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>Clone the response element into plasmid DNA encoding the vector genome or transcript. 
	NOTE: For two or more copy response elements using a unique restriction site for insertion is easier and more versatile.
	<ol>
		<li>Insert response element or unique restriction site using splice-overlap extension (SOE) PCR. This method has been described in detail31.</li>
		<li>If using a restriction site for insertion, purchase commercially synthesized, PAGE-purified sense and antisense ultramers encoding the insert sequences flanked by the overhang sequences of the unique restriction site (Figure 2).</li>
		<li>Combine 0.5 &#181;g sense ultramer, 0.5 &#181;g antisense ultramer, DNA ligation buffer to final concentration of 1x, and bring to 50 &#181;l with H2O. Anneal the ultramers by incubating the reaction at 85 &#176;C for 10 min and then reducing the temperature by 0.5 &#176;C every 30 sec until the reaction reaches 25 &#176;C.</li>
		<li>Combine 0.5 &#181;g of vector DNA encoding the new unique restriction site, enzyme buffer to a 1x final concentration, 1 &#181;l of the appropriate restriction enzyme, and bring to a final volume of 20 &#181;l with H2O. Digest the vector at 37 &#176;C for 2 hr. Purify the linearized DNA by agarose gel purification32.</li>
		<li>Ligate the annealed ultramers into the digested vector overnight at 16 &#176;C using a 3:1 ultramers:vector molar ratio and standard ligation techniques33. 
		NOTE: When using a single restriction enzyme site for insertion it can be more efficient to dephosphorylate the ends of the digested vector and phosphorylate the ends of the annealed oligonucleotides.</li>
		<li>Transform the ligated DNA into E. coli using the heat shock method34. 
		NOTE: Plasmids encoding viral genomes are generally large, therefore using competent cells optimized to uptake large DNA constructs may increase transformation efficiency.</li>
		<li>Purify plasmid DNA from individual colonies using a commercially available purification kit35. Identify an appropriate clone with the microRNA response element in the correct orientation (Figure 2) by sequencing the insert region36.</li>
	</ol>
	</li>
</ol>
2. Rescuing microRNA-targeted Picornavirus from Plasmid DNA
<ol>
	<li>Rescue microRNA-targeted picornavirus in a cell line permissive for virus replication that does not express the cognate microRNA(s). This protocol uses H1-HeLa cells because they do not express miR-142, miR-124, or miR-125, however it is not required to use H1-HeLa cells for rescue. 
	CAUTION: All guidelines for the safe handling and disposal of infectious agents should be followed accordingly.
	<ol>
		<li>Plate H1-HeLa cells in a 6-well plate such that they are ~80% confluent at the time of transfection (24 h post seeding). Plate cells in 2 mL per well of DMEM supplemented with 10% fetal bovine serum.</li>
		<li>Warm the transfection reagent to room temperature. Combine 250 &#181;L serum-free media, 2.5 &#181;g of plasmid DNA encoding microRNA-targeted genome, and 7.5 &#181;L of transfection reagent in a sterile microcentrifuge tube and pipet gently to mix. Incubate the mixture at room temperature for 15-30 min.</li>
		<li>Aspirate the media from the plated cells and add 2 mL of fresh complete media.</li>
		<li>Add the entire transfection mixture from step 2.1.2 to one well of the 6-well plate drop-wise. Add each drop to a different area of the well.</li>
		<li>Incubate the cells at 37 &#176;C until cytopathic effects (CPE) or reporter proteins are detectable (~24-72 h).</li>
	</ol>
	</li>
	<li>Harvest and passage the rescued virus onto fresh cells.
	<ol>
		<li>Plate cells in a 6-well plate such that they are 80-90% confluent at the time of infection (24 h post seeding). 
		NOTE: Scale up or down depending on the amount of virus needed for all subsequent experiments.</li>
		<li>Harvest each rescue well by scraping the cells into the supernatant using a rubber cell scraper or rubber policeman. Gently drag the scraper across the bottom of the well. Transfer the cells and supernatant into a cryogenic storage tube.</li>
		<li>Freeze/thaw the samples two times and then pellet the cellular debris by centrifuging at 1,200 x g for 5 min at 4 &#176;C.</li>
		<li>Filter the cleared supernatant twice using 0.2 &#181;m syringe filters. The filtered rescue supernatant contains the virus. 
		NOTE: Filter pore size may vary depending on the size of virus particles and may not be applicable for large viruses.</li>
