system. To study RNase L (endonuclease L) in intact cells independently of intracellular 2-5A and of its activity, we have developed polyclonal antibodies against RNase L. RNase L from mouse spleen was purified on a column of 2-5A-Sepharose and used to immunize rabbits in co-injection with polyadenylic-polyuridylic acid as adjuvant. Antibodies were purified by chromatography on Affi-Gel blue and 2-5A-Sepharose-immobilized RNase L. These polyclonal antibodies immunoprecipitate the 80- and 40-kDa forms of RNase L in mouse spleen. In Western blot, only the 80-kDa form of RNase L is recognized by these antibodies. These purified antibodies were used to localize RNase L in the cytoplasm of intact mouse NIH 3T3 cells by immunofluorescence. The cytoplasmic localization of RNase L was confirmed by its 2-5A binding activity after cellular fractionation.

Metabolically stable phosphorothioate tetramer analogues of (2'-5')(A)n with Rp and/or Sp chirality in the 2'-5'-phosphodiester linkages constitute a new class of antiviral agents since they mimic the effects of interferons. Three of the diastereomeric 5'-monophosphates (i.e., pRpRpRp, pSpRpRp, and pRpSpSp) bind to and activate RNase L from extracts of HeLa cells. However, the pSpSpSp (2'-5')-(A)4-phosphorothioate is unique in that it binds to, but cannot activate, RNase L to cleave rRNA. When microinjected into the cytoplasm of HeLa cells followed by virus infection, the pRpRpRp, pSpRpRp, and pRpSpSp (2'-5')(A)4-phosphorothioates demonstrate antiviral activity, as does (2'-5')(A)4ox-red, an active (2'-5')(A)n analogue. When microinjected simultaneously with (2'-5')(A)nox-red, an active the pSpSpSp (2'-5')(A)4-phosphorothioate inhibits activation of RNase L in HeLa cells, thereby blocking direct protection of vesicular stomatitis virus. The agonist and antagonist properties of pRpRpRp and pSpSpSp, respectively, are transient probably as a consequence of the hydrolysis of the 5'-monophosphate and formation of the less active (2'-5')(A)4-phosphorothioate cores. The possible use of these (2'-5')(A)4-phosphorothioates as tools for dissecting the biological significance of the (2'-5')(A)n system or in antiviral chemotherapy is discussed.

RNase L is considered as the major if not unique target of (2'-5')(A)n and therefore as an important intracellular mediator of interferon action. It behaves as an 185-kDa species in various cell extracts when analyzed by gel filtration. SDS/PAGE analysis of the polypeptides covalently labelled with a (2'-5')(A)4-3'-[32P]pCp probe reveals a single 80-kDa species, thus attesting a multimeric form of the 185-kDa protein. At variance with such data, mouse spleen extracts reveal an additional 40-kDa polypeptide with (2'-5')(A)n-dependent ribonucleolytic activity. This seemingly new form of RNase L migrates as a 40-kDa polypeptide when analyzed under native or denaturing conditions. It bears some structural similarity with the larger-molecular-mass RNase L as revealed by partial proteolysis. It is probably not generated through proteolytic degradation of the 185-kDa RNase L during extract preparation, although its physiological significance is unknown. Indeed various protease inhibitors do not significantly alter the ratio of 40-kDa and 185-kDa (2'-5')(A)n-dependent ribonucleases; moreover, the (2'-5')(A)n-binding capacity of the 40-kDa polypeptide is more stable than that of the 185-kDa one.

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)

Poly(rI).poly(rC) induces a lytic reaction in interferon-treated mouse LM cells. We have attempted to determine whether the intracellular penetration of poly(rI).poly(rC) is a prerequisite for cell lysis and to gain some insight into the pathway followed. We found that poly(rI).poly(rC) coupled to Sepharose beads was unable to generate lysis of interferon-treated cells whereas the cells underwent lysis after microinjection of poly(rI).poly(rC). Some inhibitors of endocytosis were found to inhibit the development of the lytic reaction. Lysosomotropic amines or a low temperature (19 degrees C) blocked endocytosis of poly(rI).poly(rC) but did not prevent its uptake. The internalization of poly(rI).poly(rC) was energy-dependent and was blocked when sodium azide and 2-deoxyglucose were added simultaneously. We conclude that poly(rI).poly(rC) is internalized and reaches an acidic compartment before triggering the lytic reaction in the cell.

This short review summarizes available evidence for (i) growth regulatory properties of exogenous as well as recently described autocrine IFNs, (ii) down-regulation of cellular oncogene expression with emphasis on c-myc and (iii) the possible involvement of the IFN-regulated 2-5A pathway at these levels. Initially described as a part of the IFN-induced antiviral mechanism, this double-stranded RNA-activated pathway leads to the preferential degradation of viral mRNAs in IFN-treated virus-infected cells probably through localized activation at the site of virus replication. Such mechanisms could be involved in the regulation of the stability of rapidly turning over mRNAs as for instance c-myc mRNA in IFN-treated cells. Whatever the elegance of the concept, however, experimental evidence is essentially circumstantial; tools developed in our group to strengthen the demonstration are briefly described.