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. 2020 May 4;94(10):e00148-20. doi: 10.1128/JVI.00148-20

FIG 8.

FIG 8

Duration of the effect of the BMS and AEG compounds on the VLP Env conformation. (A) VLPs prepared from A549-Gag/Env cells expressing HIV-1AD8 Env and Gag-mCherry were incubated with DMSO or 10 μM BMS-806 or BMS-529. The VLPs were pelleted and washed twice, resuspended in PBS with 2% DMSO or fresh compound, and incubated at room temperature for the indicated times. Then, the pelleted VLPs were lysed and incubated with antibodies and protein A-Sepharose beads. The precipitated proteins were analyzed by Western blotting with a rabbit anti-gp120 antiserum. The results are shown for the absence of the compound (−), in the continuous presence of the compound (+/+), or after initial exposure to the compound, washing, and incubation in PBS with 2% DMSO for the indicated times (+/−). (B) VLPs with the HIV-1AD8 Env were incubated with DMSO or the indicated AEG compound and, in some cases, irradiated with UV light. The VLPs were pelleted and washed twice and then resuspended in either PBS with 2% DMSO or fresh compound at room temperature for 3 weeks. The VLPs were then pelleted and lysed. The VLP lysates were directly analyzed by Western blotting (Input) or incubated with the 19b or 2G12 antibody and protein A-Sepharose beads. The precipitated proteins were analyzed by Western blotting with a rabbit anti-gp120 antiserum. Results are shown for the absence of the compound (−), in the continuous presence of the compound (+/+), or after initial exposure to the compound, washing, and incubation in PBS with 2% DMSO for 3 weeks (+/−). The results shown are typical of those obtained in two independent experiments. Lanes M, molecular mass markers. The numbers to the left are molecular masses (in kilodaltons). (C) The binding of the BMS and AEG compounds to the VLP Env is shown as a function of the time of incubation of the virus-compound complex in PBS with 2% DMSO at room temperature. The compound-VLP Env binding index was calculated as follows: compound-VLP Env binding index = [(−) – (+/−)]/[(−) – (+/+)] × 100%, where the symbols in parentheses represent the total amount of gp160 and gp120 recognized by the 19b antibody, as defined in the legend to panel B.