RNase L: Effector Nuclease of an Activatable RNA Degradation System in Mammals

1997 ◽  
pp. 19-34 ◽  
Author(s):  
C. Bisbal
Keyword(s):  
Rna Degradation ◽  
Rnase L

scholarly journals CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Chao Sui ◽  
Dandan Jiang ◽  
Xiangju Wu ◽  
Xiaoyan Cong ◽  
Feng Li ◽  
...  
Keyword(s):  
Cell Line ◽  
Gene Editing ◽  
Rna Degradation ◽  
Type I ◽  
Rnase L ◽  
Agarose Gel Electrophoresis ◽  
Biological Functions ◽  
Wild Type ◽  
Induction Of Apoptosis

Ribonuclease L (RNase L) is an important antiviral endoribonuclease regulated by type I IFN. RNase L is activated by viral infection and dsRNA. Because the role of swine RNase L (sRNase L) is not fully understood, in this study, we generated a sRNase L knockout PK-15 (KO-PK) cell line through the CRISPR/Cas9 gene editing system to evaluate the function of sRNase L. After transfection with CRISPR-Cas9 followed by selection using puromycin, sRNase L knockout in PK-15 cells was further validated by agarose gel electrophoresis, DNA sequencing, and Western blotting. The sRNase L KO-PK cells failed to trigger RNA degradation and induced less apoptosis than the parental PK-15 cells after transfected with poly (I: C). Furthermore, the levels of ISGs mRNA in sRNase L KO-PK cells were higher than those in the parental PK-15 cells after treated with poly (I: C). Finally, both wild type and attenuated pseudorabies viruses (PRV) replicated more efficiently in sRNase L KO-PK cells than the parental PK-15 cells. Taken together, these findings suggest that sRNase L has multiple biological functions including cellular single-stranded RNA degradation, induction of apoptosis, downregulation of transcript levels of ISGs, and antiviral activity against PRV. The sRNase L KO-PK cell line will be a valuable tool for studying functions of sRNase L as well as for producing PRV attenuated vaccine.

scholarly journals RNase L Mediates the Antiviral Effect of Interferon through a Selective Reduction in Viral RNA during Encephalomyocarditis Virus Infection

1998 ◽  
Vol 72 (4) ◽  
pp. 2752-2759 ◽  
Author(s):  
Xiao-Ling Li ◽  
John A. Blackford ◽  
Bret A. Hassel
Keyword(s):  
Antiviral Activity ◽  
Selective Reduction ◽  
Antiviral Effect ◽  
Rna Degradation ◽  
Degradation Pathway ◽  
Encephalomyocarditis Virus ◽  
Viral Rna ◽  
Rnase L ◽  
Intact Cells ◽  
Cellular Mrnas

ABSTRACT The 2′,5′-oligoadenylate (2-5A) system is an RNA degradation pathway which plays an important role in the antipicornavirus effects of interferon (IFN). RNase L, the terminal component of the 2-5A system, is thought to mediate this antiviral activity through the degradation of viral RNA; however, the capacity of RNase L to selectively target viral RNA has not been carefully examined in intact cells. Therefore, the mechanism of RNase L-mediated antiviral activity was investigated following encephalomyocarditis virus (EMCV) infection of cell lines in which expression of transfected RNase L was induced or endogenous RNase L activity was inhibited. RNase L induction markedly enhanced the anti-EMCV activity of IFN via a reduction in EMCV RNA. Inhibition of endogenous RNase L activity inhibited this reduction in viral RNA. RNase L had no effect on IFN-mediated protection from vesicular stomatitis virus. RNase L induction reduced the rate of EMCV RNA synthesis, suggesting that RNase L may target viral RNAs involved in replication early in the virus life cycle. The RNase L-mediated reduction in viral RNA occurred in the absence of detectable effects on specific cellular mRNAs and without any global alteration in the cellular RNA profile. Extensive rRNA cleavage, indicative of high levels of 2-5A, was not observed in RNase L-induced, EMCV-infected cells; however, transfection of 2-5A into cells resulted in widespread degradation of cellular RNAs. These findings provide the first demonstration of the selective capacity of RNase L in intact cells and link this selective activity to cellular levels of 2-5A.

scholarly journals Association of Zinc Finger Antiviral Protein Binding to Viral Genomic RNA with Attenuation of Replication of Echovirus 7

