scholarly journals Terminal uridylyltransferases target RNA viruses as part of the innate immune system in animals

2017 ◽  
Author(s):  
Jérémie Le Pen ◽  
Hongbing Jiang ◽  
Tomás Di Domenico ◽  
Emma Kneuss ◽  
Joanna Kosałka ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Influenza A ◽  
Large Scale ◽  
Defense Mechanism ◽  
Rna Viruses ◽  
Interferon Response ◽  
Rnai Pathway ◽  
Antiviral Defense ◽  
C Elegans ◽  
Protein Levels

RNA viruses are a major threat to animals and plants. RNA interference (RNAi) and the interferon response provide innate antiviral defense against RNA viruses. Here we performed a large-scale screen using C. elegans and its natural pathogen, the Orsay virus (OrV), and identified cde-1 as important for antiviral defense. CDE-1 is a homologue of the mammalian TUT4/7 terminal uridylyltransferases; its catalytic activity is required for its antiviral function. CDE-1 uridylates the 3′ end of the OrV RNA genome and promotes its degradation, independently of the RNAi pathway. Likewise, TUT4/7 uridylate influenza A virus (IAV) mRNAs in mammalian cells. Deletion of TUT4/7 leads to increased IAV mRNA and protein levels. We have defined 3′ terminal uridylation of viral RNAs as a conserved antiviral defense mechanism.

scholarly journals Deletion of Cytoplasmic Double-Stranded RNA Sensors Does Not Uncover Viral Small Interfering RNA Production in Human Cells

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Susan Schuster ◽  
Lotte E. Tholen ◽  
Gijs J. Overheul ◽  
Frank J. M. van Kuppeveld ◽  
Ronald P. van Rij
Keyword(s):  
Rna Interference ◽  
Antiviral Activity ◽  
Mammalian Cells ◽  
Rna Viruses ◽  
Antiviral Immunity ◽  
Antiviral Effect ◽  
Interferon Response ◽  
Rnai Pathway ◽  
Differentiated Cells ◽  
Positive Sense

ABSTRACT The contribution of the RNA interference (RNAi) pathway in antiviral immunity in vertebrates has been widely debated. It has been proposed that RNAi possesses antiviral activity in mammalian systems but that its antiviral effect is masked by the potent antiviral interferon response in differentiated mammalian cells. In this study, we show that inactivation of the interferon response is not sufficient to uncover antiviral activity of RNAi in human epithelial cells infected with three wild-type positive-sense RNA viruses. Antiviral immunity in insects and plants is mediated by the RNA interference (RNAi) pathway in which viral long double-stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer enzymes. Although this pathway is evolutionarily conserved, its involvement in antiviral defense in mammals is the subject of debate. In vertebrates, recognition of viral RNA induces a sophisticated type I interferon (IFN)-based immune response, and it has been proposed that this response masks or inhibits antiviral RNAi. To test this hypothesis, we analyzed viral small RNA production in differentiated cells deficient in the cytoplasmic RNA sensors RIG-I and MDA5. We did not detect 22-nucleotide (nt) viral siRNAs upon infection with three different positive-sense RNA viruses. Our data suggest that the depletion of cytoplasmic RIG-I-like sensors is not sufficient to uncover viral siRNAs in differentiated cells. IMPORTANCE The contribution of the RNA interference (RNAi) pathway in antiviral immunity in vertebrates has been widely debated. It has been proposed that RNAi possesses antiviral activity in mammalian systems but that its antiviral effect is masked by the potent antiviral interferon response in differentiated mammalian cells. In this study, we show that inactivation of the interferon response is not sufficient to uncover antiviral activity of RNAi in human epithelial cells infected with three wild-type positive-sense RNA viruses.

scholarly journals Dicer is involved in protection against influenza A virus infection

2007 ◽  
Vol 88 (10) ◽  
pp. 2627-2635 ◽  
Author(s):  
Alexey A. Matskevich ◽  
Karin Moelling
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Virus Production ◽  
Vero Cells ◽  
Antiviral Response ◽  
Protein Levels ◽  
Infected Cells ◽  
Influenza A Virus Infection