		<li>Aspirate media from the plated cells, wash once with serum-free media and add 1 ml of fresh serum-free media to each well.</li>
		<li>Add 50 &#181;L per well of filtered rescue supernatant and gently rock the plate to evenly distribute virus.</li>
		<li>Incubate the plate at 37 &#176;C for 2 h and then aspirate media from each well to remove unincorporated virus.</li>
		<li>Add 1.5-2 mL of fresh complete media per well and incubate at 37 &#176;C until CPE or reporter proteins are apparent (~24-48 h).</li>
		<li>Repeat steps 2.2.2 to 2.2.4. Filtered rescue supernatant contains virus stock. Store virus stocks in single-use aliquots in cryogenic storage tubes at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Rescuing microRNA-targeted Virus from In Vitro Transcribed RNA Transcripts
<ol>
	<li>Generate RNA transcripts from plasmid DNA encoding the microRNA-targeted viral genome using an upstream promoter. 
	NOTE: Standard practices for generating and maintaining a nuclease-free environment should be used for all subsequent steps.</li>
	<li>Linearize plasmid DNA using an enzyme restriction site downstream of the transcript. Combine 5 &#181;g plasmid DNA, 1x final concentration enzyme buffer, 3 &#181;l restriction enzyme and bring to 50 &#181;l with H2O. Incubate the reaction at 37 &#176;C for 3 hr.</li>
	<li>Add 1/20th volume of 0.5 M EDTA, 1/10th volume 5 M NH4 acetate, and 2 volumes of 100% ethanol to the digested reaction and mix. Incubate at -20 &#176;C for 1 h up to overnight.</li>
	<li>Pellet the precipitated DNA for 10 min at 17,000 x g at 4 &#176;C. Pour off the supernatant, resuspend the pellet in 500 &#181;L of cold 70% ethanol and pellet the DNA by centrifuging at 17,000 x g for 10 min at 4 &#176;C.</li>
	<li>Pour off the supernatant and centrifuge again for 30 sec. Remove the residual supernatant with a pipet and air-dry the pellet. Resuspend the DNA pellet in sterile nuclease-free H2O at a concentration of 0.5-1 &#181;g/&#181;L.</li>
	<li>Thaw the in vitro transcription reagents at room temperature and place the ribonucleotides on ice (enzymes in glycerol do not freeze and should go directly on ice). Keep the reaction buffer at room temperature.</li>
	<li>Assemble the transcription reaction in a PCR tube by combining 2 &#181;L each of ATP, CTP, GTP, and UTP solutions, 2 &#181;L 10x reaction buffer, 1 &#181;g linearized DNA, 2 &#181;l enzyme, and bring to a final volume of 20 &#181;L with nuclease-free H2O.</li>
	<li>Incubate the reaction at 37 &#176;C for 2 hr.</li>
	<li>Purify the RNA transcripts. 
	NOTE: Before starting, prepare wash solution with ethanol as directed and preheat elution solution to 95 &#176;C for elution step.
	<ol>
		<li>Bring the transcription reaction to 100 &#181;L with the elution solution (not-preheated). Add 350 &#181;L of binding solution concentrate and 250 &#181;L of 100% ethanol to reaction and mix.</li>
		<li>Transfer the sample to a filter cartridge inserted into a collection tube and centrifuge for 1 min at 12,000 x g. Discard the flow-through.</li>
		<li>Add 500 &#181;L of wash solution to the filter cartridge and centrifuge for 1 min at 12,000 x g. Discard flow-through. Repeat this wash step one more time. Discard flow-through. Centrifuge for 1 min at 12,0000 x g to remove residual ethanol.</li>
		<li>Transfer the filter cartridge to a clean collection tube and add 50 &#181;L of the preheated elution solution to the filter cartridge. Centrifuge for 1 min at 12,000 x g. Repeat this elution step for a total volume of 100 &#181;l of eluent containing purified RNA transcripts. Discard filter-cartridge.</li>
	</ol>
	</li>
	<li>Determine the RNA concentration in the eluent by measuring the absorbance of the sample at 260 and 280 nm and using the Beer-Lambert Law.</li>
	<li>Assess the integrity of the RNA using RNA gel electrophoresis37. See Figure 3 for examples of good and bad RNA integrity. RNA should be aliquotted into single-use tubes and stored at -80 &#176;C to maintain integrity.</li>
	<li>Warm transfection reagents to room temperature. Combine 250 &#181;l serum-free media, 2.5 &#181;g of purified RNA transcripts, 5 &#181;L of boost solution, and 5 &#181;L of transfection reagent in a sterile microcentrifuge tube and pipet gently to mix. Incubate the mixture at room temperature for 2-5 min.</li>
	<li>Plate H1-HeLa cells in a 6-well plate such that they are ~80% confluent at the time of transfection (24 h post seeding). Plate cells in 2 mL per well of DMEM supplemented with 10% fetal bovine serum.</li>