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Niluka Goonawardane ◽  
Dung Nguyen ◽  
Peter Simmonds
Keyword(s):  
Zinc Finger ◽  
Specific Binding ◽  
Stress Granules ◽  
Rna Degradation ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Antiviral Protein ◽  
Rna Sequences ◽  
Cpg Dinucleotides ◽  
Cellular Compartments

ABSTRACT Previous studies have implicated both zinc finger antiviral protein (ZAP) and oligoadenylate synthetase 3 (OAS3)/RNase L in the attenuation of RNA viruses with elevated CpG and UpA dinucleotides. Mechanisms and interrelationships between these two pathways were investigated using an echovirus 7 (E7) replicon with compositionally modified sequences inserted into the 3′ untranslated region. ZAP and OAS3 immunoprecipitation (IP) assays provided complementary data on dinucleotide composition effects on binding. Elevated frequencies of alternative pyrimidine/purine (CpA and UpG) and reversed (GpC and ApU) dinucleotides showed no attenuating effect on replication or specific binding to ZAP by IP. However, the bases 3′ and 5′ of CpG motifs influenced replication and ZAP binding; UCGU enhanced CpG-mediated attenuation and ZAP binding, while A residues shielded CpGs from ZAP recognition. Attenuating effects of elevated frequencies of UpA on replication occurred independently of CpG dinucleotides and bound noncompetitively with CpG-enriched RNA, consistent with a separate recognition site from CpG. Remarkably, immunoprecipitation with OAS3 antibody reproduced the specific binding to CpG- and UpA-enriched RNA sequences. However, OAS3 and ZAP were coimmunoprecipitated in both ZAP and OAS3 IP and colocalized with E7 and stress granules (SGs) by confocal microscopy analysis of infected cells. ZAP’s association with larger cellular complexes may mediate the recruitment of OAS3/RNase L, KHNYN, and other RNA degradation pathways. IMPORTANCE We recently discovered that the OAS3/RNase L antiviral pathway is essential for restriction of CpG- and UpA-enriched viruses, in addition to the requirement for zinc finger antiviral protein (ZAP). The current study provides evidence for the specific dinucleotide and wider recognition contexts associated with virus recognition and attenuation. It further documents the association of ZAP and OAS3 and association with stress granules and a wider protein interactome that may mediate antiviral effects in different cellular compartments. The study provides a striking reconceptualization of the pathways associated with this aspect of antiviral defense.

scholarly journals A Study of the Interferon Antiviral Mechanism: Apoptosis Activation by the 2–5A System

1997 ◽  
Vol 186 (6) ◽  
pp. 967-972 ◽  
Author(s):  
JoAnn C. Castelli ◽  
Bret A. Hassel ◽  
Katherine A. Wood ◽  
Xiao-Ling Li ◽  
Kei Amemiya ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
Antiviral Effect ◽  
Inducible Promoter ◽  
Rna Degradation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Rnase L ◽  
Apoptosis Inhibition ◽  
Human Rnase ◽  
Induced Apoptosis

The 2–5A system contributes to the antiviral effect of interferons through the synthesis of 2–5A and its activation of the ribonuclease, RNase L. RNase L degrades viral and cellular RNA after activation by unique, 2′–5′ phosphodiester-linked, oligoadenylates [2–5A, (pp)p5′ A2′(P5′A2′)]n, n ⩾2. Because both the 2–5A system and apoptosis can serve as viral defense mechanisms and RNA degradation occurs during both processes, we investigated the potential role of RNase L in apoptosis. Overexpression of human RNase L by an inducible promoter in NIH3T3 fibroblasts decreased cell viability and triggered apoptosis. Activation of endogenous RNase L, specifically with 2–5A or with dsRNA, induced apoptosis. Inhibition of RNase L with a dominant negative mutant suppressed poly (I)·poly (C)–induced apoptosis in interferon-primed fibroblasts. Moreover, inhibition of RNase L suppressed apoptosis induced by poliovirus. Thus, increased RNase L levels induced apoptosis and inhibition of RNase L activity blocked viral-induced apoptosis. Apoptosis may be one of the antiviral mechanisms regulated by the 2–5A system.