In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the involvement of RNA interference (RNAi) in antiviral response against RNA viruses is uncertain. Here, we tested whether RNAi is involved in the antiviral response in mammalian cells. To investigate the role of RNAi in influenza A virus-infected cells in the absence of IFN, we used Vero cells that lack IFN-α and IFN-β genes. Our results demonstrate that knockdown of a key RNAi component, Dicer, led to a modest increase of virus production and accelerated apoptosis of influenza A virus-infected cells. These effects were much weaker in the presence of IFN. The results also show that in both Vero cells and the IFN-producing alveolar epithelial A549 cell line influenza A virus targets Dicer at mRNA and protein levels. Thus, RNAi is involved in antiviral response, and Dicer is important for protection against influenza A virus infection.

scholarly journals Caenorhabditis elegans Deficient in DOT-1.1 Exhibit Increases in H3K9me2 at Enhancer and Certain RNAi-Regulated Regions

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1846 ◽  
Author(s):  
Ruben Esse ◽  
Alla Grishok
Keyword(s):  
Small Rnas ◽  
Mammalian Cells ◽  
Leukemic Cells ◽  
Rnai Pathway ◽  
Open Chromatin ◽  
Loss Of Function ◽  
Promoter Regions ◽  
Heterochromatin Formation ◽  
C Elegans ◽  
H3k79 Methylation

The methylation of histone H3 at lysine 79 is a feature of open chromatin. It is deposited by the conserved histone methyltransferase DOT1. Recently, DOT1 localization and H3K79 methylation (H3K79me) have been correlated with enhancers in C. elegans and mammalian cells. Since earlier research implicated H3K79me in preventing heterochromatin formation both in yeast and leukemic cells, we sought to inquire whether a H3K79me deficiency would lead to higher levels of heterochromatic histone modifications, specifically H3K9me2, at developmental enhancers in C. elegans. Therefore, we used H3K9me2 ChIP-seq to compare its abundance in control and dot-1.1 loss-of-function mutant worms, as well as in rde-4; dot-1.1 and rde-1; dot-1.1 double mutants. The rde-1 and rde-4 genes are components of the RNAi pathway in C. elegans, and RNAi is known to initiate H3K9 methylation in many organisms, including C. elegans. We have previously shown that dot-1.1(−) lethality is rescued by rde-1 and rde-4 loss-of-function. Here we found that H3K9me2 was elevated in enhancer, but not promoter, regions bound by the DOT-1.1/ZFP-1 complex in dot-1.1(−) worms. We also found increased H3K9me2 at genes targeted by the ALG-3/4-dependent small RNAs and repeat regions. Our results suggest that ectopic H3K9me2 in dot-1.1(−) could, in some cases, be induced by small RNAs.

scholarly journals Homologous recombination is an intrinsic defense against antiviral RNA interference

2018 ◽  
Vol 115 (39) ◽  
pp. E9211-E9219 ◽  
Author(s):  
Lauren C. Aguado ◽  
Tristan X. Jordan ◽  
Emily Hsieh ◽  
Daniel Blanco-Melo ◽  
John Heard ◽  
...  
Keyword(s):  
Rna Interference ◽  
Homologous Recombination ◽  
Virus Replication ◽  
Defense Mechanism ◽  
Selective Pressure ◽  
Rna Viruses ◽  
Antiviral Defense ◽  
Dynamic Relationship ◽  
Pathogen Prevalence ◽  
Genomic Material