	<li>Aspirate the media from the plated cells and add 2 mL of fresh complete media.</li>
	<li>Add the entire transfection mixture from step 3.12 to one well of the 6-well plate drop-wise. Add each drop to a different area of the well.</li>
	<li>Incubate the cells at 37 &#176;C until cytopathic effects (CPE) or reporter proteins are detectable (~24-72 h).</li>
	<li>Continue rescue of microRNA-targeted virus as described in steps 2.2 to 2.2.9.</li>
</ol>
4. Titrating Virus Stocks by Calculating 50% Tissue-culture Infectious Dose (TCID50)
<ol>
	<li>Plate H1-HeLa cells in a 96-well plate at 104 cells per well in DMEM supplemented with 10% fetal bovine serum. Incubate cells at 37 &#176;C overnight. 
	NOTE: Titrate virus in cells permissive for virus replication that do not express cognate microRNAs.</li>
	<li>Make 10-fold serial dilutions of virus each in a total volume of 1 mL of serum-free media. 
	NOTE: Use lower dilutions if more precision is required. 
	NOTE: Change tips after each dilution to prevent overestimation of titer as virus can stick to tips.</li>
	<li>Tip 96-well plate to the side and aspirate media from plated HeLa cells. Add 100 &#181;L of each virus dilution per well to 8-wells of 96-well plate (1 row per dilution). Add 100 &#181;L of serum-free media without virus to row 1 and row 12 on plate for controls. 
	NOTE: Always aspirate and add media to wells by touching tip to side of well to prevent cell detachment.</li>
	<li>Incubate plate at 37 &#176;C for 2 hr. Tip plate to the side and aspirate the media from the wells. 
	NOTE: Change tips between each dilution row.</li>
	<li>Add 100 &#181;l of complete media to each well. Incubate plate at 37 &#176;C for 72 hr.</li>
	<li>Visualize the wells under a microscope and mark each well positive or negative for CPE.</li>
	<li>Calculate each virus titer with the following equation: Log10 (TCID50/ml) = L + D(S-0.5) + log10 (1/V). L is the negative log10 of the most concentrated virus dilution tested in which all wells are positive. D is the log10 of the dilution factor. S is the sum of individual proportions (pi). pi is the calculated proportion of an individual dilution (amount of positive wells/total amount of wells per dilution. V is the volume of inoculum (ml/well).</li>
</ol>
<img alt="Table 1" src="/files/ftp_upload/55033/55033tbl1.jpg" /> 
Table 1: Quantifying infectious virus particles by calculating the TCID50. Representative results from cells infected with a series of ten-fold dilutions of a virus stock. &#34;L&#34; equates to 4 because the last dilution where all the wells are positive for CPE is 10-4. &#34;D&#34; is the log10 of 10 since 10-fold dilutions were used and is therefore equal to 1. &#34;S&#34; is the sum of the individual proportions. In this example the individual proportions are 1.0, 0.875, 0.375, 0.125, and 0. The sum of these (S) is 2.375. &#34;V&#34; is the volume of inoculum in ml used to infect the cells initially.
5. Evaluating microRNA-targeting Efficacy: Single-step Growth Kinetics
<ol>
	<li>Controls for this assay include mock-infected cells, unmodified virus and virus containing a non-targeted or non-functional response element.</li>
	<li>Plate H1-HeLa cells for a time course experiment in 12-well tissue culture plates such that they are 80-90% confluent at the time of infection (24 h post seeding). Plate cells in DMEM supplemented with 10% fetal bovine serum at 1 mL per well. 
	NOTE: Recommended to plate a separate plate for each time point. This protocol uses 7 different time points for a virus with an 8-12 hr replication cycle. H1-HeLa cells are not required to perform this assay. This assay should be performed in permissive cells that do not express the targeted microRNAs. This assay can also be performed in cells expressing the cognate microRNAs to evaluate growth kinetics under selective pressure.</li>
	<li>Aspirate media from wells. Wash wells by adding 0.5 mL of serum-free medium to each well, swirl the plate, and aspirate media from wells. Add 0.5 mL of fresh serum-free media to each well.</li>
	<li>Infect each well at a high multiplicity of infection (MOI; number of infectious particles per cell) to ensure all cells get infected. This protocol uses an MOI of 3.
	<ol>
		<li>Dilute virus stocks in serum-free media to a concentration of MOI = 3 per 100 &#181;L. Add 100 &#181;L of virus dilution each to seven different wells and incubate at 37 &#176;C for 2 h.</li>
		<li>Aspirate the media from all wells and wash two times by adding 0.5 mL complete media per well, rocking gently and then aspirating.</li>
