scholarly journals The 3A protein of coxsackievirus B3 acts as a viral suppressor of RNA interference

2020 ◽  
Vol 101 (10) ◽  
pp. 1069-1078
Author(s):  
Jingfang Mu ◽  
Haobo Zhang ◽  
Tao Li ◽  
Ting Shu ◽  
Yang Qiu ◽  
...  
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Viral Myocarditis ◽  
Coxsackievirus B3 ◽  
Small Interfering Rnas ◽  
The Family ◽  
Viral Suppressors

RNA interference (RNAi) is a potent antiviral defence mechanism in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs). Coxsackievirus B3 (CVB3) belongs to the genus Enterovirus in the family Picornaviridae, and has been reported to be a major causative pathogen for viral myocarditis. Despite the importance of CVB3, it is unclear whether CVB3 can also encode proteins that suppress RNAi. Here, we showed that the CVB3 nonstructural protein 3A suppressed RNAi triggered by either small hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) in mammalian cells. We further uncovered that CVB3 3A interacted directly with double-stranded RNAs (dsRNAs) and siRNAs in vitro. Through mutational analysis, we found that the VSR activity of CVB3 3A was significantly reduced by mutations of D24A/L25A/L26A, Y37A/C38A and R60A in conserved residues. In addition, the 3A protein encoded by coxsackievirus B5 (CVB5), another member of Enterovirus, also showed VSR activity. Taken together, our findings showed that CVB3 3A has in vitro VSR activity, thereby providing insights into the pathogenesis of CVB3 and other enteroviruses.

scholarly journals Conditional Suppression of Cellular Genes: Lentivirus Vector-Mediated Drug-Inducible RNA Interference

2003 ◽  
Vol 77 (16) ◽  
pp. 8957-8951 ◽  
Author(s):  
Maciej Wiznerowicz ◽  
Didier Trono
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Developmental Genetics ◽  
Present System ◽  
Small Interfering Rnas ◽  
Lentivirus Vector ◽  
Molecular Therapeutics ◽  
Cellular Genes ◽  
Conditional Suppression

ABSTRACT RNA interference has emerged as a powerful technique to downregulate the expression of specific genes in cells and in animals, thus opening new perspectives in fields ranging from developmental genetics to molecular therapeutics. Here, we describe a method that significantly expands the potential of RNA interference by permitting the conditional suppression of genes in mammalian cells. Within a lentivirus vector background, we subjected the polymerase III promoter-dependent production of small interfering RNAs to doxycycline-controllable transcriptional repression. The resulting system can achieve the highly efficient and completely drug-inducible knockdown of cellular genes. As lentivirus vectors can stably transduce a wide variety of targets both in vitro and in vivo and can be used to generate transgenic animals, the present system should have broad applications.

Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus-adenovirus receptor

2005 ◽  
Vol 386 (9) ◽  
Author(s):  
Denise Werk ◽  
Steffen Schubert ◽  
Vanessa Lindig ◽  
Hans-Peter Grunert ◽  
Heinz Zeichhardt ◽  
...  
Keyword(s):  
Rna Interference ◽  
Fluorescent Protein ◽  
Rna Virus ◽  
Viral Myocarditis ◽  
Specific Treatment ◽  
Coxsackievirus B3 ◽  
Small Interfering Rnas ◽  
Virus Propagation ◽  
Green Fluorescent ◽  
Adenovirus Receptor

AbstractCoxsackievirus B3 (CVB-3) is a plus-strand RNA virus that is believed to be the most common causal agent of viral myocarditis. Since no specific treatment for CVB-3 infections is available to date, we and others have recently started to develop RNA interference (RNAi) approaches to prevent virus propagation. Here we describe our strategy for the development of efficient small interfering RNAs (siRNAs) against viral genomes. Initially, fusion constructs of a reporter (green fluorescent protein) and viral subgenomic fragments were employed to select active siRNAs against the virus. Moreover, in an attempt to achieve sustained virus silencing and reduce the risk of generating escape mutants, only highly efficient siRNAs directed against regions of the viral genome that are unlikely to tolerate mutations were considered for virus inhibition. Two siRNAs directed against the 3D RNA-dependent RNA polymerase were found to inhibit virus propagation by 80–90%. The protective effect of the efficient siRNAs lasted for several days. Furthermore, we have first evidence that inhibition of the cellular coxsackievirus-adenovirus receptor (CAR) by RNAi also reduces the virus titre. Our strategy is likely to be applicable to other (RNA) viruses as well.

