Name: microrna-based regulation of picornavirus tropism
Uploaded: 2017-02-06T10:00:00
Description: Watch this Scientific Journal Video about MicroRNA-based Regulation of Picornavirus Tropism at JoVE.com

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

1. Cloning microRNA Response Elements into the Viral Genome
<ol>
	<li>Design microRNA response element inserts.
	<ol>
		<li>Identify the desired microRNA and its corresponding target sequence. Several databases are available with mature microRNA sequences. Recommended: http://www.mirbase.org/9,27,28,29,30.</li>
	</ol>
	</li>
	<li>Clo...

The design, composition and localization of the microRNA response elements within the viral genome will dictate targeting efficacy and specificity. Optimizing these will require trial and error. However, rational design based on RNA structural analysis and previous studies of viral replication and microRNA signatures aids in the implementation of this technique with minimal optimization10,11,12,13</s...

The development of a broadly applicable, easy and effective method for engineering a vector with restricted tropism provides a major opportunity to enhance the safety, biological understanding and therapeutic utility of viruses. Several mechanisms exist to target viral tropism including transductional, transcriptional, and translational-based techniques. However, these methods are not generally applicable to all vector systems, may require defective signaling pathways in targeted cells or require insertion of large coding sequences into the viral genome. Additionally, these methods can result in attenuation of the virus, significantly hindering their therapeutic activity and limiting insight into the unmodified system.
MicroRNAs are small (22-25 nucleotides), non-coding RNAs that mediate gene silencing in eukaryotic cells. MicroRNAs function by binding complementary target sequences (response elements) in messenger RNAs (mRNA) resulting in transcript destabilization, degradation or translational repression. MicroRNAs normally bind response elements with partial complementarity and yield small modifications in gene expression1,2,3,4,5. More significant alterations in gene expression can be achieved by increasing complementarity of the response element6. Thousands of mature microRNAs have been identified in a variety of species and many exhibit differential expression patterns in a variety of cell and tissue types7,8,9. These microRNA signatures can be exploited for cell-specific restriction of virus amplification by incorporating perfectly complementary response elements into the viral genome10,11,12,13. The overall goal of this microRNA-targeting technique is to control the tropism of a vector genome without additional attenuation.
The utility of this method for regulating viral tropism was originally demonstrated in lentiviral vectors to restrict transgene expression in specific tissues14,15,16. This technique has subsequently been applied to a vast array of replicating and non-replicating viral vectors for enhanced gene therapy as well as to improve the safety profiles of many oncolytic viruses by eliminating undesired toxicities in normal tissues10,11,12,13,17. It has also been utilized to generate safe and effective live-attenuated vaccines as well as to improve virus and vaccine manufacturing processes18,19,20,21. MicroRNA-targeting of a vector can allow for attenuation in vaccinated hosts or targeted systems while maintaining wild-type growth levels in producer systems. MicroRNA-targeting can also be used to improve the biosafety of viruses for research purposes by restricting transmission in one species (e.g. humans) while maintaining transmission in other hosts22. Finally, microRNA-targeting can allow for in-depth analyses of viral life cycles and specific roles of cell types in pathogenesis and immunity by segregating viral growth23,24,25,26.
This technique offers an alternative targeting method that is easily implemented and applicable for all virus systems. Additionally, the ever-expanding collection of mature microRNAs with differential expression patterns in specific cell types makes this technique highly versatile. MicroRNA-based targeting has proven efficacious for a variety of virus systems without compromising system function. The major limitations of this technique include trial and error optimization, the potential for escape mutations, and potential off-target effects on endogenous transcripts. However, these limitations can generally be overcome with optimized and rational response element design. Positive-sense RNA viruses tend to be particularly responsive to microRNA-targeting due to the positive-sense orientation of their genome and the availability of the transcripts to the microRNA machinery during the completely cytoplasmic replication cycle. Here we describe a protocol for generating a microRNA-targeted picornavirus and the experimental methods to verify the efficiency and specificity of that targeting in vitro.

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		<tr height="40">
			<td height="40" style="height:40px;width:247px;">RE encoding Oligonucleotides</td>
			<td style="width:119px;">IDT</td>
			<td style="width:167px;">PAGE-Purified Ultramer</td>
			<td style="width:247px;">Sequence Designed by Investigator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Oligonucleotides encoding unique restriction site</td>
			<td style="width:119px;">IDT</td>
			<td style="width:167px;">25nM</td>
			<td style="width:247px;">Sequence Designed by Investigator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Expand High Fidelity PCR Kit</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:167px;">11732641001</td>
			<td style="width:247px;">Many other High Fidelity Polymerase PCR kits available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">T4 DNA Ligase System</td>
			<td style="width:119px;">NEB</td>
			<td style="width:167px;">M0202S</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">MEGAscript Kit</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:167px;">AM1333</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">MEGAclear Kit</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:167px;">AM1908</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">0.5 M EDTA</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:167px;">AM9260G</td>
			<td style="width:247px;">RNase-free</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">5 M NH4 Acetate</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:167px;">N/A</td>
			<td style="width:247px;">Comes in MEGAclear Kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Ethanol</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:167px;">BP2818100</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Nuclease-free Water</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td style="width:167px;">AM9938</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">TransIT-2020 Transfection Reagent</td>
			<td style="width:119px;">Mirus</td>
			<td style="width:167px;">MIR 5404</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">TransIT-mRNA Transfection Reagent</td>
			<td style="width:119px;">Mirus</td>
			<td style="width:167px;">MIR 2225</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">0.2 &#956;m syringe filter</td>
			<td style="width:119px;">Millipore</td>
			<td style="width:167px;">SLGP033RS</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">2mL Screw-Cap Tubes</td>
			<td style="width:119px;">Sarstedt</td>
			<td style="width:167px;">72.694.005</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Cell Scrapers</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td style="width:167px;">08-100-241</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">MicroRNA Mimics</td>
			<td style="width:119px;">Dharmacon</td>
			<td style="width:167px;">Varied</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">MTT Cell Proliferation Assay</td>
			<td style="width:119px;">ATCC</td>
			<td style="width:167px;">30-1010K</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Subcloning Efficiency DH5&#945; Competent Cells</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:167px;">18265017</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:247px;">pBlueScript II Vectors</td>
			<td style="width:119px;">Agilent Technologies</td>
			<td style="width:167px;">Variable (e.g. 212205)</td>
			<td style="width:247px;">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.

Table 1 represents results typical of a titration assay for a picornavirus and describes how to calculate the 50% tissue culture infectious dose. A schematic representation of the overall concept of microRNA-based regulation of viral tropism described in this manuscript is shown in Figure 1. The orientation of microRNA to response element during intracellular interactions, proper design of response element oligonucleotides for annealing and plasmid insert...

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.

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

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