	</ol>
	</li>
	<li>Add 1 mL of complete media to each well and incubate at 37 &#176;C until desired time point.</li>
	<li>Collect samples for virus titration at 2, 4, 6, 8, 10, 24 and 48 h post-infection (1 well per time point). Transfer 700 &#181;L of the media in the well to a cryogenic storage tube. Scrape the cells into the remaining supernatant by gently moving a rubber scraper across the entire well. Transfer the cell/supernatant mixture to the corresponding cryogenic storage tube containing the 700 &#181;L of supernatant. 
	NOTE: Make sure not to splash supernatant from one well into another during scraping resulting in contaminated samples. Transferring the 700 &#181;L prior to scraping will minimize splashing.</li>
	<li>Place samples at -80 &#176;C until all samples are collected.</li>
	<li>Freeze/thaw the samples three times and remove cellular debris by centrifuging the samples at 1,200 x g for 5 min at 4 &#176;C.</li>
	<li>Titrate the virus as described in section 4 and compare growth kinetics over time.</li>
</ol>
6. Evaluating microRNA-targeting Specificity: Virus-spreading Assay Using Synthetic microRNA Mimics
NOTE: Use of a synthetic microRNA mimic is performed to show specificity of the microRNA:microRNA-target interaction.
<ol>
	<li>Include mock transfection, negative control microRNA mimic and experimental microRNA mimic only controls for every assay to evaluate microRNA-mediated toxicity. It is also ideal to include a positive control (i.e. microRNA-targeted gene or genome) as low transfection efficiency of microRNA mimics can result in false negatives.</li>
	<li>Plate H1-HeLa cells in a 96-well tissue culture plate such that they are 80-90% confluent at the time of transfection (24 h post-seeding). Plate cells in DMEM supplemented with 10% fetal bovine serum at 0.1 ml per well. 
	NOTE: Perform this assay in cells permissive for viral replication that do not express the cognate microRNAs.</li>
	<li>Warm the transfection reagents to room temperature. Combine 9 &#181;L serum-free media, 200 nM final concentration per well of microRNA mimic stock, 0.18 &#181;L boost reagent, 0.18 &#181;L transfection reagent and mix. Incubate the mixture at room temperature for 2-5 min. 
	NOTE: Volumes listed are per well. It is recommended that a master mix be assembled for transfection of all wells to maintain consistency and minimize pipetting error. The optimal concentration of microRNA mimic will vary. Consult the manufacturer&#39;s instructions accompanying the microRNA mimic for a reasonable starting concentration. This will generally range from 5-200 nM final concentrations.</li>
	<li>Aspirate the media from the wells and add 92 &#181;l of fresh complete medium. 
	NOTE: If there are a significant number of samples, complete this step prior to assembling the transfection solution and store the plate at 37 &#176;C until ready.</li>
	<li>Add the entire transfection mixture in step 6.3 to the cells drop-wise.</li>
	<li>Incubate at 37 &#176;C for 6 h.</li>
	<li>Infect each well at a low MOI to ensure a low percentage of cells get infected to allow analysis of virus spread. This protocol uses an MOI of 0.2.
	<ol>
		<li>Dilute virus stocks in serum-free media to a concentration of MOI = 0.2 per 100 &#181;L. Remove media from the wells and add 100 &#181;L of virus dilution per well.</li>
		<li>Incubate at 37 &#176;C for 2 h.</li>
	</ol>
	</li>
	<li>Aspirate media from each well and add 100 &#181;L of fresh complete media.</li>
	<li>Incubate at 37 &#176;C for 20-22 h.</li>
	<li>Determine the virus titration in the supernatants.
	<ol>
		<li>Collect the supernatant from each well and replace with 100 &#181;L of fresh complete media. 
		NOTE: If using suspension cells, resuspend the cell pellet from the following step in 100 &#181;L of fresh complete media and return to sample well for viability assay.</li>
		<li>Remove cellular debris from the collected supernatants by centrifuging at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Transfer the cleared supernatant to a fresh tube and titrate infectious virus on permissive cells that do not express the cognate microRNAs as described in section 4. 
		NOTE: Keep all samples on ice to prevent loss of infectivity.</li>
	</ol>
	</li>
	<li>Determine the viability of the cells.
	<ol>
		<li>Add 10 &#181;L of MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) reagent per well.</li>
		<li>Incubate at 37 &#176;C for 2-4 h until purple precipitate is visible.</li>