RNA interference (RNAi) is the major antiviral defense mechanism of plants and invertebrates, rendering the capacity to evade it a defining factor in shaping the viral landscape. Here we sought to determine whether different virus replication strategies provided any inherent capacity to evade RNAi in the absence of an antagonist. Through the exploitation of host microRNAs, we recreated an RNAi-like environment in vertebrates and directly compared the capacity of positive- and negative-stranded RNA viruses to cope with this selective pressure. Applying this defense against four distinct viral families revealed that the capacity to undergo homologous recombination was the defining attribute that enabled evasion of this defense. Independent of gene expression strategy, positive-stranded RNA viruses that could undergo strand switching rapidly excised genomic material, while negative-stranded viruses were effectively targeted and cleared upon RNAi-based selection. These data suggest a dynamic relationship between host antiviral defenses and the biology of virus replication in shaping pathogen prevalence.

scholarly journals The Capsid Protein of Semliki Forest Virus Antagonizes RNA Interference in Mammalian Cells

2019 ◽  
Vol 94 (3) ◽  
Author(s):  
Qi Qian ◽  
Hui Zhou ◽  
Ting Shu ◽  
Jingfang Mu ◽  
Yuan Fang ◽  
...  
Keyword(s):  
Rna Interference ◽  
Capsid Protein ◽  
Mammalian Cells ◽  
Semliki Forest Virus ◽  
Rna Viruses ◽  
Strong Support ◽  
Life Cycles ◽  
Immune Defense ◽  
Rnai Pathway ◽  
Viral Suppressors

ABSTRACT RNA interference (RNAi) is a conserved antiviral immune defense in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs) to counteract antiviral RNAi. Alphaviruses are a large group of positive-stranded RNA viruses that maintain their transmission and life cycles in both mosquitoes and mammals. However, there is little knowledge about how alphaviruses antagonize RNAi in both host organisms. In this study, we identified that Semliki Forest virus (SFV) capsid protein can efficiently suppress RNAi in both insect and mammalian cells by sequestrating double-stranded RNA and small interfering RNA. More importantly, when the VSR activity of SFV capsid was inactivated by reverse genetics, the resulting VSR-deficient SFV mutant showed severe replication defects in mammalian cells, which could be rescued by blocking the RNAi pathway. Besides, capsid protein of Sindbis virus also inhibited RNAi in cells. Together, our findings show that SFV uses capsid protein as VSR to antagonize RNAi in infected mammalian cells, and this mechanism is probably used by other alphaviruses, which shed new light on the knowledge of SFV and alphavirus. IMPORTANCE Alphaviruses are a genus of positive-stranded RNA viruses and include numerous important human pathogens, such as Chikungunya virus, Ross River virus, Western equine encephalitis virus, etc., which create the emerging and reemerging public health threat worldwide. RNA interference (RNAi) is one of the most important antiviral mechanisms in plants and insects. Accumulating evidence has provided strong support for the existence of antiviral RNAi in mammals. In response to antiviral RNAi, viruses have evolved to encode viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. It is unclear whether alphaviruses encode VSRs that can suppress antiviral RNAi during their infection in mammals. In this study, we first uncovered that capsid protein encoded by Semliki Forest virus (SFV), a prototypic alphavirus, had a potent VSR activity that can antagonize antiviral RNAi in the context of SFV infection in mammalian cells, and this mechanism is probably used by other alphaviruses.

scholarly journals Magnitude and timing of the antiviral response determine SARS-CoV-2 replication early in infection

2021 ◽  
Author(s):  
Nagarjuna R. Cheemarla ◽  
Timothy A. Watkins ◽  
Valia T. Mihaylova ◽  
Bao Wang ◽  
Dejian Zhao ◽  
...  
Keyword(s):  
Viral Replication ◽  
Host Response ◽  
Defense Mechanism ◽  
Interferon Response ◽  
Antiviral Defense ◽  
Airway Epithelial ◽  
Replication Rate ◽  
Interferon Stimulated Genes ◽  
Biological Variables ◽  
The Impact