scholarly journals A Virus-Encoded Inhibitor That Blocks RNA Interference in Mammalian Cells

2005 ◽  
Vol 79 (12) ◽  
pp. 7371-7379 ◽  
Author(s):  
Christopher S. Sullivan ◽  
Don Ganem
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Rna Virus ◽  
Small Interfering Rnas ◽  
Mechanism Of Inhibition ◽  
Highly Sensitive ◽  
Protein Functions ◽  
Dicer Cleavage ◽  
Short Hairpin Rnas

ABSTRACT Nodamura virus (NoV) is a small RNA virus that is infectious for insect and mammalian hosts. We have developed a highly sensitive assay of RNA interference (RNAi) in mammalian cells that shows that the NoV B2 protein functions as an inhibitor of RNAi triggered by either short hairpin RNAs or small interfering RNAs. In the cell, NoV B2 binds to pre-Dicer substrate RNA and RNA-induced silencing complex (RISC)-processed RNAs and inhibits the Dicer cleavage reaction and, potentially, one or more post-Dicer activities. In vitro, NoV B2 inhibits Dicer-mediated RNA cleavage in the absence of any other host factors and specifically binds double-stranded RNAs corresponding in structure to Dicer substrates and products. Its abilities to bind to Dicer precursor and post-Dicer RISC-processed RNAs suggest a mechanism of inhibition that is unique among known viral inhibitors of RNAi.

scholarly journals Flavivirus induces and antagonizes antiviral RNA interference in both mammals and mosquitoes

2020 ◽  
Vol 6 (6) ◽  
pp. eaax7989 ◽  
Author(s):  
Yang Qiu ◽  
Yan-Peng Xu ◽  
Miao Wang ◽  
Meng Miao ◽  
Hui Zhou ◽  
...  
Keyword(s):  
Rna Interference ◽  
Dengue Virus ◽  
Viral Infection ◽  
Mammalian Cells ◽  
Nonstructural Protein ◽  
Life Cycles ◽  
Viral Suppressor ◽  
Bona Fide ◽  
Host Life

Mosquito-borne flaviviruses infect both mammals and mosquitoes. RNA interference (RNAi) has been demonstrated as an anti-flavivirus mechanism in mosquitoes; however, whether and how flaviviruses induce and antagonize RNAi-mediated antiviral immunity in mammals remains unknown. We show that the nonstructural protein NS2A of dengue virus-2 (DENV2) act as a viral suppressor of RNAi (VSR). When NS2A-mediated RNAi suppression was disabled, the resulting mutant DENV2 induced Dicer-dependent production of abundant DENV2-derived siRNAs in differentiated mammalian cells. VSR-disabled DENV2 showed severe replication defects in mosquito and mammalian cells and in mice that were rescued by RNAi deficiency. Moreover, NS2As of multiple flaviviruses act as VSRs in vitro and during viral infection in both organisms. Overall, our findings demonstrate that antiviral RNAi can be induced by flavivirus, while flavivirus uses NS2A as a bona fide VSR to evade RNAi in mammals and mosquitoes, highlighting the importance of RNAi in flaviviral vector-host life cycles.

scholarly journals Small interfering RNAs are highly effective inhibitors regarding Crimean-Congo hemorrhagic fever virus replication in vitro

2020 ◽  
Author(s):  
Fanni Földes ◽  
Mónika Madai ◽  
Henrietta Papp ◽  
Gábor Kemenesi ◽  
Brigitta Zana ◽  
...  
Keyword(s):  
Rna Interference ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
World Health ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus ◽  
Antiviral Therapies

AbstractCrimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of World Health Organization, considering its potential to create a public health emergency and more importantly, the absence of efficacious drugs and/or vaccines regarding treatment. The highly lethal nature characteristic to CCHFV restricts research to BSL-4 laboratories, which complicates effective research and developmental strategies. In consideration of antiviral therapies, RNA interference can be used to suppress viral replication by targeting viral genes. RNA interference uses small interfering RNAs (siRNAs) to silence genes. The aim of our study was to design siRNAs that inhibit CCHFV replication and can serve as a basis for further antiviral therapies. A549 cells were infected with CCHFV after transfection with the siRNAs. Following 72 hours, nucleic acid from the supernatant was extracted for Droplet Digital PCR analysis. Among the investigated siRNAs we identified four effective candidates against all three segments of CCHF genome: one for the S and M segments, whilst two for the L segment. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs in vitro. The most active siRNAs were demonstrated a specific inhibitory effect against CCHFV in a dose-dependent manner. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be used in future anti-CCHFV therapy developments.

scholarly journals Optimizing RNA interference for application in mammalian cells

2004 ◽  
Vol 380 (3) ◽  
pp. 593-603 ◽  
Author(s):  
René H. MEDEMA
Keyword(s):  
Rna Interference ◽  
Gene Silencing ◽  
Mammalian Cells ◽  
Reverse Genetics ◽  
Mutational Analysis ◽  
Life Sciences ◽  
Novel Technologies ◽  
Genome Wide ◽  
Technological Developments ◽  
Interfering Rna

Over the last 2 years, the scientific community has rapidly embraced novel technologies that allow gene silencing in vertebrates. Ease of application, cost effectiveness and the possibilities for genome-wide reverse genetics have quickly turned this approach into a widely accepted, almost mandatory asset for a self-respecting laboratory in life sciences. This review discusses some of the recent technological developments that allow the application of RNAi (RNA interference) in mammalian cells. In addition, the advantages of applying RNAi to study cell cycle events and the emerging approaches to perform mutational analysis by complementation in mammalian cells are evaluated. In addition, common pitfalls and drawbacks of RNAi will be reviewed, as well as the possible ways to get around these shortcomings of gene silencing by small interfering RNA.

scholarly journals Reovirus Nonstructural Protein σNS Acts as an RNA Stability Factor Promoting Viral Genome Replication

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Paula F. Zamora ◽  
Liya Hu ◽  
Jonathan J. Knowlton ◽  
Roni M. Lahr ◽  
Rodolfo A. Moreno ◽  
...  
Keyword(s):  
Virus Replication ◽  
Viral Genome ◽  
Nonstructural Protein ◽  
Dsrna Virus ◽  
Genome Replication ◽  
Nonstructural Proteins ◽  
Content Type ◽  
The Family ◽  
Viral Genome Replication

ABSTRACTViral nonstructural proteins, which are not packaged into virions, are essential for the replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. The reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, the activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered, usingin vitroand cell-based RNA degradation experiments, that σNS increases the RNA half-life. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases the viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication.IMPORTANCEFollowing infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the familyReoviridaeencode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different viruses in the familyReoviridaediverge in primary sequence, they are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Usingin vitroand cell culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the familyReoviridaeassort and replicate their genomes.

scholarly journals Small Interfering RNAs Are Highly Effective Inhibitors of Crimean-Congo Hemorrhagic Fever Virus Replication In Vitro

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5771
Author(s):  
Fanni Földes ◽  
Mónika Madai ◽  
Henrietta Papp ◽  
Gábor Kemenesi ◽  
Brigitta Zana ◽  
...  
Keyword(s):  
Rna Interference ◽  
A549 Cells ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
World Health ◽  
Small Interfering Rnas ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus ◽  
Antiviral Therapies