		<li>Add 100 &#181;L of detergent reagent.</li>
		<li>Incubate at room temperature in the dark for 2 h.</li>
		<li>Read absorbance of all wells at 570 nm. 
		NOTE: Normalize all samples to mock transfected cells for comparison of percent cell viability.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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 optimization10,11,12,13,38.
When initiating the design of REs, investigators should begin by compiling a set of microRNAs that are expressed in the target cells of interest, are diminished in the non-target cells of interest, and that have been experimentally validated. Following this compilation, several prediction-based analyses should be conducted to address the possibility of competing microRNA-target interactions. Many viruses encode microRNAs or non-coding RNAs that sequester cellular microRNAs in order to manipulate viral and host gene expression. Moreover, several RNA viruses have been shown to interact with cellular microRNAs in order to enhance their replication capacity39,40. Therefore, when designing REs, be sure to investigate whether there are known interactions of the virus with cellular microRNAs or if they express viral microRNAs that could potentially recognize the chosen microRNA target or alter the endogenous expression of the chosen microRNA. In addition to literature searches, the reader should consider online bioinformatics prediction tools and databases that include viral microRNA target information to aid in the rational design of REs. Such databases and prediction tools can also facilitate identification of target sequences that can be recognized by multiple microRNAs or overlapping seed sequences present within the viral genome. For a more in-depth discussion on methods of identifying microRNA targets and the pros and cons of some of the online prediction tools the reader is referred to references 41,42. It is recommended that these prediction tools serve only as guides as many times predictions can yield false positives or negatives. To this end, there may be seed sequences that overlap, however the 3&#39; end of the microRNA will also influence the targeting efficiency. Using too stringent of a rule set can sometimes be detrimental.
Once a set of potential microRNA targets have been identified, the investigator should continue by ranking the targets based on the biological properties of the microRNAs and RNA structural predictions10,11,12,13,38. The absolute abundance of the mature microRNA in targeted cells and its level of association with an Argonaute protein will dictate the degree of gene silencing. Several studies have demonstrated that only abundantly expressed microRNAs will significantly regulate gene expression16,43,44. Moreover, while some microRNAs are abundantly expressed their interaction with Argonaute proteins and formation of RISC is insufficient to repress translation44. Using a highly abundant microRNA with validated function will also minimize the potential for microRNA saturation in the target cell and subsequent off-target toxicities. A different approach may be to use REs that are targeted by multiple miRNAs45,46, which could allow for a reduced copy number while still minimizing the potential for off-target toxicities. The ratio of target mRNA to microRNA will also have a significant impact on the success of this approach. A high target to microRNA ratio will reduce the level of repression42,43,47,48. This is especially critical with viruses that replicate rapidly when if improperly regulated early during infection will accumulate to levels beyond the control of endogenous microRNAs. It is essential to consider these properties when choosing a microRNA for targeting; ideally, using a microRNA whose function has been experimentally validated.
