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 Enhanced Gene Silencing in Cells Cured of Persistent Virus Infection by RNA Interference

2010 ◽  
Vol 84 (13) ◽  
pp. 6880-6885 ◽  
Author(s):  
Isabelle Pelletier ◽  
Aure Saulnier ◽  
Cynthia Brisac ◽  
Sophie Jegouic ◽  
Nicolas Vabret ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Interference ◽  
Virus Infection ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
Small Interfering Rnas ◽  
Rnai Machinery ◽  
Persistent Virus Infection ◽  
Persistent Virus ◽  
Green Fluorescent

ABSTRACT We compared HEp-2-derived cells cured of persistent poliovirus infection by RNA interference (RNAi) with parental cells, to investigate possible changes in the efficiency of RNAi. Lower levels of poliovirus replication were observed in cured cells, possibly facilitating virus silencing by antiviral small interfering RNAs (siRNAs). However, green fluorescent protein (GFP) produced from a measles virus vector and also GFP and luciferase produced from plasmids that do not replicate in human cells were more effectively silenced by specific siRNAs in cured than in control cells. Thus, cells displaying enhanced silencing were selected during curing by RNAi. Our results strongly suggest that the RNAi machinery of cured cells is more efficient than that of parental cells.

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.

A novel linker histone-like protein is associated with cytoplasmic filaments inCaenorhabditis elegans

2002 ◽  
Vol 115 (14) ◽  
pp. 2881-2891
Author(s):  
Monika A. Jedrusik ◽  
Stefan Vogt ◽  
Peter Claus ◽  
Ekkehard Schulze
Keyword(s):  
Rna Interference ◽  
Fluorescent Protein ◽  
Muscle Growth ◽  
Egg Laying ◽  
Globular Domain ◽  
Protein Fusion ◽  
Linker Histones ◽  
C Elegans ◽  
Fusion Protein Expression ◽  
Green Fluorescent

The histone H1 complement of Caenorhabditis elegans contains a single unusual protein, H1.X. Although H1.X possesses the globular domain and the canonical three-domain structure of linker histones, the amino acid composition of H1.X is distinctly different from conventional linker histones in both terminal domains. We have characterized H1.X in C. elegans by antibody labeling, green fluorescent protein fusion protein expression and RNA interference. Unlike normal linker histones, H1.X is a cytoplasmic as well as a nuclear protein and is not associated with chromosomes. H1.X is most prominently expressed in the marginal cells of the pharynx and is associated with a peculiar cytoplasmic cytoskeletal structure therein, the tonofilaments. Additionally H1.X::GFP is expressed in the cytoplasm of body and vulva muscle cells, neurons, excretory cells and in the nucleoli of embryonic blastomeres and adult gut cells. RNA interference with H1.X results in uncoordinated and egg laying defective animals, as well as in a longitudinally enlarged pharynx. These phenotypes indicate a cytoplasmic role of H1.X in muscle growth and muscle function.

scholarly journals Development and Application of a Green Fluorescent Protein Sentinel System for Identification of RNA Interference in Blastomyces dermatitidis Illuminates the Role of Septin in Morphogenesis and Sporulation

2007 ◽  
Vol 6 (8) ◽  
pp. 1299-1309 ◽  
Author(s):  
T. Krajaejun ◽  
G. M. Gauthier ◽  
C. A. Rappleye ◽  
T. D. Sullivan ◽  
B. S. Klein
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Interference ◽  
Gene Silencing ◽  
Rapid Detection ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Blastomyces Dermatitidis ◽  
Dimorphic Fungus ◽  
Green Fluorescent

ABSTRACT A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus Blastomyces dermatitidis, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, “X,” next to GFP to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in B. dermatitidis, we first used LACZ and the virulence gene BAD1 as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A CDC11 septin homolog in B. dermatitidis localized to the neck of budding yeast cells. CDC11-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of B. dermatitidis.

scholarly journals Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays

Insects ◽  
2013 ◽  
Vol 4 (1) ◽  
pp. 90-103 ◽  
Author(s):  
Francis Nunes ◽  
Aline Aleixo ◽  
Angel Barchuk ◽  
Ana Bomtorin ◽  
Christina Grozinger ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Interference ◽  
Honey Bee ◽  
Fluorescent Protein ◽  
Double Stranded Rna ◽  
Green Fluorescent ◽  
Target Effects

scholarly journals Application of RNA Interference Technology to Acroporid Juvenile Corals

2021 ◽  
Vol 8 ◽  
Author(s):  
Ikuko Yuyama ◽  
Tomihiko Higuchi ◽  
Michio Hidaka
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Stress Response ◽  
Large Scale ◽  
Elevated Temperatures ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Pcr Analysis ◽  
Genes Encoding ◽  
Green Fluorescent

