PS2-72 Extracellular 2′-5′ Oligoadenylate synthetase stimulates RNase L-independent antiviral activity: a novel mechanism of virus-induced innate immunity

The oligoadenylate synthetase–ribonuclease L (OAS–RNase L) system is a potent antiviral pathway that severely limits the pathogenesis of many viruses. Upon sensing dsRNA, OASs produce 2′,5′-oligoadenylates (2-5A) that activate RNase L to cleave both host and viral single-stranded RNA, thereby limiting protein production, virus replication and spread, leading to apoptotic cell death. Endogenous host dsRNA, which accumulates in the absence of adenosine deaminase acting on RNA (ADAR)1, can also activate RNase L and lead to apoptotic cell death. RNase L activation and antiviral activity during infections with several types of viruses in human and bat cells is dependent on OAS3 but independent of virus-induced interferon (IFN) and, thus, RNase L can be activated even in the presence of IFN antagonists. Differently from other human viruses examined, Zika virus is resistant to the antiviral activity of RNase L and instead utilizes RNase L to enhance its replication factories to produce more infectious virus. Some betacoronaviruses antagonize RNase L activation by expressing 2′,5′-phosphodiesterases (PDEs) that cleave 2-5A and thereby antagonize activation of RNase L. The best characterized of these PDEs is the murine coronavirus (MHV) NS2 accessory protein. Enzymatically active NS2 is required for replication in myeloid cells and in the liver. Interestingly, while wild type mice clear MHV from the liver by 7–10 days post-infection, RNase L knockout mice fail to effectively clear MHV, probably due to diminished apoptotic death of infected cells. We suggest that RNase L antiviral activity stems from direct cleavage of viral genomes and cessation of protein synthesis as well as through promoting death of infected cells, limiting the spread of virus. Importantly, OASs are pattern recognition receptors and the OAS–RNase L pathway is a primary innate response pathway to viruses, capable of early response, coming into play before IFN is induced or when the virus shuts down IFN signaling.

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Zika Virus Production Is Resistant to RNase L Antiviral Activity

Journal of Virology ◽

10.1128/jvi.00313-19 ◽

2019 ◽

Vol 93 (16) ◽

Cited By ~ 9

Author(s):

Jillian N. Whelan ◽

Yize Li ◽

Robert H. Silverman ◽

Susan R. Weiss

Keyword(s):

Antiviral Activity ◽

Zika Virus ◽

Viral Genome ◽

Molecular Mechanisms ◽

Virus Production ◽

Type I ◽

Rnase L ◽

Oligoadenylate Synthetase ◽

Zikv Infection ◽

Initial Report

SUMMARYThere is currently no knowledge of how the emerging human pathogen Zika virus (ZIKV) interacts with the antiviral endoribonuclease L (RNase L) pathway during infection. Since activation of RNase L during infection typically limits virus production dramatically, we used CRISPR-Cas9 gene editing technology to knockout (KO) targeted host genes involved in the RNase L pathway to evaluate the effects of RNase L on ZIKV infection in human A549 cells. RNase L was activated in response to ZIKV infection, which degraded ZIKV genomic RNA. Surprisingly, despite viral genome reduction, RNase L activity did not reduce ZIKV infectious titers. In contrast, both the flavivirus dengue virus and the alphavirus Sindbis virus replicated to significantly higher titers in RNase L KO cells compared to wild-type (WT) cells. Using MAVS/RNase L double KO cells, we demonstrated that the absence of increased ZIKV production in RNase L KO cells was not due to compensation by enhanced type I interferon transcripts to thus inhibit virus production. Finally, when synthetic double-stranded RNA was detected by OAS3 to induce RNase L antiviral activity prior to ZIKV infection, we observed reduced ZIKV replication factory formation, as well as a 42-fold reduction in virus yield in WT but not RNase L KO cells. This study proposes that ZIKV evades RNase L antiviral activity by generating a viral genome reservoir protected from RNase L cleavage during early infection, allowing for sufficient virus production before RNase L activation is detectable.IMPORTANCEWith the onset of the 2015 ZIKV outbreak, ZIKV pathogenesis has been of extreme global public health interest, and a better understanding of interactions with the host would provide insight into molecular mechanisms driving the severe neurological outcomes of ZIKV disease. Here is the initial report on the relationship between ZIKV and the host oligoadenylate synthetase-RNase L (OAS-RNase L) system, a potent antiviral pathway effective at restricting replication of diverse viruses. Our study elucidated a unique mechanism whereby ZIKV production is impervious to antiviral RNase L activity, through a mechanism of viral RNA protection that is not mimicked during infection with numerous other RNase L-activating viruses, thus identifying a distinct replication strategy potentially important for ZIKV pathogenesis.