The efficiency of targeted gene silencing is also affected by the percent complementarity of the target to the mature microRNA as well as the number of copies of microRNA-target sequences inserted. The 5&#39; region of the microRNA (nucleotides 2-8) constitutes the seed sequence. It is generally accepted that this region must be perfectly complementary for successful targeting48,49. The majority of studies use response elements with perfect complementarity because it can increase gene-silencing activity by promoting endonucleolytic cleavage of the transcript and rapid recycling of the microRNA. This endonucleolytic cleavage only occurs when a microRNA interacts with an Argonaute protein that has endonucleolytic activity, which in mammalian cells is restricted to Argonaute-2. Other Argonaute proteins promote mRNA degradation by deadenylation and exonucleolytic attack of the transcript. The reader is referred to references4,5,13,50,51,52 for an in-depth description of microRNA biogenesis and function. It is generally thought that increasing the copy number will further enhance the targeting efficiency. However, this can also potentiate microRNA saturation and has not always proven more efficacious. It is sometimes better to incorporate target sequences for multiple different microRNAs enriched in the target cells or to use target sequences that are recognized by multiple microRNAs. The copy number required is also highly dependent on the amount of transcript that will need silencing and the relative abundance of the microRNA in the target cells. Transcripts that will accumulate quickly may require more copies to prevent them from outcompeting the microRNA levels. Experimentally determining the optimal copy number for each microRNA target that results in sufficient targeting, maintains viral fitness, and that minimizes the rate of escape mutations is recommended.
Localization of the microRNA response element within the viral genome is extremely important. A negative result when analyzing the targeting efficiency does not always mean the virus cannot be microRNA-targeted. It may simply mean the target is not accessible in that location or that repression of the targeted gene is not sufficient to inhibit pathogenicity. The majority of response elements are inserted into the 3&#39; untranslated regions (UTR) of the targeted transcript. However, successful targeting of viral genomes has been accomplished and sometimes exhibits enhanced genetic stability when response elements are inserted into the 5&#39; UTR or within the coding regions of essential genes38. Optimal insertion sites will allow high accessibility of the target sequence to the microRNA. Accessibility can be hindered by RNA secondary structures and stoichiometric interference. Therefore, localizing response elements in unstructured regions that are highly conserved is a good starting point. The sequences being inserted may also influence and be influenced by the surrounding sequences. Thus, an optimal location for one microRNA target sequence is not necessarily optimal for other response elements. While experimental validation and optimization of RE localization is necessary, using prediction software (e.g. http://unafold.rna.albany.edu/; http://rna.urmc.rochester.edu/software.html) to screen potential insert sites for perturbation of the surrounding RNA structures is highly recommended53,54.
The use of clean nucleic acid preparations of high integrity is also critical for obtaining the best results. Aseptic technique and maintenance of an RNase-free environment when handling RNA transcripts or microRNA mimics is essential. For the best results RNA transcripts, microRNA mimics, and final titrated virus stocks should be stored at -80 &#176;C in small aliquots to avoid repeated freezing and thawing resulting in RNA degradation and loss of virus infectivity. When in use, these reagents should be kept on ice at all times.
It is important to note that many microRNAs are members of a family of microRNAs that share complementarity. Therefore when conducting studies it may be beneficial to include members of the immediate microRNA family to improve targeting-specificity evaluation. This becomes critical when the cells within which virus replication is desired, express members of the family (e.g. miR-Let7 family). It should also be noted that the specificity assay using microRNA mimics is an artificial system and over-expression of a microRNA in some cells may result in off-target effects55. If this occurs, the specificity can also be evaluated in cells that express the appropriate microRNAs using microRNA inhibitors instead. This assay would allow analysis of targeting-specificity based on virus titration and cell viability readouts in the presence of physiologically relevant levels of microRNAs.