scholarly journals Antiviral Actions of Interferons

2001 ◽  
Vol 14 (4) ◽  
pp. 778-809 ◽  
Author(s):  
Charles E. Samuel
Keyword(s):  
Signal Transduction ◽  
Translational Control ◽  
Cell Biology ◽  
Molecular Basis ◽  
Expression Profiles ◽  
Basic Research ◽  
Rna Degradation ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Host Interaction

SUMMARY Tremendous progress has been made in understanding the molecular basis of the antiviral actions of interferons (IFNs), as well as strategies evolved by viruses to antagonize the actions of IFNs. Furthermore, advances made while elucidating the IFN system have contributed significantly to our understanding in multiple areas of virology and molecular cell biology, ranging from pathways of signal transduction to the biochemical mechanisms of transcriptional and translational control to the molecular basis of viral pathogenesis. IFNs are approved therapeutics and have moved from the basic research laboratory to the clinic. Among the IFN-induced proteins important in the antiviral actions of IFNs are the RNA-dependent protein kinase (PKR), the 2′,5′-oligoadenylate synthetase (OAS) and RNase L, and the Mx protein GTPases. Double-stranded RNA plays a central role in modulating protein phosphorylation and RNA degradation catalyzed by the IFN-inducible PKR kinase and the 2′-5′-oligoadenylate-dependent RNase L, respectively, and also in RNA editing by the IFN-inducible RNA-specific adenosine deaminase (ADAR1). IFN also induces a form of inducible nitric oxide synthase (iNOS2) and the major histocompatibility complex class I and II proteins, all of which play important roles in immune response to infections. Several additional genes whose expression profiles are altered in response to IFN treatment and virus infection have been identified by microarray analyses. The availability of cDNA and genomic clones for many of the components of the IFN system, including IFN-α, IFN-β, and IFN-γ, their receptors, Jak and Stat and IRF signal transduction components, and proteins such as PKR, 2′,5′-OAS, Mx, and ADAR, whose expression is regulated by IFNs, has permitted the generation of mutant proteins, cells that overexpress different forms of the proteins, and animals in which their expression has been disrupted by targeted gene disruption. The use of these IFN system reagents, both in cell culture and in whole animals, continues to provide important contributions to our understanding of the virus-host interaction and cellular antiviral response.

scholarly journals Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and limits apoptosis in macrophages

2017 ◽  
Vol 114 (21) ◽  
pp. E4251-E4260 ◽  
Author(s):  
Xufang Deng ◽  
Matthew Hackbart ◽  
Robert C. Mettelman ◽  
Amornrat O’Brien ◽  
Anna M. Mielech ◽  
...  
Keyword(s):  
Mutant Mouse ◽  
Nonstructural Protein ◽  
Rna Degradation ◽  
Innate Immune ◽  
Mouse Bone Marrow ◽  
Type I ◽  
Rnase L ◽  
Double Stranded Rna ◽  
L System ◽  
Infected Cells

Coronaviruses are positive-sense RNA viruses that generate double-stranded RNA (dsRNA) intermediates during replication, yet evade detection by host innate immune sensors. Here we report that coronavirus nonstructural protein 15 (nsp15), an endoribonuclease, is required for evasion of dsRNA sensors. We evaluated two independent nsp15 mutant mouse coronaviruses, designated N15m1 and N15m3, and found that these viruses replicated poorly and induced rapid cell death in mouse bone marrow-derived macrophages. Infection of macrophages with N15m1, which expresses an unstable nsp15, or N15m3, which expresses a catalysis-deficient nsp15, activated MDA5, PKR, and the OAS/RNase L system, resulting in an early, robust induction of type I IFN, PKR-mediated apoptosis, and RNA degradation. Immunofluorescence imaging of nsp15 mutant virus-infected macrophages revealed significant dispersal of dsRNA early during infection, whereas in WT virus-infected cells, the majority of the dsRNA was associated with replication complexes. The loss of nsp15 activity also resulted in greatly attenuated disease in mice and stimulated a protective immune response. Taken together, our findings demonstrate that coronavirus nsp15 is critical for evasion of host dsRNA sensors in macrophages and reveal that modulating nsp15 stability and activity is a strategy for generating live-attenuated vaccines.

scholarly journals RNA is required for the maintenance of multiple cytoplasmic and nuclear membrane-less organelles.