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Extracellular 2′-5′ Oligoadenylate Synthetase Stimulates RNase L-Independent Antiviral Activity: a Novel Mechanism of Virus-Induced Innate Immunity

Journal of Virology ◽

10.1128/jvi.01003-10 ◽

2010 ◽

Vol 84 (22) ◽

pp. 11898-11904 ◽

Cited By ~ 49

Author(s):

Helle Kristiansen ◽

Christina A. Scherer ◽

Maralee McVean ◽

Shawn P. Iadonato ◽

Susanne Vends ◽

...

Keyword(s):

Antiviral Activity ◽

Antiviral Agent ◽

Rnase L ◽

Oligoadenylate Synthetase ◽

Antiviral Compound ◽

Clinical Prognosis ◽

Strong Antiviral Activity ◽

Infected Cells

ABSTRACT The 2′-5′ oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins. However, several studies demonstrate a correlation between the concentration of freely circulating OAS protein in sera from hepatitis C patients and their clinical prognosis. Here we demonstrate that extracellular OAS1 enters into cells and possesses a strong antiviral activity, both in vitro and in vivo, which is independent of RNase L. The OAS protein directly inhibits viral proliferation and does not require the activation of known antiviral signaling pathways. We propose that OAS produced by cells infected with viruses is released to the extracellular space, where it acts as a paracrine antiviral agent. Thus, the OAS protein represents the first direct antiviral compound released by virus-infected cells.

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RNase L Antiviral Activity Is Not a Critical Component of the Oas1b-Mediated Flavivirus Resistance Phenotype

Journal of Virology ◽

10.1128/jvi.00946-19 ◽

2019 ◽

Vol 93 (22) ◽

Author(s):

J. C. Madden ◽

Dan Cui ◽

M. A. Brinton

Keyword(s):

Antiviral Activity ◽

Full Length ◽

Viral Rna ◽

Type I ◽

Mrna Levels ◽

Rnase L ◽

Oligoadenylate Synthetase ◽

Resistant Mouse ◽

Resistance Phenotype

ABSTRACT In mice, resistance to central nervous system (CNS) disease induced by members of the genus Flavivirus is conferred by an allele of the 2′-5′ oligoadenylate synthetase 1b gene that encodes the inactive full-length protein (Oas1b-FL). The susceptibility allele encodes a C-terminally truncated protein (Oas1b-tr). We show that the efficiency of neuron infection in the brains of resistant and susceptible mice is similar after an intracranial inoculation of two flaviviruses, but amplification of viral proteins and double-stranded RNA (dsRNA) is inhibited in infected neurons in resistant mouse brains at later times. Active OAS proteins detect cytoplasmic dsRNA and synthesize short 2′-5′-linked oligoadenylates (2′-5′A) that interact with the latent endonuclease RNase L, causing it to dimerize and cleave single-stranded RNAs. To evaluate the contribution of RNase L to the resistance phenotype in vivo, we created a line of resistant RNase L−/− mice. Evidence of RNase L activation in infected RNase L+/+ mice was indicated by higher levels of viral RNA in the brains of infected RNase L−/− mice. Activation of type I interferon (IFN) signaling was detected in both resistant and susceptible brains, but Oas1a and Oas1b mRNA levels were lower in RNase L+/+ mice of both types, suggesting that activated RNase L also has a proflaviviral effect. Inhibition of virus replication was robust in resistant RNase L−/− mice, indicating that activated RNase L is not a critical factor in mediating this phenotype. IMPORTANCE The mouse genome encodes a family of Oas proteins that synthesize 2′-5′A in response to dsRNA. 2′-5′A activates the endonuclease RNase L to cleave single-stranded viral and cellular RNAs. The inactive, full-length Oas1b protein confers flavivirus-specific disease resistance. Although similar numbers of neurons were infected in resistant and susceptible brains after an intracranial virus infection, viral components amplified only in susceptible brains at later times. A line of resistant RNase L−/− mice was used to evaluate the contribution of RNase L to the resistance phenotype in vivo. Activation of RNase L antiviral activity by flavivirus infection was indicated by increased viral RNA levels in the brains of RNase L−/− mice. Oas1a and Oas1b mRNA levels were higher in infected RNase L−/− mice, indicating that activated RNase L also have a proflaviviral affect. However, the resistance phenotype was equally robust in RNase L−/− and RNase L+/+ mice.

