The overall goal of the following experiment is to screen compounds of interest to determine their cellular cytotoxicity and ability to inhibit wild type or drug resistant HIV one in a single round infectivity assay. This is achieved by first exposing the cells to the compounds for uptake and then incubating the treated cells with luciferase expressing wild type or drug resistant HIV one. The luciferase activity of the virus can then be measured.
Ultimately, the luciferase readout signal can be used to quantify the inhibition of the HIV V one viral vector replication by the compounds. This technique has therapeutic implications as it can be used to screen new compounds that exhibit better efficacy against drug resistant HIV. Such compounds could potentially be used in antiretroviral therapies in HIV one patients On the day prior to transfection plate 2 93 cells on 100 millimeter dishes at a density of 1, 500, 000 cells.
On the next day transfect the cells with 16 micrograms of wild type or mutant HIV and four micrograms of VSV using the calcium phosphate method app. Six hours later, wash the 2 93 cells twice with PBS and then incubate them with fresh media for 48 hours. Two days later, harvest the virus containing S supernatants from the 100 millimeter diameter dishes and clarify the supernatants by low speed centrifugation for 10 minutes at 1, 620 Gs at room temperature.
Next, filter the supernatants through a 45 micromolar pore size syringe filter and treat the viral suspensions with turbo DNAs. After 30 minutes at room temperature, dilute the virus with fresh media and store it at minus 80 degrees Celsius. To perform the screening of the compounds in an infectivity assay on the day before the experiment, seed 4, 000 of the cells of interest into each well of a 96.
Well plate in 100 microliters of media and incubate them at 37 degrees Celsius in 5%carbon dioxide. 24 hours later, serially dilute the compound from the stock solution into fresh media. The final concentrations chosen will depend on an empirically determined range of concentrations as outlined in the table.
Next, add the serial dilution of the compounds to the wells in triplicate. At one 10th of volume of the final concentration, incubate the appropriate dilution of the compounds with the cells for a minimum of three hours. After the incubation, use a multi-channel pipette to add 100 microliters of diluted virus to each of the wells.
Accept the negative control wells. Then incubate the plates for another 48 hours. After the incubation, use a glass pipette equipped with a 200 microliter pipette tip to aspirate the media from the wells starting at the top of the media and slowly working down toward the bottom corner of the well.
Immediately add 100 microliters of PBS supplemented with 0.5 millimolar magnesium chloride to each well then to measure the luciferase activity, add 10 microliters of the substrate buffer from the luminescence reporter gene assay to each vial of supplied lyophilized reagent. Next, add 100 microliters of the reconstituted reagent to each well and incubate the plate for 20 minutes at room temperature to allow the signal to develop. Finally, read the 96 well plate using a microplate luminometer.
The luciferase values from a successful compound screening procedure are presented in the table. Note that potentially potent compounds exhibit increasing luciferase activity with the control exhibiting the highest signal in this representative Kaleido graft. The concentration of the compound versus the percent inhibition of the Lucifer activity has been plotted to determine whether the compound is effective at inhibiting the replication of the wild type or drug resistant HIV.
While attempting this procedure, it is important to accurately distribute a uniform amount of cells and virus into all wells of the plates, and to make sure to put the proper concentration of compounds into each well.
