FIG 5.

Release of p53 from MDM2 control is sufficient to reduce susceptibility of MDMs to HIV-1. (A, B, and D to F) MDMs were pretreated for 1 h with Nutlin-3, EFV, or RGV before infection with DNase-treated NL/Bal-HSA. The drug pressure was maintained for the entire period of virus infection. (A) The percentage of HSA⁺ MDMs was estimated by flow cytometry analysis at 3 dpi. (B) Cell viability was measured by flow cytometry at 3 dpi with the fixable viability dye eFluor780. No significant change in cell viability was detected (one-way ANOVA with Tukey’s multiple-comparison test, n = 5). (C) MDMs were treated with Nutlin-3 for 3 days without exposure to HIV-1. The percentage of apoptotic cells was measured by flow cytometry with annexin V and 7-AAD. Treatment with Nutlin-3 induces a small increase in apoptosis compared to the level for the DMSO control. This donor is representative of a total of 2 donors. (D and E) The number of completed reverse transcripts (D) and integrated proviral DNA copies (E) was defined by qPCR. (F) Relative mRNA levels of MDM2, CDKN1A, and Tat were measured at 24 and 72 h posttreatment by qRT-PCR. The dotted line represents the gene expression for the DMSO-treated MDMs. (G) The total protein levels of MDM2, p53, p21, SAMHD1, and phosphorylated SAMHD1 (Thr592) in MDMs after 24 h of treatment with Nutlin-3 without exposure to HIV-1 were determined by a Western blot assay. Indicated samples were treated with MG-132 for 6 h before protein extraction. Some proteins had to be exposed for a different time when treated with MG-132, as shown with the cropped membrane. The donor shown is representative of 5 distinct donors. Statistical analyses were done using the ratio-paired Student's t test (A, D, and E; n = 5) or paired Student's t test (F; n = 4) (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).