Numerous genes involved in calcification, algal endosymbiosis, and the stress response have been identified in corals by large-scale gene expression analysis, but functional analysis of those genes is lacking. There are few experimental examples of gene expression manipulation in corals, such as gene knockdown by RNA interference (RNAi). The purpose of this study is to establish an RNAi method for coral juveniles. As a first trial, the genes encoding green fluorescent protein (GFP, an endogenous fluorophore expressed by corals) and thioredoxin (TRX, a stress response gene) were selected for knockdown. Synthesized double-stranded RNAs (dsRNAs) corresponding to GFP and TRX were transformed into planula larvae by lipofection method to attempt RNAi. Real-time PCR analysis to verify knockdown showed that GFP and TRX expression levels tended to decrease with each dsRNA treatment (not significant). In addition, stress exposure experiments following RNAi treatment revealed that planulae with TRX knockdown exhibited increased mortality at elevated temperatures. In GFP-knockdown corals, decreased GFP fluorescence was observed. However, the effect of GFP-knockdown was confirmed only in the coral at the initial stages of larval metamorphosis into polyps, but not in planulae and 1 month-old budding polyps. This study showed that lipofection RNAi can be applied to coral planulae and polyps after settlement, and that this method provides a useful tool to modify expression of genes involved in stress tolerance and fluorescence emission of the corals.

scholarly journals Recombination between Two Identical Sequences within the Same Retroviral RNA Molecule

1999 ◽  
Vol 73 (7) ◽  
pp. 5912-5917 ◽  
Author(s):  
Jiayou Zhang ◽  
Christy M. Sapp
Keyword(s):  
Recombination Rate ◽  
Fluorescent Protein ◽  
Rna Virus ◽  
Leukemia Virus ◽  
Single Copy ◽  
Repeat Sequence ◽  
Green Fluorescent Protein Gene ◽  
Chimeric Rna ◽  
Retroviral Replication ◽  
Green Fluorescent

ABSTRACT As a consequence of being diploid viruses, members of theRetroviridae have a high recombination rate. To measure recombination between two identical sequences within the same RNA molecule per round of retroviral replication cycle, a murine leukemia virus based vector (JZ442 + 3′ Hyg) has been constructed. It carries a drug resistance gene, hyg, and a 290-bp repeat sequence of the 3′ hyg gene inserted into the 3′ untranslated region of the green fluorescent protein gene (gfp). Under fluorescence microscopy, Hygrcells containing the recombinant proviruses were clear, while a green color was observed in the drug-resistant cells carrying the parental proviruses. The rate of recombination was determined by the ratio of the number of clear colonies to the total number of Hygrcolonies (green and clear colonies). The rate of recombination was found to be 62% by this method. The intermolecular recombination rate between an infectious virus bearing two copies of the 290-bp segment and a noninfectious chimeric RNA virus containing only a single copy of this sequence was also measured.

Abstract 3810: Antiviral RNA Interference Mediated by a Recombinant Cardiotropic AAV9 Vector Improves Cardiac Function in Coxsackievirus B3 Cardiomyopathy

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Wolfgang Poller ◽  
Isaac Sipo ◽  
Dirk Westermann ◽  
Jens Kurreck ◽  
Roland Vetter ◽  
...  
Keyword(s):  
Rna Interference ◽  
Coxsackievirus B3 ◽  
Left Ventricular ◽  
Lv Function ◽  
Target Cells ◽  
Small Interfering Rnas ◽  
Rat Cardiomyocytes ◽  
Terminal Heart Failure ◽  
Novel Therapeutic

RNA interference (RNAi) has potential to be a novel therapeutic strategy in diverse areas of medicine. We report here on targeted RNAi for the treatment of a viral cardiomyopathy which is a major cause of sudden cardiac death or terminal heart failure in children and young adults. RNAi therapy employs small regulatory RNAs to achieve its effect but in vivo use of synthetic small interfering RNAs is limited by instability in plasma and low transfer into target cells. We instead evaluated an RNAi strategy using short hairpin RNA (shRdRp) directed at the RNA polymerase (RdRP) of Coxsackievirus B3 (CoxB3) in HeLa cells, primary rat cardiomyocytes (PNCMs), and CoxB3-infected mice in vivo. A conventional AAV2 vector expressing shRdRp protected HeLa against virus-induced death, but this vector type was unable to transduce PNCMs. In contrast, an analogous pseudotyped AAV2.6 vector was protective also in PNCMs and reduced virus replication by >3 log10 steps. Finally, we evaluated intravenous treatment of mice with an AAV2.9-shRdRp vector since AAV9 carries the most cardiotropic AAV capsid currently known for in vivo use. Mice with CoxB3 cardiomyopathy had disturbed left ventricular (LV) function with impaired parameters of contractility (dP/dtmax 3006±287 vs. 7482±487 mmHg/s, p<0.01) and diastolic relaxation (dP/dtmin -2224±195 vs. -6456±356 mmHg/s, p<0.01 and Tau 16.2±1.1 vs. 10.7±0.6 ms, p<0.01) as compared to control mice. AAV2.9-shRdRp treatment significantly attenuated the cardiac dysfunction compared to control vector-treated mice on day 10 after CoxB3 infection: dP/dtmax 3865±354 vs. 3006±287 mmHg/s (p<0.05) and dP/dtmin -3245±231 vs. −2224±195, mmHg/s (p<0.05), and Tau 11.9±0.5 vs. 16.2±1.1 ms (p<0.01). The data show, for the first time, that intravenously injected AAV9 has the potential to target RNAi to the heart and suggest AAV9-shRNA vectors as a novel therapeutic approach for cardiac disorders.

