Al and Mary Agnes McQuinn, the Richard M. Schulze Foundation, and an NIH Relief Grant from the Mayo Clinic funded representative work described here.

The authors have nothing to disclose.

Das Design, die Zusammensetzung und die Lokalisierung der microRNA-Response-Elemente innerhalb des viralen Genoms wird diktieren, Wirksamkeit und Spezifität Targeting. Die Optimierung dieser wird Versuch und Irrtum erforderlich. Jedoch rationales Design basierend auf RNA Strukturanalyse und frühere Studien der viralen Replikation und mikroRNA Signaturen hilft bei der Durchführung dieser Technik mit minimalem Optimierungs 10, 11, <...

Die Entwicklung einer breit anwendbaren, einfache und effektive Methode, um einen Vektor mit eingeschränkten Tropismus Engineering bietet eine große Chance, um die Sicherheit, biologische Verständnis und therapeutischen Nutzen von Viren zu verbessern. Mehrere Mechanismen existieren viralen Tropismus einschließlich transductional, transkriptionale und translationale Gestützte Techniken zielen. Jedoch sind diese Verfahren nicht allgemein anwendbar auf alle Vektorsysteme, können defekte Signalwege in Zielzellen erfordern oder Insertion von großen codierenden Sequenzen in das Virusgenom erfordern. Zusätzlich können diese Verfahren in der Dämpfung des Virus führen, erheblich ihre therapeutische Aktivität zu behindern und einen Einblick in das unmodifizierte System zu begrenzen. MicroRNAs sind kleine (22-25 Nukleotide), nicht-kodierenden RNAs, die Gen-Silencing in eukaryotischen Zellen vermitteln. MicroRNAs Funktion durch komplementäre Zielsequenzen Bindung (Response-Elemente) in Boten-RNAs (mRNA) resulti<html> ng in Transkript Destabilisierung, Abbau oder translationale Repression. MicroRNAs normalerweise Response - Elemente mit partieller Komplementarität binden und kleine Veränderungen in der Genexpression 1, 2, 3, 4, 5 ergeben. Weitere signifikante Veränderungen in der Genexpression kann durch Erhöhen 6 Komplementarität des Antwortelements erreicht werden. Tausende von reifen microRNAs wurden in einer Vielzahl von Arten und viele weisen differentielle Expressionsmuster in einer Vielzahl von Zell- und Gewebetypen 7, 8, 9 identifiziert. Diese MikroRNA Signaturen können durch Einbeziehen perfekt komplementären Response - Elemente in das virale Genom 10 zur zellspezifischen Restriktions Virusamplifikation ausgenutzt werden ,= &quot;Xref&quot;&gt; 11, 12, 13. Das übergeordnete Ziel dieser microRNA-Targeting-Technik ist es, die Tropismus eines Vektorgenoms ohne zusätzliche Dämpfung zu steuern. Die Nützlichkeit dieses Verfahrens für die virale Tropismus regulierende wurde ursprünglich in lentiviralen Vektoren gezeigt Transgen - Expression in spezifischen Geweben 14, 15, 16 zu beschränken. Diese Technik hat in der Folge zu einer Vielzahl von replizierende und nicht-replizierende virale Vektoren für eine verbesserte Gentherapie als auch angewendet worden , wie die Sicherheitsprofile vieler onkolytischen Viren zu verbessern , indem eine unerwünschte Toxizitäten in normalen Geweben eliminiert 10, 11, 12, 13, 17 . Es ist auch sicher und e zu erzeugen, verwendet worden, umffective lebendes abgeschwächtes Impfstoffe sowie Virus und Impfstoffherstellung zu verbessern , Prozesse 18, 19, 20, 21. MicroRNA-Targeting eines Vektors kann für die Dämpfung in geimpften Hosts oder gezielt Systeme ermöglichen, während Wildtyp-Wachstumsraten in den Erzeugersystemen aufrechterhalten wird. MicroRNA-Targeting kann auch durch die Beschränkung Übertragung in einer Spezies (zB Menschen) , um die biologische Sicherheit von Viren zu Forschungszwecken zu verbessern verwendet werden , während 22 Übertragung in anderen Hosts zu halten. MicroRNA-Targeting Schließlich kann für viralen Lebensanalysen von Zyklen in-Tiefe ermöglichen und spezifische Rollen von Zelltypen in der Pathogenese und Immunität durch das virale Wachstum Aussortieren 23, 24, 25, 26. Diese Technik bietet ein alternative Verfahren Targeting, die leicht umgesetzt und gilt für alle Virus-Systemen. Darüber hinaus macht die ständig wachsende Sammlung von reifen microRNAs mit differentiellen Expressionsmuster in bestimmten Zelltypen diese Technik vielseitig einsetzbar. MicroRNA-basierten Targeting hat sich für eine Vielzahl von Virus-Systemen ohne Kompromisse bei der Systemfunktion wirksam erwiesen. Die wichtigsten Einschränkungen dieser Technik gehören Versuch und Irrtum-Optimierung, das Potenzial für Mutationen und mögliche Nebeneffekte auf endogenen Transkripte. Allerdings können diese Einschränkungen im Allgemeinen mit optimiertem und rational-Response-Element Design überwunden werden. Positive Sense-RNA-Viren sind in der Regel besonders als Reaktion auf microRNA-Targeting zu sein aufgrund der positiven Sense-Orientierung ihres Genoms und die Verfügbarkeit der Transkripte auf die microRNA Maschinen während des vollständig zytoplasmatischen Replikationszyklus. Hier beschreiben wir ein Protokoll zur Erzeugung eines microRNA-bezogene picornavirus und die Experimental Methoden , um die Effizienz und Spezifität , dass Targeting in vitro zu überprüfen.

