Figure 5

AdOx inhibition acts through Vpr and envelope. A) γ-irradiated or untreated HeLa MAGI-X4 cells were infected with a NL4.3 Vpr-negative HIV molecular clone (pNL4.3VprFS) produced in HEK293T cells treated with AdOx as indicated. Cells were infected with virus normalized to RT activity. Each infection was performed in duplicate. The experiment was performed 5 and 4 times for 10 μM and 30 μM AdOx, respectively, using independent virus stocks. Shown are the average results and the standard deviation of the mean. B) HEK293 cells were transfected with pNL4.3 or pNL4.3env- co-transfected with a plasmid expressing VSV-G envelope. HeLa MAGI-X4 cells were infected with equal amounts of each virus normalized to 1 ng virion RT. Shown are the average % infectivity values for three independent experiments and the standard deviation of the mean. The % infectivity value for each AdOx treated virus is shown relative to its respective untreated virus infectivity, with the infectivity of each untreated virus being expressed as 100%. C) HIV-1 infected CEM cells were treated with AdOx as previously described (Fig. 1). ERT reactions were performed using AdOx-treated or control HIV-1. In addition as a control, untreated HIV-1 were supplied exogenous 1 mM AdOx to show that AdOx does not affect ERT. Reverse transcription was initiated by addition of deoxynucleotides and 0.1 mM Triton X-100. The HIV-1 cDNA products were recovered and negative strand strong stop DNA was quantitated by real-time PCR. The copy number indicated was normalized to total RT activity in the virus supernatant. The experiment was performed three times and the standard deviation of the mean is indicated. D) Pelleted virus produced in AdOx -treated or control HEK293T cells were resuspended in Berman lysis buffer. Equal amounts of each virus normalized for RT activity were analyzed by western blot using an anti-gp120 envelope antibody.