2021 ◽  
Author(s):  
Carolyn J Decker ◽  
James M Burke ◽  
Patrick K Mulvaney ◽  
Roy Parker
Keyword(s):  
Stress Granules ◽  
Rna Degradation ◽  
Cajal Bodies ◽  
Rnase L ◽  
Nuclear Speckles ◽  
Processing Bodies ◽  
Rna Molecules ◽  
Super Enhancer ◽  
Rnp Granules ◽  
The Impact

Numerous membrane-less organelles composed of a combination of RNA and proteins, referred to as RNP granules, are observed in the nucleus and cytoplasm of eukaryotic cells, including stress granules, processing bodies, Cajal bodies, and nuclear speckles. An unresolved issue is how frequently RNA molecules are required for the maintenance of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. Strikingly, we find the majority of RNP granules, including stress granules, processing bodies, Cajal bodies, nuclear speckles and the nucleolus are altered by the degradation of their RNA components. In contrast, super-enhancer complexes and TIS granules were largely unaffected by widespread intracellular RNA degradation. This highlights a critical and widespread role of RNA in the organization of many, but not all, RNP granules.

RAR Gamma Activation Sensitizes Human Myeloma Cells to Carfilzomib Treatment through OAS-RNase L Innate Immune Pathway

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-37
Author(s):  
Qiang Wang ◽  
Zhijuan Lin ◽  
Qing Yi
Keyword(s):  
Retinoic Acid ◽  
Conflicts Of Interest ◽  
Rna Degradation ◽  
Gene Profiling ◽  
Therapeutic Effects ◽  
Rnase L ◽  
Atra Treatment ◽  
Induced Apoptosis ◽  
The Impact

Proteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz) kill MM cells by disrupting the degradation of misfolded proteins, presumably derived from high-level immunoglobulin production, which provides an appealing explanation for why MM cells are so uniquely sensitive to PIs. However, relapses are frequent and acquired resistance to PI treatment emerges in most patients. Therefore, identifying novel and safe drugs overcoming PI resistance in MM will aid in chemo-(re)sensitization, reducing PI-induced side effects, and maximizing the outcomes of PI therapy. Here we performed a high-throughput screen of 1855 FDA-approved drugs (Figure 1) and identified all-trans retinoic acid (ATRA), a classic pan-retinoic acid receptor (RAR) agonist used successfully to treat acute promyelocytic leukemia (APL), as a potent drug that enhanced MM sensitivity to Cfz-induced cytotoxicity and re-sensitized Cfz-resistant MM cells to Cfz in vitro. To determine which RARs are important for ATRA enhancement of Cfz-induced apoptosis in MM, esiRNAs of RARs for knocking down RARs and selective agonists of RARs were used in Cfz-treated MM cells. We identified that RARγ activation is important for ATRA sensitizing MM cells to Cfz treatment. To determine which signaling pathways are involved in ATRA-treated MM cells, gene-profiling data analysis of Cfz- versus ATRA+Cfz-treated MM cells was performed and real-time PCR was used to validate the microarray results. We found that ATRA treatment activated IFN-β response pathway (Figure 2), leading to upregulated expression of IRF1 and OAS1-3. Interestingly, similar to ATRA, IFN-β, which alone did not induce MM apoptosis, enhanced Cfz-induced MM cell apoptosis. Furthermore, using RNA integrity assay and dsRNA detection assay, we identified that ATRA treatment elevated the expression of OASs, which synthesized 2-5A upon binding to dsRNA induced by Cfz and resulted in cellular RNA degradation by RNase L and cell death (Figure 3). By knocking down each gene of OAS1-3, we demonstrated that OAS1 is essential for ATRA to sensitize MM cells to Cfz-induced apoptosis. Furthermore, we determined the impact and significance of RARγ and OAS1 in human MM pathogenesis and drug response by analyzing the gene-profiling data of 264 MM patients from Mulligan et al. datasets and 1,143 MM patients from MMRF coMMpass study IA13. In support of these findings, analyses of the large patient's gene-profiling datasets showed a strong and positive correlation between RARγ and OAS1 expression and patient's response to PI treatment (Figure 4). Finally, BMS961, a selective RARγ agonist, similar to ATRA, could also (re)sensitize MM cells to Cfz in vitro, and both ATRA and BMS961 significantly enhanced the therapeutic effects of Cfz in established MM in vivo (Figure 5). Thus, this study highlights the potential for RARγ agonists to sensitize MM and overcome MM resistance to Cfz treatment in patients. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document