Our protocol provides alternative methods for the identification and characterization of viral, nucleolar, and non-nucleolar host factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways. Troubleshooting techniques are described for modification to other protein models.
Demonstrating the procedure will be Haitang Li and Pritsana Chomchan, scientists in the Molecular and Cellular Biology Department at the Beckman Research Institute at the City of Hope, and Roger Moore, members of the City of Hope Proteomics Core and the Department of Molecular Immunology. Begin by growing an HLfB stably expressing HeLa cell line culture in three, 100-millimeter culture plates to a two-times-10-to-the-six-cells-per-milliliter density. When the cells reach the appropriate concentration, mix 20 micrograms of plasmid containing the Rev nucleolar localization signal three-prime flag mutation of interest with 1.76 milliliters of TE 79/10 in a 15-milliliter tube, followed by the addition of 240 microliters of two-molar calcium chloride, with thorough mixing.
Next, transfer the contents of the tube dropwise into a new 15-milliliter tube containing two milliliters of 2x HBS, followed by mixing on a vortex machine. After 30 minutes at room temperature, vortex the precipitation. Add one milliliter of the suspension dropwise to each of the three, 100-millimeter HLfB culture plates while gently swirling the medium.
After a six-hour incubation in the cell culture incubator, replace the transfection mixture with 10 milliliters of fresh culture medium, and return the cells to the incubator for another 42 hours. 48 hours after the transfection, place each plate on a bed of ice within a biohazard level two tissue culture hood. Remove the cell culture media, and gently rinse the cells with 10 milliliters of prechilled PBS, without disrupting the cell layers.
Discard the PBS after rinsing the cells. Next, treat the cells with one milliliter of lysis buffer, supplemented with protease inhibitor cocktail, per plate. Tilting the plates to gather the cells into a pool, use a cell scraper to manually disrupt the layers.
Using a 1, 000-microliter micropipette, pool the lysate from each plate into a prechilled, 15-milliliter tube for a 15-minute incubation on ice, with vortexing every five minutes. At the end of the incubation, aliquot the lysates into three, 1.5-milliliter microcentrifuge tubes, and collect the lysates by centrifugation. Transfer the supernatant into a new 15-milliliter tube, and obtain the viral protein lysate concentration, as described in the manuscript.
For coimmunoprecipitation of the Rev nucleolar localization signal three-prime flag, rinse 25 microliters of M2 affinity gel beads three times with 500 microliters of fresh lysis buffer, supplemented with protease inhibitor cocktail, per wash. Perform these steps in a cold room or with the reagents on ice. After the last rinse, add a one-milligram-per-milliliter concentration of viral protein lysate to the rinsed beads in a final volume of five milliliters of lysis buffer.
Incubate the reaction for three hours at four degrees Celsius with rotation. At the end of the incubation, pellet the viral protein lysate-conjugated beads by centrifugation. Collect the supernatant for measurement of the post-immunoprecipitation lysate protein concentration.
See the manuscript for sample storage instructions. Add 750 microliters of lysis buffer to the viral protein lysate-conjugated bead pellet, transfer the beads into a 1.5-milliliter microcentrifuge tube, and wash the beads on a rotator for five minutes at four degrees Celsius. Collect the beads by centrifugation for an additional two washes on the rotator, as just demonstrated.
After the last wash, use a pinched, long, gel-loading tip to remove any traces of lysis buffer from the bead-coimmununoprecipitation complex. Resuspend the beads in 55 microliters of 2x loading buffer. Then, boil the sample at 95 degrees Celsius for 10 minutes.
Load 25 microliters of eluate onto one western blot gel and one Coomassie staining SDS-PAGE gel. After completion of SDS-PAGE gel electrophoresis, rinse the gel three times in 25 milliliters of ultrapure water for 15 minutes. After the last wash, add 100 milliliters of Coomassie stain reagent onto the gel.
See the manuscript for the complete staining protocol. For digestion of the gel bands, first use a clean razor blade to cut the gel bands from the entire lane of the gel, representing each mutation, into approximately five-millimeter cubes. Place each band in a clean, 0.5-milliliter microcentrifuge tube.
Cover the bands two times with 100-millimolar ammonium bicarbonate in a one-to-one acetonitrile-to-water solution at room temperature for 15 minutes per wash. Then, dry the gel pieces for five minutes in a vacuum centrifuge. To reduce the proteins, cover the dried gel pieces with 10-millimolar dithiothreitol in 100-millimolar ammonium bicarbonate for a one-hour incubation at 56 degrees Celsius, followed by alkylation in 100-millimolar iodoacetamide in water for one hour at room temperature in the dark.
Next, shrink and reswell the gel pieces two times with acetonitrile, followed by 100-millimolar ammonium bicarbonate, both with gentle shaking at room temperature for 15 minutes. After the second reswelling, dry the gel pieces for five minutes in a vacuum centrifuge. Swell the gel pieces with 50 nanograms per microliter of sequencing-grade modified trypsin in 100-millimolar ammonium bicarbonate.
After five minutes, cover the gel pieces with 100-millimolar ammonium bicarbonate, and allow them to reswell completely, adding additional 100-millimolar ammonium bicarbonate so the swollen gel pieces are completely covered. Then, incubate the gel pieces overnight at 37 degrees Celsius, stopping the reaction with 1/10 of the volume of the samples with 10%formic acid in water the next morning, and collect the supernatant from each tube. Rev nucleolar localization signal three-prime flag is detectable under three different lysis buffer conditions containing various concentrations of sodium chloride.
B23 is also detectable in lysis buffer containing the lower salt concentration, barely detectable in lysis buffer containing the medium salt concentration, and undetectable in lysis buffer containing a high salt concentration. B23 detection is optimal in lysis buffer containing the low sodium chloride concentration with wild-type Rev three-prime flag and loses affinity with Rev nucleolar localization signal M1 three-prime flag. B23 affinity with wild-type Rev three-prime flag also decreases with a higher salt concentration, and the pcDNA negative control does not yield nonspecific immunodetection after immunoprecipitation under both lysis buffer conditions.
Rev nucleolar localization signal three-prime flag is most detectable after elution through boiling in 2x sample loading buffer, while B23 is detectable under both the 37-and 95-degree Celsius sample buffer incubation conditions. After immunoprecipitation and silver staining, as demonstrated, wild-type Rev and Rev nucleolar localization signals M2, M6, and M9 are detectable at 18 kilodaltons. Bands corresponding to B23 protein are also observed at the 37-kilodalton marker.
Staining with Coomassie reagent further demonstrates the detectability of Rev nucleolar localization signal three-prime flag in the presence and absence of mutations at 18 kilodaltons. Use as many controls, both positive and negative, as necessary for references that pertain to your disease model during the troubleshooting and screening process for potential binding factors. All deletion and single-point mutations can be examined for dominant-negative activity through multimerization with wild-type Rev and utilized in the arrest of HIV-1 replication.