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Activation of RNase L by Murine Coronavirus in Myeloid Cells Is Dependent on BasalOasGene Expression and Independent of Virus-Induced Interferon

Journal of Virology ◽

10.1128/jvi.03036-15 ◽

2016 ◽

Vol 90 (6) ◽

pp. 3160-3172 ◽

Cited By ~ 24

Author(s):

L. Dillon Birdwell ◽

Zachary B. Zalinger ◽

Yize Li ◽

Patrick W. Wright ◽

Ruth Elliott ◽

...

Keyword(s):

Gene Expression ◽

Antiviral Activity ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Myeloid Cells ◽

Type I ◽

Rnase L ◽

Oligoadenylate Synthetase ◽

Double Stranded Rna ◽

Murine Coronavirus

ABSTRACTThe oligoadenylate synthetase (OAS)-RNase L pathway is a potent interferon (IFN)-induced antiviral activity. Upon sensing double-stranded RNA, OAS produces 2′,5′-oligoadenylates (2-5A), which activate RNase L. Murine coronavirus (mouse hepatitis virus [MHV]) nonstructural protein 2 (ns2) is a 2′,5′-phosphodiesterase (PDE) that cleaves 2-5A, thereby antagonizing RNase L activation. PDE activity is required for robust replication in myeloid cells, as a mutant of MHV (ns2H126R) encoding an inactive PDE fails to antagonize RNase L activation and replicates poorly in bone marrow-derived macrophages (BMM), while ns2H126Rreplicates to high titer in several types of nonmyeloid cells, as well as in IFN receptor-deficient (Ifnar1−/−) BMM. We reported previously that myeloid cells express significantly higher basal levels of OAS transcripts than nonmyeloid cells. Here, we investigated the contributions ofOasgene expression, basal IFN signaling, and virus-induced IFN to RNase L activation. Infection with ns2H126Ractivated RNase L inIfih1−/−BMM to a similar extent as in wild-type (WT) BMM, despite the lack of IFN induction in the absence of MDA5 expression. However, ns2H126Rfailed to induce RNase L activation in BMM treated with IFNAR1-blocking antibody, as well as inIfnar1−/−BMM, both expressing low basal levels ofOasgenes. Thus, activation of RNase L does not require virus-induced IFN but rather correlates with adequate levels of basalOasgene expression, maintained by basal IFN signaling. Finally, overexpression of RNase L is not sufficient to compensate for inadequate basal OAS levels.IMPORTANCEThe oligoadenylate synthetase (OAS)-RNase L pathway is a potent antiviral activity. Activation of RNase L during murine coronavirus (mouse hepatitis virus [MHV]) infection of myeloid cells correlates with high basalOasgene expression and is independent of virus-induced interferon secretion. Thus, our data suggest that cells with high basalOasgene expression levels can activate RNase L and thereby inhibit virus replication early in infection upon exposure to viral double-stranded RNA (dsRNA) before the induction of interferon and prior to transcription of interferon-stimulated antiviral genes. These findings challenge the notion that activation of the OAS-RNase L pathway requires virus to induce type I IFN, which in turn upregulates OAS gene expression, as well as to provide dsRNA to activate OAS. Our data further suggest that myeloid cells may serve as sentinels to restrict viral replication, thus protecting other cell types from infection.

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MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells

10.1101/2021.12.20.473564 ◽

2021 ◽

Author(s):

Courtney Comar ◽

Clayton Otter ◽

Jessica Pfannenstiel ◽

Ethan Doerger ◽

David Renner ◽

...

Keyword(s):

Innate Immunity ◽

Innate Immune ◽

Genome Replication ◽

Accessory Proteins ◽

Rnase L ◽

Oligoadenylate Synthetase ◽

Protein Kinase R ◽

Recombinant Viruses ◽

Dsrna Binding Protein ◽

Immune Pathways

Middle East respiratory syndrome coronavirus (MERS CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be in part because MERS CoV is adept at antagonizing early innate immune pathways; these include interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase ribonuclease L (OAS/RNase L), all activated in response to viral double stranded (ds)RNA generated during genome replication. This is in contrast to SARS CoV 2, which we recently reported activates PKR and RNase L and to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of the dsRNA-induced innate immune pathways. This resulted in ten-fold attenuation of replication in human lung derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of WT MERS CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA induced innate immunity during MERS CoV infection in order to optimize viral replication.

