The overall goal of this procedure is to compare the efficacy and toxicity of new RNAs targeting HIV-1 production. This method can help identify new RNA molecules for using HIV-1 gene or drug therapy, such as short hairpin or small interfering RNAs, ribozymes, and RNA decoys. The main advantage of this technique is that several new RNA designs can be screened simultaneously with a quick turnaround time.
Although this method was designed to evaluate new RNA drug or gene therapy for HIV, it can also be applied to other systems, such as small molecules or CRISPR tasks. We first had the idea for this method when we were setting up experiments to screen a large number of ribozymes for anti-HIV effects. To begin, prepare a suspension of human embryonic kidney 293T cells at two times 10 to the fifth cells per milliliter in DMEM with 10%FPS and 1%penicillin streptomycin.
Add 500, 100 and 1, 000 microliters of the cell suspension to each of the wells of 24, 96, and 12-well plates for viral production, cell viability, and immune activation assays, respectively. Gently swirl the plates and incubate them overnight at 37 degrees Celsius with 5%carbon dioxide to 50%to 70%confluency. For the viral production assay, prepare a 10 nanogram per microliter dilution of an HIV-1 expression plasmid and add 10 microliters to each assay tube.
Next, prepare five micromolar dilutions of test RNAs and a negative control RNA. Then add 2.5 microliters or 10 microliters of each test RNA and negative control dilution to the corresponding tubes for 25 nanomolar or 100 nanomolar final concentrations. For the cell viability assay, prepare five micromolar dilutions of test RNAs and a 10 milligram per milliliter dilution of the positive control RNA, low molecular weight poly(I:C)Add two microliters of the test RNA or the positive control RNA dilutions to the corresponding tubes for 100 nanomolar or 200 micrograms per milliliter final concentrations, respectively.
For the immune activation assay, add 20 microliters of test RNA or positive control RNA dilutions, as prepared for the cell viability assay, to the corresponding tubes for 100 nanomolar or 200 micrograms per milliliter final concentrations, respectively. Then add 50, 25, or 75 microliters of DMEM to each transfection tube for viral production, cell viability, and immune activation assays, respectively. For viral production assays, bring cells and prepared transfection tubes to a biosafety level three, or BSL-3, laboratory before the next step.
Add two microliters of the transfection reagent sequentially to the transfection tubes, and incubate 15 to 20 minutes to allow complexes to form. Add the entire transfection mixture from each microtube drop-wise to the corresponding positions in the cell culture plates. Gently swirl, and incubate the plates for 48 hours at 37 degrees Celsius and 5%carbon dioxide.
To carry out the viral production assay, after removing the 24-well cell culture plates from the incubator, transfer them to the BSL-3 cell culture hood. Gently swirl the plates and then transfer 150 microliters of the supernatant from each well to a corresponding well in a 96-well flat-bottom plate to be used to quantify HIV-1 production. Transfer five microliters of supernatant to the corresponding wells in a 96-well plate containing 25 microliters of a viral disruption cocktail.
After incubating the mixture for five minutes at room temperature, transfer the plate to a radioactivity work station. Next, prepare a radioactive cocktail, and add 25 microliters to each well of viral supernatant and disruption cocktail. Incubate the plates at 37 degrees Celsius for two hours.
Spot five microliters of the reaction mixture onto corresponding squares in a glass fiber/DEAE filter mat paper, and allow the spots to dry for 10 minutes. Use 2x SSC buffer to wash the papers five times for five minutes each, followed by two one-minute washes with 95%ethanol. Allow the papers to dry before sealing them in sample bags.
Clip sample bags containing the filter mat paper into a cassette, and insert the cassette into a microplate scintillation counter. Then start the counter. For any viral quantification method, divide the values obtained for each test RNA by the adjacent negative control and multiply this value by 100 to get the percent inhibition of HIV-1 production for each replicate of the test RNAs.
To carry out a cell viability assay, after removing the 96-well plates from the incubator, add 20 microliters of five milligrams per milliliter MTT and DPBS to each well. Then incubate the plates for three hours at 37 degrees Celsius. Add 150 microliters of acidified isopropanol with detergent to each well, and incubate the plates at room temperature for two hours.
