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.

Il design, la composizione e la localizzazione degli elementi di risposta microRNA all&#39;interno del genoma virale detterà mira efficacia e specificità. Ottimizzazione questi richiederà tentativi ed errori. Tuttavia, progettazione razionale sulla base di RNA analisi strutturale e studi precedenti di replicazione virale e microRNA firme aiuti per l&#39;attuazione di questa tecnica con l&#39;ottimizzazione minimo 10, 11, 12,<...

Lo sviluppo di un metodo ampiamente applicabile, facile ed efficace per ingegneria un vettore con tropismo limitata offre una grande opportunità per migliorare la sicurezza, la comprensione biologica e l&#39;utilità terapeutica del virus. Diversi meccanismi esistono per indirizzare tropismo virale tra cui trasduzionali, trascrizione, e le tecniche di traslazionali-based. Tuttavia, questi metodi non sono generalmente applicabili a tutti i sistemi vettoriali, può richiedere vie di segnalazione difettosi in cellule bersaglio o richiedere l&#39;inserimento di grandi sequenze codificanti nel genoma virale. Inoltre, questi metodi possono provocare un&#39;attenuazione del virus, ostacolando significativamente la loro attività terapeutica e limitando la visibilità nel sistema non modificato. I microRNA sono piccole (22-25 nucleotidi), RNA non codificanti che mediano silenziamento genico nelle cellule eucariotiche. funzione di microRNA legandosi sequenze target complementari (elementi di risposta) in RNA messaggero (mRNA) resulti<html> ng in trascrizione di destabilizzazione, il degrado o la repressione traslazionale. I microRNA normalmente si legano elementi di risposta con complementarità parziale e cedono piccole modifiche nell&#39;espressione genica 1, 2, 3, 4, 5. Più significative alterazioni nell&#39;espressione genica possono essere realizzati aumentando complementarietà dell&#39;elemento risposta 6. Migliaia di microRNAs mature sono state identificate in una varietà di specie e molti modelli di espressione mostra differenziali in una varietà di cellule e tessuti tipi 7, 8, 9. Queste firme microRNA possono essere sfruttati per la restrizione specifica delle cellule di amplificazione del virus, incorporando elementi di risposta perfettamente complementari nel genoma virale 10,= &quot;xref&quot;&gt; 11, 12, 13. L&#39;obiettivo generale di questa tecnica di microRNA-targeting è quello di controllare il tropismo di un genoma vettore senza attenuazione supplementare. L&#39;utilità di questo metodo per regolare tropismo virale è stato originariamente dimostrata in vettori lentivirali per limitare l&#39;espressione del transgene in tessuti specifici 14, 15, 16. Questa tecnica è stata successivamente applicato ad un&#39;ampia gamma di replicare e non replicanti vettori virali per la terapia genica intenso nonché per migliorare i profili di sicurezza di molti virus oncolitici eliminando tossicità indesiderati nei tessuti normali 10, 11, 12, 13, 17 . E &#39;stato anche utilizzato per generare e sicura effective vaccini vivi attenuati, nonché per migliorare virus e produzione di vaccini processi 18, 19, 20, 21. MicroRNA-targeting di un vettore può consentire per l&#39;attenuazione in ospiti vaccinati o sistemi mirati, pur mantenendo livelli di crescita wild-type in sistemi di produttori. MicroRNA targeting può essere utilizzato anche per migliorare la sicurezza biologica del virus per scopi di ricerca, limitando la trasmissione in una specie (ad esempio, gli esseri umani), pur mantenendo la trasmissione in altri host 22. Infine, microRNA-targeting possono consentire approfondite analisi del ciclo di vita virali e ruoli specifici di tipi di cellule nella patogenesi e immunità segregando crescita virale 23, 24, 25, 26. Questa tecnica offre un alternative metodo che viene facilmente implementata e applicabile il targeting per tutti i sistemi di virus. Inoltre, la collezione in continua espansione dei microRNA maturi con pattern di espressione differenziali in specifici tipi cellulari rende questa tecnica molto versatile. MicroRNA basata Targeting è dimostrato efficace per una varietà di sistemi di virus senza compromettere la funzione di sistema. I principali limiti di questa tecnica sono tentativi ed errori di ottimizzazione, il potenziale di mutazioni di fuga, e potenziali effetti fuori bersaglio sulla trascrizioni endogene. Tuttavia, queste limitazioni possono generalmente essere superati con design ottimizzato e razionale elemento di risposta. virus a RNA positivo-senso tendono ad essere particolarmente sensibili alle microRNA-targeting causa dell&#39;orientamento positivo-senso della loro genoma e la disponibilità dei trascritti ai macchinari microRNA durante il ciclo di replica completamente citoplasmatica. Qui si descrive un protocollo per la generazione di Picornavirus microRNA-mirata e la sperimMetodi ental per verificare l&#39;efficienza e la specificità di tale targeting in vitro.

<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.

La tabella 1 rappresenta i risultati tipici di un test di titolazione per Picornavirus e descrive come calcolare la dose infettante coltura tissutale del 50%. Una rappresentazione schematica del concetto generale di regolazione microRNA a base di tropismo virale descritto in questo manoscritto è mostrato in Figura 1. L&#39;orientamento di microRNA all&#39;elemento di risposta durante le interazioni intracellulari, corretta progettazione di oligonucleoti...

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.

Regolamento MicroRNA basata su Picornavirus Tropismo

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

microrna-based-regulation-of-picornavirus-tropism

Research

JoVE Journal

Immunology and Infection

7.5K Views.  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. 

Video: 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 recupero mediato del Infettive Norovirus Murine

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

Ricerca

Immunologia e infezioni

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.

Utilizzo di una zanzara ricombinante Densovirus come un vettore di consegna del Gene per l'analisi funzionale di geni in larve di zanzara

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

Biologia dello sviluppo

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.

Virus della patata Silenziamento del microRNA basato sull'X (VbMS) nella patata.

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

Ambiente

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.

Salvataggio di virus ricombinante malattia di Newcastle da 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.

Attivazione Monitoraggio del Pattern Recognition antivirale recettori RIG-I E PKR By limitata proteasi Digestione e PAGE Native

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.

Un saggio reporter per analizzare la maturazione dei microRNA intronici in cellule di mammifero

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

Ricerca sul cancro

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.

Genome-Wide Screen per le destinazioni miRNA utilizzo di target Biblioteca MISSION ID

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

Biologia

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.

Obiettivi Identificazione di microRNA umani con il sistema di analisi Luciferasi LightSwitch utilizzando 3&#39;UTR-reporter Costruisce e un Mimic microRNA in cellule aderenti

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.

Infezioni da arbovirus come strumenti di Screening per l'identificazione di fattori virali immunomodulatori e Host antivirale

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.

Formazione immagine confocal di RNA Double-Stranded e Pattern Recognition Receptors in infezione Virus RNA negativo

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