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of the World Health Organization, considering its potential to create a public health emergency and, more importantly, the absence of efficacious drugs and/or vaccines for treatment. The highly pathogenic characteristic of CCHFV restricts research to BSL-4 laboratories, which complicates effective research and developmental strategies. In consideration of antiviral therapies, RNA interference can be used to suppress viral replication by targeting viral genes. RNA interference uses small interfering RNAs (siRNAs) to silence genes. The aim of our study was to design and test siRNAs in vitro that inhibit CCHFV replication and can serve as a basis for further antiviral therapies. A549 cells were infected with CCHFV after transfection with the siRNAs. Following 72 h, nucleic acid from the supernatant was extracted for RT Droplet Digital PCR analysis. Among the investigated siRNAs we identified effective candidates against all three segments of the CCHF genome. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be integrated into future anti-CCHFV therapy developments.

scholarly journals p180 Is Involved in the Interaction between the Endoplasmic Reticulum and Microtubules through a Novel Microtubule-binding and Bundling Domain

2007 ◽  
Vol 18 (10) ◽  
pp. 3741-3751 ◽  
Author(s):  
Kiyoko Ogawa-Goto ◽  
Keiko Tanaka ◽  
Tomonori Ueno ◽  
Keisuke Tanaka ◽  
Takeshi Kurata ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Specific Interaction ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Animal Cells ◽  
Microtubule Binding

p180 was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum membrane, although its precise role in animal cells has not yet been elucidated. Here, we characterized a new function of human p180 as a microtubule-binding and -modulating protein. Overexpression of p180 in mammalian cells induced an elongated morphology and enhanced acetylated microtubules. Consistently, electron microscopic analysis clearly revealed microtubule bundles in p180-overexpressing cells. Targeted depletion of endogenous p180 by small interfering RNAs led to aberrant patterns of microtubules and endoplasmic reticulum in mammalian cells, suggesting a specific interaction between p180 and microtubules. In vitro sedimentation assays using recombinant polypeptides revealed that p180 bound to microtubules directly and possessed a novel microtubule-binding domain (designated MTB-1). MTB-1 consists of a predicted coiled-coil region and repeat domain, and strongly promoted bundle formation both in vitro and in vivo when expressed alone. Overexpression of p180 induced acetylated microtubules in cultured cells in an MTB-1-dependent manner. Thus, our data suggest that p180 mediates interactions between the endoplasmic reticulum and microtubules mainly through the novel microtubule-binding and -bundling domain MTB-1.

scholarly journals Spongiform Neurodegeneration-associated E3 Ligase Mahogunin Ubiquitylates TSG101 and Regulates Endosomal Trafficking

2007 ◽  
Vol 18 (4) ◽  
pp. 1129-1142 ◽  
Author(s):  
Bong Yoon Kim ◽  
James A. Olzmann ◽  
Gregory S. Barsh ◽  
Lih-Shen Chin ◽  
Lian Li
Keyword(s):  
Mammalian Cells ◽  
Growth Factor Receptor ◽  
Endosomal Trafficking ◽  
Small Interfering Rnas ◽  
Gene Encoding ◽  
Endosomal Sorting ◽  
Ubiquitin Protein Ligase ◽  
Downstream Signaling

A null mutation in the gene encoding the putative E3 ubiquitin–protein ligase Mahogunin causes spongiform neurodegeneration, a recessively transmitted prion-like disease in mice. However, no substrates of Mahogunin have been identified, and the cellular role of Mahogunin is unknown. Here, we report the identification of TSG101, a key component of the endosomal sorting complex required for transport (ESCRT)-I, as a specific Mahogunin substrate. We find that Mahogunin interacts with the ubiquitin E2 variant (UEV) domain of TSG101 via its PSAP motif and that it catalyzes monoubiquitylation of TSG101 both in vivo and in vitro. Depletion of Mahogunin by small interfering RNAs in mammalian cells disrupts endosome-to-lysosome trafficking of epidermal growth factor receptor, resulting in prolonged activation of a downstream signaling cascade. Our findings support a role for Mahogunin in a proteasome-independent ubiquitylation pathway and suggest a link between dysregulation of endosomal trafficking and spongiform neurodegeneration.

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