The overall goal of the following experiments is to assess the efficiency and specificity of microRNA-based regulation of picornavirus tropism in vitro. This is achieved by preparing picornavirus encoding microRNA response elements in their genome. As a second step, use the viruses to infect cells that express or don't express the cognate microRNAs.
Then, evaluate the viral replication genetics, propagation, and toxicity to determine the efficiency and specificity of targeting. This targeting method can be used in the field of virology to improve virus safety, manufacturing and utility, or to facilitate research focused on understanding the biological processes involved in a virus infection. The main advantages of this technique are that it requires minimal engineering of the viral genome, and it's highly versatile because a significant number of cellular microRNA signatures have been identified.
To begin this procedure, plate the permissive cells in a 96-well plate and complete growth media. Add serum-free media without virus to row one and row 12 for uninfected controls. Then, incubate the cells at 37 degrees Celsius overnight.
Next, make ten-fold serial dilutions of virus, each in a total volume of one milliliter of serum-free media. Add 100 microliters of serum-free media without virus to row one and row 12 on the plate for controls. Tip the 96-row plate to a side and aspirate the media from the plated cells.
After that, add 100 microliters of each virus dilution to eight wells of the 96-well plate. Incubate the plate at 37 degrees celsius for two hours. After two hours, tip the plate to a side and aspirate the media from the wells.
Add 100 microliters of complete media to each well, and incubate the plate at 37 degrees celsius for 72 hours. After 72 hours, visualize the wells under a microscope, mark each well positive or negative for the cytopathic effects, and calculate the virus titer. In this procedure, plate the cells for a time course experiment in the 12-well tissue culture plates such that they are 80 to 90%confluent at the time of infection.
After 24 hours, aspirate the media, and wash the wells by adding 0.5 milliliters of serum-free medium to each well, swirl the plate, and aspirate the media. Then, add 0.5 milliliters of fresh, serum-free media to each well. Next, dilute the virus stocks in serum-free media.
Add 100 microliters of each virus dilution to seven different wells, and incubate them at 37 degrees Celsius for two hours. After two hours, aspirate the media from all the wells, and wash the cells two times by adding 0.5 milliliters of complete media to each well, rocking gently, and then aspirating the media. Subsequently, add one milliliter of complete media to each well, and incubate the samples at 37 degrees celsius until the desired time point.
Transfer 700 microliters of the media from each well to a cryogenic storage tube. Scrape the cells into the remaining supernatant by gently moving a rubber scraper across the entire well. Next, transfer the cells'supernatant mixture to the corresponding cryogenic storage tube containing 700 microliters of supernatant.
Place the samples at 80 degrees celsius until all of them are collected. Then, freeze and thaw the samples three times. Remove the cellular debris by centrifuging the samples at 1, 200 x g for 5 minutes at four degrees celsius and titrate the virus.
In this step, plate the permissive cells in a 96-well tissue culture plate such that they are 80 to 90%confluent at the time of transfection. Then, warm the transfection reagents to room temperature. Make a master mix by combing nine microliters of serum-free media, 200 nanomolar microRNA mimic stock, 0.18 microliters of boost reagent, and 0.18 microliters of transfection reagent in each well, and mix.
Incubate the mixture at room temperature for two to five minutes. Afterward, aspirate the media from the wells, and add 92 microliters of fresh complete medium. Then, add the appropriate volume of transfection mixture to the cells drop wise, and incubate them at 37 degrees celsius for six hours.
MicroRNA mimic transfections can be incubated for 24 hours prior to infection to improve transfection efficiency if necessary. Next, dilute the virus stocks in serum-free media. Remove the media, and add 100 microliters of virus dilution to each well.
Subsequently, incubate the cells at 37 degrees celsius for two hours. After two hours, aspirate the media from each well, and add 100 microliters of fresh complete media. Then, incubate the cells at 37 degrees celsius for 20 to 22 hours.
To determine the virus titration in the supernatants, collect the supernatant from each well, and replace it with 100 microliters of fresh complete media. After that, remove the cellular debris from the collected supernatants by centrifuging at 300 x g for five minutes at four degrees celsius. Then, transfer the cleared supernatants to a fresh tube, and titrate the infectious virus on permissive cells that do not express the cognate microRNAs.
To determine the viability of the cells, add 10 microliters of MTT reagent to each well. Incubate the cells at 37 degrees celsius for two to four hours until purple precipitate is visible. After that, add 100 microliters of detergent reagent, and incubate the cells at room temperature in the dark for two hours.
Subsequently, read the absorbance of all wells at 570 nanometers. The representative results for microRNA-targeted virus in the presence and absence of the cognate microRNAs compared to unmodified virus are showed here. Mango viruses containing microRNA response elements replicated with similar kinetics to unmodified virus, shown in red, and H1-HeLa cells, which did not express the cognate microRNAs.
In contrast, viruses containing response elements complementary to microRNAs 142 and 125 had restricted replication in RAW 264.7 cells, which express high levels of microRNA-142 and intermediate levels of microRNA-125. The replication kinetics of mango virus containing microRNA-124 response elements remained similar to unmodified virus because RAW 264.7 cells expressed little to no microRNA-124. The representative results for determining the specificity of microRNA targeting are shown here.
The cell viability of H1-HeLa cells infected with microRNA-targeted viruses was only improved when the cells were transfected with the corresponding microRNA mimic. Correspondingly, a decrease in virus titer is observed in these samples as well, which demonstrates the specificity of targeting. While attempting this procedure ensured that the permissive cells do not express the targeted microRNAs, and non-permissive cells express an appropriate level of a microRNA with validated function, as permissive and non-permissive cells will be different for different viruses.
Don't forget, working with viruses can be extremely hazardous, and all guidelines for the safe handling and disposal of infectious agents should be followed accordingly.