AbstractThe interferon response is a potent antiviral defense mechanism, but its effectiveness depends on its timing relative to viral replication. Here, we report viral replication and host response kinetics in patients at the start of SARS-CoV-2 infection and explore the impact of these kinetics experimentally. In both longitudinal patient nasopharyngeal samples and airway epithelial organoids, we found that SARS-CoV-2 initially replicated exponentially with a doubling time of ∼6hr, and induced interferon stimulated genes (ISGs) with delayed timing relative to viral replication. Prior exposure to rhinovirus increased ISG levels and blocked SARS-CoV-2 replication. Conversely, inhibiting ISG induction abrogated interference by rhinovirus and enhanced SARS-CoV-2 replication rate. These results demonstrate the importance of initial interferon-mediated defenses in determining the extent to which SARS-CoV-2 can replicate at the start of infection and indicate that biological variables that alter the airway interferon response, including heterologous induction of innate immunity by other viruses, could profoundly impact SARS-CoV-2 susceptibility and transmission.

scholarly journals Antiviral Defense Involves AGO4 in an Arabidopsis–Potexvirus Interaction

2016 ◽  
Vol 29 (11) ◽  
pp. 878-888 ◽  
Author(s):  
Chantal Brosseau ◽  
Mohamed El Oirdi ◽  
Ayooluwa Adurogbangba ◽  
Xiaofang Ma ◽  
Peter Moffett
Keyword(s):  
Gene Expression ◽  
Nuclear Localization ◽  
Rna Silencing ◽  
Defense Mechanism ◽  
Rna Viruses ◽  
Antiviral Defense ◽  
Double Stranded Rna ◽  
Micro Rnas ◽  
Plantago Asiatica ◽  
Antiviral Activities

In plants, RNA silencing regulates gene expression through the action of Dicer-like (DCL) and Argonaute (AGO) proteins via micro RNAs and RNA-dependent DNA methylation (RdDM). In addition, RNA silencing functions as an antiviral defense mechanism by targeting virus-derived double-stranded RNA. Plants encode multiple AGO proteins with specialized functions, including AGO4-like proteins that affect RdDM and AGO2, AGO5, and AGO1, which have antiviral activities. Here, we show that AGO4 is also required for defense against the potexvirus Plantago asiatica mosaic virus (PlAMV), most likely independent of RdDM components such as DCL3, Pol IV, and Pol V. Transient assays showed that AGO4 has direct antiviral activity on PlAMV and, unlike RdDM, this activity does not require nuclear localization of AGO4. Furthermore, although PlAMV infection causes a decrease in AGO4 expression, PlAMV causes a change in AGO4 localization from a largely nuclear to a largely cytoplasmic distribution. These results indicate an important role for AGO4 in targeting plant RNA viruses as well as demonstrating novel mechanisms of regulation of and by AGO4, independent of its canonical role in regulating gene expression by RdDM.

scholarly journals Antiviral RNAi in Insects and Mammals: Parallels and Differences

Viruses ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 448 ◽  
Author(s):  
Susan Schuster ◽  
Pascal Miesen ◽  
Ronald P. van Rij
Keyword(s):  
Defense Mechanism ◽  
Rnai Pathway ◽  
Future Research ◽  
Type I ◽  
Antiviral Defense ◽  
First Line ◽  
Differentiated Cells ◽  
Open Questions ◽  
Interferon System ◽  
Viral Suppressors

The RNA interference (RNAi) pathway is a potent antiviral defense mechanism in plants and invertebrates, in response to which viruses evolved suppressors of RNAi. In mammals, the first line of defense is mediated by the type I interferon system (IFN); however, the degree to which RNAi contributes to antiviral defense is still not completely understood. Recent work suggests that antiviral RNAi is active in undifferentiated stem cells and that antiviral RNAi can be uncovered in differentiated cells in which the IFN system is inactive or in infections with viruses lacking putative viral suppressors of RNAi. In this review, we describe the mechanism of RNAi and its antiviral functions in insects and mammals. We draw parallels and highlight differences between (antiviral) RNAi in these classes of animals and discuss open questions for future research.