The main concerns when using microRNA-targeting as a method to control viral tropism are genetic stability, potential for off-target effects, and virus-mediated effects on the cellular microRNA environment10,11,12,13. Many viruses exhibit high mutation rates and escape mutants may arise quickly. Including multiple copies of a target sequence, including targets for more than one microRNA, localizing the targets within multiple highly conserved regions, or the presence of additional antiviral factors including an immune response can alleviate this. Additionally, insertion of foreign genetic material into a viral genome will often result in diminished replication capacity of the virus. If this occurs, the microRNA target is likely to be genetically instable and escape mutants will arise more rapidly. Inclusion of multiple microRNA target copies can also increase the potential for recombinatorial deletion of the microRNA targets. Therefore, experimental determination of the minimum copy number required for sufficient targeting is necessary. Localizing microRNA target copies in various locations throughout the genome versus using tandem repeats can aid in bypassing this constraint. However, identifying optimal RE configurations with the minimum number of target copies needed and insert sites that do not result in altered replication kinetics of the virus is critical for minimizing the rate of recombination and target mutation. Inclusion of too many copies of a target sequence may also increase the potential for off-target toxicities if it results in microRNA saturation. A major alteration in the amount of microRNA available for regulating normal cellular proteins could result in undesired effects55. To this end, many viruses alter the microRNA environment within a cell56 and if this includes the targeted microRNA, the efficiency of regulation may be decreased if enough virus replication is maintained. All of these concerns may be addressed by optimizing response element composition and/or localization and with a thorough understanding of the system being targeted and its limitations.
Typical problems associated with other targeting methods include off-target attenuation of the virus, size constraints for genetic targeting material in viruses with limited carrying capacities, and safety concerns associated with engineering viruses to target cells that are normally not infected. MicroRNA-targeting allows for regulation of viral tropism with minimal modification of the virus. It requires minimal space within a viral genome and can be tailored based on an expansive array of cellular microRNA signatures. This technique does not introduce new tropisms for the virus and therefore does not introduce any new safety concerns. Additionally, this method can be used to regulate multiple tropisms simultaneously by using target sequences for multiple different microRNAs enriched within different cell types16,17,57,58,59,60,61. This can all be accomplished without attenuating the virus in cells that do not express the corresponding microRNAs and therefore can provide a mechanism for improving the safety of therapeutic viruses with enhanced potency.
Exploitation of cellular microRNA machinery can be used for regulating the tropism of many different classes of viruses. The protocols detailed here are designed for the rescue and characterization of a microRNA-targeted picornavirus, however they can be adapted according to a virus&#39; replication cycle and specific reporter readouts. Although different viruses have specific rescue strategies, microRNA target sequences can be inserted into the viral genome regardless of whether the entire genome is encoded in a single plasmid or whether the virus has a DNA or RNA genome. As long as the producer cells do not express the cognate microRNAs, optimized microRNA targets should not disrupt virus rescue. Experiments to analyze virus growth kinetics and microRNA-targeting specificity can be modified by changing the time points collected based on the length of a single round of virus replication and on specific readouts for virus replication/toxicity (e.g. reporter proteins, cytotoxicity, genome quantification, etc.). It is important to note that although this technique can theoretically be applied to all classes of viruses, many factors can affect the efficiency of this method. For example, negative-sense RNA viruses have not proven as responsive as positive-sense RNA viruses likely due to limited accessibility of the genomic RNA to the miRNA machinery13. Thus, the biological properties of each class of viruses will impart additional constraints on RE optimization. Despite these constraints, this technique offers an alternative method for targeting viral tropism facilitating research involving the safety, utility, and basic understanding of biological processes for all classes of viruses.

