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Immunology and Infection

Regolamento MicroRNA basata su Picornavirus Tropismo

Published: February 06, 2017
doi:
10.3791/55033
,
1Department of Molecular Medicine,Mayo Clinic College of Medicine

Summary

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.

Abstract

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.

Introduction

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'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'inserimento di grandi sequenze codificanti nel genoma virale. Inoltre, questi metodi possono provocare un'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 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'espressione genica 1, 2, 3, 4, 5. Più significative alterazioni nell'espressione genica possono essere realizzati aumentando complementarietà dell'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,= "xref"> 11, 12, 13. L'obiettivo generale di questa tecnica di microRNA-targeting è quello di controllare il tropismo di un genoma vettore senza attenuazione supplementare.

L'utilità di questo metodo per regolare tropismo virale è stato originariamente dimostrata in vettori lentivirali per limitare l'espressione del transgene in tessuti specifici 14, 15, 16. Questa tecnica è stata successivamente applicato ad un'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 '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'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'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'efficienza e la specificità di tale targeting in vitro.

Protocol

1. La clonazione microRNA Risposta elementi nel genoma virale Progettazione microRNA elemento di risposta inserti. Identificare il microRNA desiderata e la corrispondente sequenza bersaglio. Diverse banche dati sono disponibili con sequenze microRNA maturi. Consigliato: http://www.mirbase.org/ 9, 27, 28, 29, 30. …

Representative Results

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'orientamento di microRNA all'elemento di risposta durante le interazioni intracellulari, corretta progettazione di oligonucleoti…

Discussion

Il design, la composizione e la localizzazione degli elementi di risposta microRNA all'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'attuazione di questa tecnica con l'ottimizzazione minimo 10, 11, 12,<…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

Materials

RE encoding Oligonucleotides IDT PAGE-Purified Ultramer Sequence Designed by Investigator
Oligonucleotides encoding unique restriction site IDT 25nM Sequence Designed by Investigator
Expand High Fidelity PCR Kit Sigma Aldrich 11732641001 Many other High Fidelity Polymerase PCR kits available
T4 DNA Ligase System NEB M0202S
MEGAscript Kit ThermoFisher Scientific AM1333
MEGAclear Kit ThermoFisher Scientific AM1908
0.5 M EDTA ThermoFisher Scientific AM9260G RNase-free
5 M NH4 Acetate ThermoFisher Scientific N/A Comes in MEGAclear Kit
Ethanol ThermoFisher Scientific BP2818100
Nuclease-free Water Fisher Scientific AM9938
TransIT-2020 Transfection Reagent Mirus MIR 5404
TransIT-mRNA Transfection Reagent Mirus MIR 2225
0.2 μm syringe filter Millipore SLGP033RS
2mL Screw-Cap Tubes Sarstedt 72.694.005
Cell Scrapers Fisher Scientific 08-100-241
MicroRNA Mimics Dharmacon Varied
MTT Cell Proliferation Assay ATCC 30-1010K
Subcloning Efficiency DH5α Competent Cells ThermoFisher Scientific 18265017
pBlueScript II Vectors Agilent Technologies Variable (e.g. 212205) There are different plasmids with T7 or T3 promoters and variable cloning sites to enable cloning and RNA transcription.
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Tags

MicroRNAPicornavirusTropismVirus TargetingVirus SafetyVirus ManufacturingVirus UtilityVirus InfectionVirus ReplicationVirus PropagationVirus ToxicityVirus TiterCell InfectionCytopathic Effects96-well Plate12-well PlateSerum-free MediaComplete Media

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Ruiz, A. J., Russell, S. J. MicroRNA-based Regulation of Picornavirus Tropism. J. Vis. Exp. (120), e55033, doi:10.3791/55033 (2017).
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