Then use a microplate spectrophotometer to determine the absorbance at 570 nanometers. Calculate the relative MTT metabolism for each positive control and test RNA by dividing the value obtained for each sample by its adjacent transfection control. For the immune activation assay, after removing the 12-well plates from the incubator, aspirate the culture medium and use DPBS to gently wash the cells twice.
Then add 70 microliters of cold lysis buffer containing protease and phosphotase inhibitors to each well, and incubate the plates on ice for 10 minutes. Transfer cell lysates to microtubes, and fast freeze them by immersing the tubes in liquid nitrogen. After allowing the samples to thaw, freeze-thaw them two more times, for a total of three freeze-thaw cycles.
Next, centrifuge the lysates at 15, 700 times g and 4 degrees Celsius for 15 minutes to pellet the cell debris. Then, following electrophoresis and transfer to a membrane, reveal protein bands by incubating the membrane in Ponceau S for one minute. Then wash it with double-distilled water.
Use the bands and protein ladder as a guide to cut the membrane at 80 and 55 kilodaltons. Now, use TBS with 0.05%TWEEN 20 or TBST to wash out the Ponceau S staining. Add TBST with 5%non-fat milk to completely cover the membranes, and incubate them at room temperature with agitation for one hour.
Transfer the membranes into TBST with 3%BSA and antibodies diluted 1:1, 000 for ADAR, phosphor-PKR, and phosphor-IRF3 for the top, middle, and bottom pieces of the membrane, respectively. Incubate at 4 degrees Celsius with agitation overnight. The following morning, use TBST to wash the membranes five times for five minutes each, before transferring to TBST with 5%non-fat milk and 1:5, 000 peroxidase labeled goat anti-rabbit secondary antibodies for one hour.
After visualizing the protein bands on films, use a stripping solution to wash the middle and bottom pieces of the membrane for 10 minutes, before incubating the middle piece of the membrane in PKR antibodies and the bottom piece in IRF3 antibodies. As a positive control, probe the bottom membrane with antibodies to Actin according to the text protocol. A general schematic of the assays is shown here with an example transfection plan for three test RNAs and a control RNA.
For viral production and cell viability assays, the readout for each test construct is normalized to a negative control. Using the HIV-1 production assay in cells transfected with a plasmid expressing HIV-1 strain NL4-3, the percent of RT activity in cells co-transfected with one of the siRNAs shown here, compared to cells transfected with a long Dicer substrate nonsense siRNA, was calculated to identify the optimal length of RNA interference molecules targeting a conserved site in HIV-1 RNA. Using the most effective siRNAs identified, the percent metabolism of MTT was determined for transfected cells.
Along double-stranded RNA, poly(I:C)reduced cell viability only at the highest dose. No significant reduction in cell viability was observed for siRNAs targeting the 1498 site in HIV-1 RNA, regardless of their length. As demonstrated here by Western blot, the same set of RNAs was evaluated in the immune activation assay.
Under conditions where poly(I:C)activated the expression of ADAR1-p150 and induced phosphorilation of PKR and IRF3, no significant effect on any inactivation markers could be observed for any of the test RNAs. Once mastered, this technique can be done in seven days, if it's performed properly. While attempting this procedure, it is important to use high quality preparations of both RNA and DNA for transfection.
Furthermore, to include the proper negative and positive controls. Following this procedure, other methods like HIV infection of transduced cells, can be used to evaluate the long-term efficacy and toxicity of potential anti-HIV candidates. After its development, this technique allowed us to identify an accessible and conserved target site in HIV RNA and optimize the formats of ribozymes, shRNAs and siRNAs targeting it.
After watching this video, you should have a good understanding of how to quickly screen new RNA candidates targeting HIV production for efficacy using the viral production assay, and potential toxicity using the immune activation and cell viability assays. Don't forget that working with HIV and radiolabeled nucleotides can be extremely hazardous. Precautions such as using certified biosafety and radioactivity laboratories should always be taken while performing this procedure.