scholarly journals The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation

2020 ◽  
Vol 295 (33) ◽  
pp. 11776-11788
Author(s):  
Molly Hodul ◽  
Rakesh Ganji ◽  
Caroline L. Dahlberg ◽  
Malavika Raman ◽  
Peter Juo
Keyword(s):  
Mammalian Cells ◽  
Deubiquitinating Enzymes ◽  
Post Translational Modification ◽  
Wd40 Repeat ◽  
C Elegans ◽  
Protein Levels ◽  
Specific Protease ◽  
The Stability ◽  
And Function

Ubiquitination is a reversible post-translational modification that has emerged as a critical regulator of synapse development and function. However, the mechanisms that regulate the deubiquitinating enzymes (DUBs) responsible for the removal of ubiquitin from target proteins are poorly understood. We have previously shown that the DUB ubiquitin-specific protease 46 (USP-46) removes ubiquitin from the glutamate receptor GLR-1 and regulates its trafficking and degradation in Caenorhabditis elegans. We found that the WD40-repeat proteins WDR-20 and WDR-48 bind and stimulate the catalytic activity of USP-46. Here, we identified another mechanism by which WDR-48 regulates USP-46. We found that increased expression of WDR-48, but not WDR-20, promotes USP-46 abundance in mammalian cells in culture and in C. elegans neurons in vivo. Inhibition of the proteasome increased USP-46 abundance, and this effect was nonadditive with increased WDR-48 expression. We found that USP-46 is ubiquitinated and that expression of WDR-48 reduces the levels of ubiquitin–USP-46 conjugates and increases the t1/2 of USP-46. A point-mutated WDR-48 variant that disrupts binding to USP-46 was unable to promote USP-46 abundance in vivo. Finally, siRNA-mediated knockdown of wdr48 destabilizes USP46 in mammalian cells. Together, these results support a model in which WDR-48 binds and stabilizes USP-46 protein levels by preventing the ubiquitination and degradation of USP-46 in the proteasome. Given that a large number of USPs interact with WDR proteins, we propose that stabilization of DUBs by their interacting WDR proteins may be a conserved and widely used mechanism that controls DUB availability and function.

scholarly journals Tetramerizing tGCN4 domain facilitates production of Influenza A H1N1 M2e higher order soluble oligomers that show enhanced immunogenicity in vivo

2020 ◽  
Vol 295 (42) ◽  
pp. 14352-14366
Author(s):  
Sweety Samal ◽  
Tripti Shrivastava ◽  
Praveen Sonkusre ◽  
Zaigham Abbas Rizvi ◽  
Rajesh Kumar ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Influenza A ◽  
Large Scale ◽  
Vaccine Development ◽  
Higher Order ◽  
Amino Terminus ◽  
Th2 Response ◽  
Influenza A H1n1 ◽  
Mammalian System

One strategy for the development of a next generation influenza vaccine centers upon using conserved domains of the virus to induce broader and long-lasting immune responses. The production of artificial proteins by mimicking native-like structures has shown to be a promising approach for vaccine design against diverse enveloped viruses. The amino terminus of influenza A virus matrix 2 ectodomain (M2e) is highly conserved among influenza subtypes, and previous studies have shown M2e-based vaccines are strongly immunogenic, making it an attractive target for further exploration. We hypothesized that stabilizing M2e protein in the mammalian system might influence the immunogenicity of M2e with the added advantage to robustly produce the large scale of proteins with native-like fold and hence can act as an efficient vaccine candidate. In this study, we created an engineered construct in which the amino terminus of M2e is linked to the tetramerizing domain tGCN4, expressed the construct in a mammalian system, and tested for immunogenicity in BALB/c mice. We have also constructed a stand-alone M2e construct (without tGCN4) and compared the protein expressed in mammalian cells and in Escherichia coli using in vitro and in vivo methods. The mammalian-expressed protein was found to be more stable, more antigenic than the E. coli protein, and form higher-order oligomers. In an intramuscular protein priming and boosting regimen in mice, these proteins induced high titers of antibodies and elicited a mixed Th1/Th2 response. These results highlight the mammalian-expressed M2e soluble proteins as a promising vaccine development platform.

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