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.

<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.

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 insertion, and a map of plasmid DNA encoding a microRNA-targeted viral genome for in vitro transcription is depicted in Figure 2. Figure 3 shows RNA transcripts at varying degrees of integrity visualized by agarose gel electrophoresis. Proper handling and the use of DNA templates devoid of impurities will result in properly transcribed RNA transcripts as shown in 3A lane 2. Residual impurities within the linearized DNA templates or trace amounts of nuclease can result in low levels of improperly transcribed RNA and/or degradation products similar to that observed in 3A lane 3. Incomplete linearization of plasmid DNA or DNA containing high amounts of impurities such as residual ethanol will result in improper transcription and degradation products represented in 3A lanes 4 and 5. Figure 3 also shows an image of Mengovirus-mediated cytopathic effects (CPE) following transfection of clean RNA transcripts. Figure 4 displays data representative of a time course experiment evaluating the growth kinetics of unmodified and microRNA-targeted Mengovirus. The results show that microRNA response elements engineered into the Mengovirus genome do not alter the kinetics of virus replication in H1-HeLa cells, which do not express any of the cognate microRNAs. However, in RAW 264.7 macrophages, which express intermediate levels of microRNA-125 and high levels of microRNA-142, viruses encoding the corresponding response elements display inhibited replication kinetics that correlates with the level of microRNA expressed. Data in Figure 5 shows the specificity of microRNA-based regulation of Mengovirus tropism. Overexpression of each individual microRNA in H1-HeLa cells specifically inhibits propagation (lower virus titers) and cytotoxicity (increased cell viability) of Mengovirus encoding the cognate microRNA response element.
<img alt="Figure 1" src="/files/ftp_upload/55033/55033fig1.jpg" /> 
Figure 1: Schematic representation of microRNA-based regulation of viral tropism. MicroRNA response elements (RE) incorporated into a viral genome will be recognized by cognate microRNAs enriched within specific cell types and result in targeted degradation of the viral transcripts/genomes. This will prevent virus replication, spread and toxicity to the surrounding cells. However, viral replication will be maintained in cells that do not express the cognate microRNAs allowing for virus propagation, spread and cell death. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55033/55033fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55033/55033fig2.jpg" /> 
Figure 2: MicroRNA response element design. (A) For successful targeting the seed sequence region of the RE should be perfectly complementary. The RE should be oriented within the viral genome such that it can be recognized by the cognate mature microRNA in mRNA transcripts and/or the viral genome. The majority of REs are placed within the 3&#39; UTR of the transcripts. (B) Depiction of properly designed and annealed oligonucleotides encoding REs containing tandem repeats of the microRNA-target sequences (miRT) flanked by the overhanging nucleotides of the XhoI restriction enzyme site. When designing tandem repeat REs, be sure to include spacer nucleotides (generally 4-6) between each microRNA-target copy. (C) Plasmid DNA encoding a full-length viral genome with a RE incorporated into the 3&#39; UTR, a T7 promoter, and a unique restriction site for linearization for in vitro transcription. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55033/55033fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55033/55033fig3.jpg" /> 
Figure 3: Rescue of virus from genome-encoding RNA transcripts. (A) RNA gel electrophoresis of in vitro transcribed RNAs encoding picornavirus genomes to evaluate transcript integrity. Lane 1: RNA ladder. Lane 2: Properly transcribed RNA with high integrity. Lane 3: RNA transcripts with moderate integrity. Higher band is correct size and lower band is potentially a degradation product or an undesired RNA transcript. Lane 4: Improperly transcribed RNA. Lane 5: Low integrity RNA. 500 ng of in vitro transcribed RNA was loaded per well. Improper transcription or degradation products are likely due to the use of low purity or incompletely linearized DNA. (B) Image of mock transfected H1-HeLa cells and cells transfected with RNA transcripts encoding Mengovirus. Administration of transfection reagents only (Mock) will maintain cell viability following transfection as well as passage onto fresh cells. Transfection of RNA transcripts encoding a Mengovirus genome (vMC24) results in cytopathic effects, which are maintained following filtration of the supernatant and passage onto fresh H1-HeLa cells. Scale bar = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55033/55033fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55033/55033fig4.jpg" /> 
Figure 4: Growth kinetics of unmodified and microRNA-targeted Mengoviruses in the presence or absence of cognate microRNAs. Modified from reference17. (A) H1-HeLa cells do not express miR-124, -125, or -142. All three Mengovirus encoding microRNA REs replicate with similar kinetics as the unmodified virus (vMC24) indicating that insertion of the microRNAs at this location (5&#39; UTR) do not alter virus replication. (B) RAW 264.7 macrophages express intermediate levels of miR-125b and high levels of miR-142-3p, but do not express miR-124. Incorporation of the corresponding microRNA-target sequences into the genome results in virus attenuation consistent with the level of microRNA expression. Data is represented as mean viral titers +/- SD. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55033/55033fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55033/55033fig5.jpg" /> 
Figure 5: Analysis of microRNA-targeting specificity using synthetic microRNA mimics. Modified from reference17. H1-HeLa cells transfected with individual microRNA mimics were infected with unmodified virus (vMC24) or microRNA-targeted virus (miRT-vMC24) at an MOI of 0.2. (A) The cell viability normalized to mock-treated cells was determined at 24 hr post-infection via MTT cell proliferation assay. (B) Virus titer in the supernatant of all samples was also determined at 24 hr post-infection. The data is represented as mean viability or viral titer +/- SD. Two-tailed unpaired Student t tests with Welch&#39;s correction (for unequal variances) were used for statistical analysis. A p value &#60;0.01 was considered significant. (**, p &#60;0.01, ***, p &#60;0.001). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55033/55033fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

microrna-based-regulation-of-picornavirus-tropism

Nanyang Technological University

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.

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

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.

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

Developmental Biology

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

Environment

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.

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.

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.

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

Cancer Research

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.

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

Biology

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.

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.

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.

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

MicroRNA-based Regulation of Picornavirus Tropism

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