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Faculty Opinions recommendation of Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon-induced antiviral activity.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718246713.793490405 ◽

2014 ◽

Author(s):

Bryan Williams

Keyword(s):

Antiviral Activity ◽

Rnase L ◽

Dimeric Structure

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Heterozygous OAS1 gain-of-function variants cause an autoinflammatory immunodeficiency

Science Immunology ◽

10.1126/sciimmunol.abf9564 ◽

2021 ◽

Vol 6 (60) ◽

pp. eabf9564

Author(s):

Thomas Magg ◽

Tsubasa Okano ◽

Lars M. Koenig ◽

Daniel F.R. Boehmer ◽

Samantha L. Schwartz ◽

...

Keyword(s):

B Cells ◽

Hematopoietic Cell Transplantation ◽

De Novo ◽

Dynamics Simulation ◽

Recurrent Fever ◽

Type I ◽

Rnase L ◽

Oligoadenylate Synthetase ◽

Gain Of Function ◽

Translational Arrest

Analysis of autoinflammatory and immunodeficiency disorders elucidates human immunity and fosters the development of targeted therapies. Oligoadenylate synthetase 1 is a type I interferon–induced, intracellular double-stranded RNA (dsRNA) sensor that generates 2′-5′-oligoadenylate to activate ribonuclease L (RNase L) as a means of antiviral defense. We identified four de novo heterozygous OAS1 gain-of-function variants in six patients with a polymorphic autoinflammatory immunodeficiency characterized by recurrent fever, dermatitis, inflammatory bowel disease, pulmonary alveolar proteinosis, and hypogammaglobulinemia. To establish causality, we applied genetic, molecular dynamics simulation, biochemical, and cellular functional analyses in heterologous, autologous, and inducible pluripotent stem cell–derived macrophages and/or monocytes and B cells. We found that upon interferon-induced expression, OAS1 variant proteins displayed dsRNA-independent activity, which resulted in RNase L–mediated RNA cleavage, transcriptomic alteration, translational arrest, and dysfunction and apoptosis of monocytes, macrophages, and B cells. RNase L inhibition with curcumin modulated and allogeneic hematopoietic cell transplantation cured the disorder. Together, these data suggest that human OAS1 is a regulator of interferon-induced hyperinflammatory monocyte, macrophage, and B cell pathophysiology.

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Introns encode dsRNAs undetected by RIG-I/MDA5/interferons and sensed via RNase L

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2102134118 ◽

2021 ◽

Vol 118 (46) ◽

pp. e2102134118

Author(s):

Alisha Chitrakar ◽

Kristina Solorio-Kirpichyan ◽

Eliza Prangley ◽

Sneha Rath ◽

Jin Du ◽

...

Keyword(s):

Innate Immunity ◽

Immune Responses ◽

Innate Immune ◽

Rna Decay ◽

Antiviral Defense ◽

Rnase L ◽

Innate Immune Responses ◽

Double Stranded Rna ◽

Turn On ◽

Nuclear Rna

Double-stranded RNA (dsRNA), a hallmark viral material that activates antiviral interferon (IFN) responses, can appear in human cells also in the absence of viruses. We identify phosphorothioate DNAs (PS DNAs) as triggers of such endogenous dsRNA (endo-dsRNA). PS DNAs inhibit decay of nuclear RNAs and induce endo-dsRNA via accumulation of high levels of intronic and intergenic inverted retroelements (IIIR). IIIRs activate endo-dsRNA responses distinct from antiviral defense programs. IIIRs do not turn on transcriptional RIG-I/MDA5/IFN signaling, but they trigger the dsRNA-sensing pathways of OAS3/RNase L and PKR. Thus, nuclear RNA decay and nuclear-cytosolic RNA sorting actively protect from these innate immune responses to self. Our data suggest that the OAS3/RNase L and PKR arms of innate immunity diverge from antiviral IFN responses and monitor nuclear RNA decay by sensing cytosolic escape of IIIRs. OAS3 provides a receptor for IIIRs, whereas RNase L cleaves IIIR-carrying introns and intergenic RNAs.

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