JoVE Journal > Immunology and Infection
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Abstract
Introduction
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Immunology and Infection

小核糖核酸病毒取向的微小RNA基于调控

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

一个广泛适用，方便，有效的方法，为工程受限取向载体的发展提供了重大机遇，以提高安全性，生物的理解和病毒的治疗效用。一些机制来瞄准病毒嗜包括转导，转录和翻译基础的技术。然而，这些方法都没有普遍适用于所有的载体系统，可能需要在靶细胞缺陷信号传导途径，或需要大的编码序列插入到病毒基因组中。此外，可能会导致该病毒的衰减这些方法中，显著妨碍它们的治疗活性和限制性洞察未修改的系统。

微小RNA是小的（22-25个核苷酸），介导基因沉默在真核细胞中的非编码RNA。通过结合在信使RNA互补的靶序列（反应元件）（mRNA）的微RNA resulti功能纳克在成绩单不稳定，降解或翻译抑制。微RNA通常结合具有部分互补性反应的元件，基因表达的1，2，3，4，5，得到小的修改。在基因表达更显著改变可通过增加反应元件6的互补性来实现。数千成熟微RNA的已在各种细胞和组织类型7，8，9的多种物种和许多表现出差异表达模式的已确定。这些微小RNA签名可通过将完全互补的反应元件到病毒基因组10被利用为病毒扩增的细胞特异性的限制，=“外部参照”> 11，12，13。这种微小RNA靶向技术的总体目标是控制一个载体基因组的取向，而不附加衰减。

这种方法用于调节病毒嗜性的实用程序最初被证实在慢病毒载体，以限制在特定的组织14，15，16的转基因表达。这种技术随后被应用于复制型或非复制型病毒载体增强基因治疗以及通过消除在正常组织10，11，12，13不希望的毒性，改善的许多溶瘤病毒的安全性概况，17的繁多。它也被用来生成安全和电子ffective减毒活疫苗，以及改善病毒和疫苗制造工艺18，19，20，21。微RNA靶向载体的可允许在接种疫苗的主机或目标系统的衰减，同时保持生产者系统的野生型的增长水平。微RNA靶向也可以使用通过在一个物种（ 例如人类）限制传输，同时保持其他主机22传输来改善病毒用于研究目的的生物安全。最后，微小RNA-定位可以允许深度通过分离病毒生长23，24，25，26分析病毒的生命周期和发病机理和免疫细胞类型的特定角色。

这种技术提供一个alternative定位方法，很容易实现的，并适用于所有病毒系统。此外，在特定的细胞类型差异表达模式成熟的microRNA的不断扩大，使收集这种技术高度灵活。微RNA为基础的定位已被证明有效的，适用于各种病毒的系统，而不会影响系统的功能。该技术的主要局限性包括试验和错误的优化，为逃逸突变的可能性，并在内源性转录物的潜在的脱靶效应。但是，这些限制一般都可以与优化，合理的反应元件设计克服。正链RNA病毒的趋向是特别响应于微小RNA-靶向由于它们的基因组的正义方向和转录到微小RNA机械的完全细胞质复制周期中的可用性。在这里，我们描述了一个协议，用于产生微小RNA靶向小核糖核酸病毒和experimental方法来验证体外靶向的效率和特异性。

Protocol

1.克隆的microRNA反应元件到病毒基因组 设计的microRNA反应元件插入。 识别所需的微RNA及其相应的靶序列。几个数据库都可以与成熟的microRNA序列。推荐：http://www.mirbase.org/ 9，27，28，29，30。 克隆应答元件到编码的载体基因组或?…

Representative Results

表1表示对于一个小核糖核酸病毒典型滴定测定的结果，并说明如何计算50％组织培养感染剂量。在这个手稿所述病毒嗜性的基于微RNA调控的整体概念的示意图期间胞内相互作用，用于退火和质粒的插入响应元件的寡核苷酸的适当的设计示于图1的微RNA的取向，以响应元件，和一个编码用于体外转录的微小RNA靶向病毒基因组的质粒DNA的图<strong…

Discussion

设计，组合物和病毒基因组中的微小RNA应答元件的定位将决定靶向功效和特异性。优化这些将需要试验和错误。然而，设计合理的基础上的RNA结构分析，并在该技术的实施病毒复制和微RNA特征助剂以最少的优化10，11，12，13，38之前的研究。

当发起RE的设…

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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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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