scholarly journals Coronavirus Spike Glycoprotein, Extended at the Carboxy Terminus with Green Fluorescent Protein, Is Assembly Competent

2004 ◽  
Vol 78 (14) ◽  
pp. 7369-7378 ◽  
Author(s):  
Berend Jan Bosch ◽  
Cornelis A. M. de Haan ◽  
Peter J. M. Rottier
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Viral Envelope ◽  
Wild Type ◽  
Virus Propagation ◽  
S Protein ◽  
Protein Functions ◽  
Geometrical Constraints ◽  
Green Fluorescent

ABSTRACT Due to the limited ultrastructural information about the coronavirion, little is known about the interactions acting at the interface between nucleocapsid and viral envelope. Knowing that subtle mutations in the carboxy-terminal endodomain of the M protein are already lethal, we have now probed the equivalent domain of the spike (S) protein by extending it terminally with a foreign sequence of 27 kDa: the green fluorescent protein (GFP). When expressed individually in murine cells, the S-GFP chimeric protein induced the formation of fluorescent syncytia, indicating that it was synthesized and folded properly, trimerized, and transported to the plasma membrane, where it exhibited the two key S protein functions, i.e., interaction with virus receptor molecules and membrane fusion. Incorporation into virus-like particles demonstrated the assembly competence of the chimeric spike protein. The wild-type S gene of mouse hepatitis coronavirus (MHV) was subsequently replaced by the chimeric construct through targeted recombination. A viable MHV-SGFP was obtained, infection by which could be visualized by the fluorescence induced. The efficiency of incorporation of the chimeric protein into particles was, however, reduced relative to that in wild-type particles which may explain, at least in part, the reduced infectivity produced by MHV-SGFP infection. We conclude that the incorporation of spikes carrying the large GFP moiety is apparently impaired by geometrical constraints and selected against during the assembly of virions. Probably due to this disadvantage, deletion mutants, having lost the foreign sequences, rapidly evolved and outcompeted the chimeric viruses during virus propagation. The fluorescent MHV-SGFP will now be a convenient tool to study coronaviral cell entry.

scholarly journals RNA1-Independent Replication and GFP Expression from Tomato marchitez virus Isolate M Cloned cDNA

2016 ◽  
Vol 106 (5) ◽  
pp. 500-509 ◽  
Author(s):  
I. Ferriol ◽  
M. Turina ◽  
E. J. Zamora-Macorra ◽  
B. W. Falk
Keyword(s):  
Fluorescent Protein ◽  
Rna Virus ◽  
Cell Movement ◽  
Full Length ◽  
Wild Type Virus ◽  
Cdna Clones ◽  
Genomic Rna ◽  
Positive Strand Rna Virus ◽  
Green Fluorescent ◽  
Positive Strand Rna

Tomato marchitez virus (ToMarV; synonymous with Tomato apex necrosis virus) is a positive-strand RNA virus in the genus Torradovirus within the family Secoviridae. ToMarV is an emergent whitefly-transmitted virus that causes important diseases in tomato (Solanum lycopersicum) in Mexico. Here, the genome sequence of the ToMarV isolate M (ToMarV-M) was determined. We engineered full-length cDNA clones of the ToMarV-M genomic RNA (RNA1 and RNA2), separately, into a binary vector. Coinfiltration of both triggered systemic infections in Nicotiana benthamiana, tomato, and tomatillo (Physalis philadelphica) plants and recapitulated the biological activity of the wild-type virus. The viral progeny generated from tomato and tomatillo plants were transmissible by the whitefly Bemisia tabaci biotype B. Also, we assessed whether these infectious clones could be used for screening tomato cultivars for resistance to ToMarV and our results allowed us to differentiate resistant and susceptible tomato lines. We demonstrated that RNA1 of ToMarV-M is required for the replication of RNA2, and it can replicate independently of RNA2. From this, ToMarV-M RNA2 was used to express the green fluorescent protein in N. benthamiana plants, which allowed us to track cell-to-cell movement. The construction of full-length infectious cDNA clones of ToMarV-M provides an excellent tool to investigate virus–host–vector interactions and elucidate the functions of torradovirus-encoded proteins or the mechanisms of replication of torradovirus genomic RNA.

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