<table><tbody><tr><td>RE encoding Oligonucleotides</td><td>IDT</td><td>PAGE-Purified Ultramer</td><td>Sequence Designed by Investigator</td></tr><tr><td>Oligonucleotides encoding unique restriction site</td><td>IDT</td><td>25nM</td><td>Sequence Designed by Investigator</td></tr><tr><td>Expand High Fidelity PCR Kit</td><td>Sigma Aldrich</td><td>11732641001</td><td>Many other High Fidelity Polymerase PCR kits available</td></tr><tr><td>T4 DNA Ligase System</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>MEGAscript Kit</td><td>ThermoFisher Scientific</td><td>AM1333</td><td></td></tr><tr><td>MEGAclear Kit</td><td>ThermoFisher Scientific</td><td>AM1908</td><td></td></tr><tr><td>0.5 M EDTA</td><td>ThermoFisher Scientific</td><td>AM9260G</td><td>RNase-free</td></tr><tr><td>5 M NH&lt;sub&gt;4&lt;sub&gt; Acetate</td><td>ThermoFisher Scientific</td><td>N/A</td><td>Comes in MEGAclear Kit</td></tr><tr><td>Ethanol</td><td>ThermoFisher Scientific</td><td>BP2818100</td><td></td></tr><tr><td>Nuclease-free Water</td><td>Fisher Scientific</td><td>AM9938</td><td></td></tr><tr><td>TransIT-2020 Transfection Reagent</td><td>Mirus</td><td>MIR 5404</td><td></td></tr><tr><td>TransIT-mRNA Transfection Reagent</td><td>Mirus</td><td>MIR 2225</td><td></td></tr><tr><td>0.2 &amp;mu;m syringe filter</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>2 ml Screw-Cap Tubes</td><td>Sarstedt</td><td>72.694.005</td><td></td></tr><tr><td>Cell Scrapers</td><td>Fisher Scientific</td><td>08-100-241</td><td></td></tr><tr><td>MicroRNA Mimics</td><td>Dharmacon</td><td>Varied</td><td></td></tr><tr><td>MTT Cell Proliferation Assay</td><td>ATCC</td><td>30-1010K</td><td></td></tr><tr><td>Subcloning Efficiency DH5&amp;alpha; Competent Cells</td><td>ThermoFisher Scientific</td><td>18265017</td><td></td></tr><tr><td>pBlueScript II Vectors</td><td>Agilent Technologies</td><td>Variable (e.g. 212205)</td><td>There are different plasmids with T7 or T3 promoters and variable cloning sites to enable cloning and RNA transcription.</td></tr></tbody></table>

microrna-based regulation of picornavirus tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

Tabelle 1 zeigt Ergebnisse typisch für einen Titrationsassay für eine Picornavirus und beschreibt , wie die 50% Gewebekultur infektiöse Dosis zu berechnen. Eine schematische Darstellung des Gesamtkonzepts von microRNA-basierten Regulation der viralen in diesem Manuskript beschrieben Tropismus ist in Abbildung 1 die Orientierung der microRNA auf Response - Element während der intrazellulären Wechselwirkungen, die richtige Gestaltung von Response - El...

Watch this Scientific Journal Video about MicroRNA-based Regulation of Picornavirus Tropism at JoVE.com

MicroRNA-based Regulation of Picornavirus Tropism

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

MicroRNA-basierten Regulation der Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

microrna-based-regulation-of-picornavirus-tropism

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.5K Aufrufe.  Mayo Clinic College of Medicine. We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle. 

Serie: MicroRNA-based Regulation of Picornavirus Tropism

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

Reverse-Genetics vermittelte Wiedergewinnung eines infektiösen Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

Forschung

Immunologie und Infektion

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors.
Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described.
This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

Verwendung von einem rekombinanten Moskito Densovirus als gen Lieferung Vektor für die funktionelle Analyse von Genen in Mückenlarven

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

Entwicklungsbiologie

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Potato Virus X-Based microRNA Silencing VbMS In Potato.

We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family.

Potato Virus X-Based microRNA Silencing (VbMS) in Kartoffeln.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

Umwelt

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Rettung von rekombinanten Newcastle Disease Virus aus cDNA

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Überwachung Aktivierung der antiviralen Pattern Recognition Receptors RIG-I und PKR durch begrenzte Protease Verdauung und Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

Ein Reporter-Assay zur Analyse der intronischen microRNA-Reifung in Säugetierzellen

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

Krebsforschung

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

Genomweiten Screen für miRNA-Targets mit dem Missionsziel ID-Bibliothek

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

Biologie

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

Identifizierung von Zielen für Menschenrechte microRNAs mit dem LightSwitch Luciferase Assay System mit 3&#39;UTR-Reporter-Konstrukte und eine microRNA Mimic in adhärenter Zellen

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

Arboviren Infektionen als Screening-Tools für die Identifizierung von viralen Immunmodulatoren und Host antiviralen Faktoren

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Konfokale Abbildung von Double-Stranded RNA und Muster Anerkennung Rezeptoren im negativen Sinn-RNA-Virusinfektion

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...