A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This method can help answer key questions in the HIV entry field such as drug discovery and identification of factors required for HIV fusion. The main advantage of this technique is that it can be scaled for high throughput screening. This technique can be used to screen large libraries of compounds to identify drugs that target early stages of the HIV-1 life cycle.

This method can also be used to study HIV fusion inhibitors in animal models such as humanized mice. Researchers should follow biosafety precautions with infectious HIV-1. This is a recombinant full-length viral clone that does not replicate in multiple rounds, but must be approved by your biosafety office.

To begin this procedure, thaw one 500 microliter vial of Jurkat RG reporter cells by placing it into a 37 degree Celsius water bath. Pipette the cells from the vial into 10 milliliters of RPMI complete medium and then centrifuge the mixture at 800 times gravity for five minutes at 23 degrees Celsius. Resuspend the pellet in 20 milliliters of RPMI complete medium and transfer the cell suspension to a T75 flask.

Incubate the flask at 37 degrees Celsius overnight. On the following day, add 0.5 micrograms per milliliter of Puromycin to the cells in the flask. Culture the cells maintaining a density of 200, 000 to 800, 000 cells per milliliter.

On the day prior to setting up the transfer assay, split the cells down to 200, 000 to 400, 000 cells per milliliter in fresh RPMI medium containing 0.5 micrograms per milliliter of Puromycin. Grow the cells overnight. To prepare the donor cells for cell-to-cell virus transmission, thaw untransduced Jurkat cells and culture them in a T75 flask with 20 milliliters of RPMI complete medium keeping the density of 200, 000 to 800, 000 cells per milliliter.

Centrifuge 7, 500, 000 cells at 800 times gravity for five minutes. Discard the supernatant and resuspend the cells in 120 microliters of Nucleofector Solution V with supplement. Transfer the cells to an electroporation cuvette and add 4.5 micrograms of Gag-iCre DNA.

Electroporate the cells using an appropriate program and then immediately transfer the cells to three milliliters of RPMI medium with 10%FBS. Allow the cells to recover by incubating them at 37 degrees Celsius with 5%CO2 overnight. On the following morning, pipette two milliliters of Ficoll into a 15 milliliter conical tube.

Slowly pipette the three milliliters of cells from the six-well plate on top of the Ficoll. Centrifuge the cells at 800 times gravity for five minutes at 23 degrees Celsius and transfer the cells from the interface Ficoll media interface to three milliliters of RPMI complete medium in a six-well plate. Allow the cells to recover at 37 degrees Celsius for two hours before proceeding with the assay setup.

Start this procedure by using a hemocytometer to count both the donor and target cells. Then spin down 50, 000 cells per well to be assayed of both donor and target cells. Resuspend one times 10 to the sixth cells per milliliter in RPMI complete medium without Puromycin.

In a 96-well plate, add 25 microliters of donor cell suspension to each well. In another 96-well plate, add 25 microliters of target cell suspension per well. To each well, add 25 microliters of the test compound diluted in RPMI complete medium at the appropriate concentration.

Incubate the donor and target cells with the compound for 30 minutes. After the 30-minute pretreatment, combine the donor cells with the target cells by pipetting the contents of one plate into the other plate and incubating the cells in a tissue culture incubator for 40 hours. To fix the cells, add paraformaldehyde to each well to a final concentration of 2%Subsequently, analyze the cells on a flow cytometer with mCherry and FITC channels.

In addition, visualize the GFP signal via fluorescence microscopy at 40X magnification on GFP-specific channels. Uninfected RG Jurkat cells exhibit a low level of background GFP signal with a very strong RFP signal. Cell-free infection with Gag-iCre causes an increase in GFP signal.

The presence of the HIV-1 fusion inhibitor AMD3100 inhibits the development of GFP signal bringing it down to uninfected background levels. When an inhibitor of a post-fusion event such as the reverse transcription inhibitor AZT is used, the signal is not affected significantly indicating that the GFP signal produced by the assay is specific to the HIV-1 fusion. In a cell-to-cell infection assay, RG Jurkat cells were mixed with Jurkat cells transfected with HIV-1 Gag-iCre.

In addition to AMD3100, a nonselective purinergic inhibitor PPADS was tested for its ability to block viral membrane fusion at 100 micromolar. The results indicate a dose-dependent inhibition of Gag-iCre fusion with PPADS. While attempting this procedure, it's important to keep the cells within exponential growth range for both virus production and infection.

Using overgrown cells will result in a dramatic decrease in signal from this assay. Don't forget that working with HIV can be hazardous. BSL two practices as indicated by your institution should be always taken while performing this procedure.