Monitoring processed, mature Human Immunodeficiency Virus type 1 particles immediately following treatment with a protease inhibitor-containing treatment regimen

Abstract

Protease inhibitors (PIs) block HIV-1 maturation into an infectious virus particle by inhibiting the protease processing of gag and gag-pol precursor proteins. We have used a simple anti-HIV-1 p24 Western blot to monitor the processing of p55gag precursor into the mature p24 capsid immediately following the first dosage of a PI-containing treatment regimen. Evidence of PI activity was observed in plasma virus as early as 72 hours post treatment-initiation and was predictive of plasma viral RNA decrease at 4 weeks.

Figure 1

Western blots for the HIV-1 gag proteins in HIV-1 produced in tissue culture following treatment with protease inhibitors. U87/CD4/CXCR4 cells were plated in 6 well plates at 80,000 cells/well and allowed to grow to confluence. The cells were infected with either HXB2 or RF/V82F/I84V (protease inhibitor resistant virus) and RT activity was monitored. On day 3 of culture, infected cells were treated with 20 nM LPV, and 1 ml of media was removed at 0, 4, 8, 24, and 72 hours post-drug treatment. The virus was pelleted, and the pellet was then lysed using sodium-dodecyl sulfate (SDS) lysis buffer and then run on a 10% SDS polyacrylamide gel. Following transfer to nylon membranes, blots were probed with the primary mouse anti-p24 antibody and the horseradish peroxidase-conjugated goat anti-mouseantiserum. Films were exposed following treatment with the ECL kit (panel A). Ratio of unprocessed p55^gag to processed CA p24 over a 72 hour time course was determined by scanning the blots and quantifying the bands (panel B).

Figure 2

Western blot analyses for the HIV-1 gag proteins in patient plasma prior to and following ARV treatment. Patient plasma was obtained at 0, 4, 8, 12, 24, 72 hours and 4 weeks following ARV treatment. Plasma was diluted with serum-free media and then centrifuged to pellet HIV-1 particles prior to the analyses (see Fig. 1). Panel A shows the Western blot results on plasma samples obtained from patient A who was treated with NVP+3TC+D4T+ABV and patient B who was treated with LPV+NVP. Panel B shows the ratios of unprocessed p55^gag to processed CA p24 in patients treated with NVP+3TC+D4T+ABV (patient A) or NVP+LPV (patient B, C, and D). Each bar at each time point represents analyses from a separate Western blot. Panel C is a plot showing the changes in CD4 cell count (cells/mm³; open squares) and viral RNA load in plasma (copies/ml, filled diamonds) following treated with either treatment regimen.

Figure 3

Comparing the drop in viral RNA load to the increase in precursor gag protein following treatment with LPV and NVP. The fold increase in the p55:p24 ratio was calculated by dividing these ratios at 3 days and 4 weeks by the observed ratio prior to treatment (time 0) (panel A). Panel B shows the fold decrease in viral RNA load at 3 days and 4 weeks following LPV+NVP treatment. This calculation involved dividing the viral load at day 3 and week 4 by that at time 0.