(A) HIV-1 replication in p21 siRNA–treated CD34⁺ primary cells. HIV-1 p24 levels were detected at days 4, 7, 10, and 14 after HIV-1 infection. Cells were treated with chemically synthesized p21 siRNA (Si-1, Si-2) or control siRNA (HPRT) or were mock treated. Culture supernatants collected at each time point were analyzed by HIV-1 p24 antigen assay. HIV-1 growth kinetics in mock-treated cells was indistinguishable from that in HPRT siRNA–treated cells. Each point represents the p24 level from 2 separate experiments run in duplicate. Values represent means ± SD (0.2–4.7). (B) HIV-1 replication in p21 siRNA–treated CMK cells. HIV-1 p24 levels were detected at days 4, 7, 10, and 14 after viral infection. Cells were treated with chemically synthesized p21 siRNA (Si-2), control siRNA, p21 shRNA (p2.3), or vector without shRNA (pU/B) or were mock treated. Each point represents the p24 level from 2 separate experiments run in duplicate. (C and D) TPA-induced p21 expression inhibited HIV-1 replication in CMK cells. Cells were treated with TPA for 1 day (T1), 2 days, or 3 days or were mock treated before HIV-1 infection. (C) p21 mRNA levels examined at day 12 compared with mock-treated control. (D) HIV-1 p24 levels determined at days 1, 7, 10, and 12. Each bar represents the p24 level from 2 separate experiments run in duplicate. Values represent means ± SD (0.3–2.6). (E) HIV-1 p24 expression from transduced p21^–/– and p21^+/+ fibroblasts. The p24 level in culture supernatant was determined at days 3 and 7 after transduction with a single cycle of HIV-1_{NLX.Luc(R–)} infection. Results are from a representative experiment, and samples run in triplicate. Values represent means ± SD (0.07–0.3).

(A–C) Detection of p21 in HIV-1 PICs. CMK (restrictive) and H9 (permissive) cells were infected with HIV-1_IIIB and cytoplasmic PICs isolated at 7 hours after infection. Viral matrix protein is a component of PICs (22, 23). (A) Crude cytoplasmic PICs were isolated from CMK cells. Antibodies against p21 or HIV-1 Gag protein matrix (MA) (50) recovered similar levels of integrase (IN) (lanes 1 and 2). Purified integrase served as a marker (lane 3). Mock-infected cell control is shown in lane 4. (B) Analyses following PIC purification by spin column chromatography. Antibodies against p21 coimmunoprecipitated p21 with integrase in CMK cells (lane 4). Antibodies against p21 failed to recover IN when CMK cells were treated with p21 siRNA (lane 6) but not when cells were treated with mutated p21-siRNA (lane 7) (15). Isotype controls IgG3k (for MA) and IgG1 (for p21) failed to recover IN (lanes 2 and 8). Purified integrase served as a marker (lane 1). (C) Anti-MA antibodies coimmunoprecipitated p21 in PICs made from CMK cells (lane 1) but not from H9 cells (lane 2). p21-containing nuclear extract served as a marker (lane 5).

(A and B) Silencing p18 or p27 did not increase HIV-1 replication. (A) The effect of siRNA specific for p18 or p27 was determined in CD34⁺ cells using a real-time TaqMan RT-PCR assay by comparing C_T values from cells treated with siRNA specific for p18 or p27 to cells treated with control siRNA. Samples run in quintuplicate were normalized to endogenous GAPDH levels. (B) HIV-1 replication in CD34⁺ cells treated with siRNA specific for p18 or p27 was determined at days 4, 7, 10, and 14 after HIV-1 infection (shown at days 4 and 14) by p24 antigen assays. No active replication was detected in cells treated with p18, p27, or control siRNA in contrast with cells treated with p21 siRNA. Values represent means ± SD (0.22–2.1).

(A) Inhibition of p21 mRNA expression by RNAi. p21 mRNA levels were decreased after transfection of chemically synthesized siRNA (Si-1, Si-2) or cell-expressed shRNA (p2.3, p3.2) specific for p21. The decrease of p21 mRNA did not occur in control cells treated with siRNA specific for hypoxanthine phosphoribosyl transferase (HPRT) or vector only (pU/B) or mock-treated cells (M) (or a mutated form of Si-2, shown in ref. 15). RNA copy number in 50 ng of total cellular RNA was detected by TaqMan real-time RT-PCR assay (Supplemental Figure 1) performed in quadruplicate. Error bars are not visible when the SD is less than 2.0%. (B) Inhibition of p21 expression with p21 siRNA examined by Western blot. The same cell aliquots used in A were examined for p21 protein expression after RNAi treatment. M, p21 protein detected in cells mock treated with siRNA; pU6/B, treated with vector only; p2.3, treated with p21 shRNA; H, treated with HPRT siRNA; and Si-2, treated with p21 siRNA. N, nuclear extract of p21 protein served as a positive control. *Percentage of control was calculated by comparing the densitometer readings of p2.3 with pU6/8 (8%) and Si-2 with H (29%). Results are representative of 3 independent experiments.

(A–C) HIV-1 DNA kinetics in p21 siRNA–treated CMK cells. Cells were infected by VSV-G–pseudotyped HIV-1 after siRNA treatment. (A) Total levels of HIV-1 cDNA were determined by real-time PCR at 7 hours after infection. (B) At 20 hours after infection, more 2-LTR circles were detected in p21 siRNA–treated cells than in control siRNA–treated cells. (C) At 24 hours after infection, p21 siRNA–treated cells had a significantly increased amount of integrated viral DNA in comparison with control cells. Each sample (500 ng) was tested in triplicate (47–49). Results are representative of 3 separate experiments. (D and E) Detection of HIV-1 2-LTR circle and provirus levels in CD34⁺ primary cells using quantitative PCR. (D) HIV-1 2-LTR circles were detected at 16 hours after infection. (E) HIV-1 integration was increased significantly in p21 siRNA–treated cells in contrast with control siRNA– or mock-treated cells. Each bar represents the mean from 4 individual tests, with each sample run in triplicate. Each sample was tested in an equal amount of total cellular DNA from 6 × 10³ CD34⁺ primary cells.

(A) Cell cycle status of CD34⁺ primary cells at 42 hours after siRNA treatment. Cells treated with p21 siRNA and control siRNAs were examined by Hoechst (DNA dye) and PY (RNA dye) staining. Cell cycle status was examined at both 10 hours (not shown) and 42 hours after siRNA treatment. No significant changes in the cell cycle status were observed at both time points among CD34⁺ primary cells that were treated with p21 siRNA or control siRNA or those that were mock treated. (B) Cell cycle status of CMK cells at 42 hours after siRNA treatment. No significant changes were observed in the cell cycle status in megakaryoblasts treated with p21 siRNA (Si-1, Si-2), control siRNA, p21 shRNA, or vector only or those that were mock treated. (C and D) Cell cycle status determined by BrdU incorporation assay. (C) Cell cycle status of CD34⁺ primary cells was examined at both 10 hours (not shown) and 42 hours after siRNA treatment. No significant changes were observed between cells treated with p21 siRNA and cells treated with control siRNA. Cells incorporating BrdU (UL + UR) consisted of 23.75% and 23.72% p21 siRNA– or control siRNA–treated cells, respectively. (D) Mean values of cells incorporating BrdU in each condition from 4 independent assays are shown.

(A and B) SIVmac-251 infection of CD34⁺ cells and CD34^– cells. (A) Silencing of p21 expression in CD34⁺ cells did not induce SIVmac-251 replication. SIV antigen p27 was detected from CD34⁺ cells treated with p21 siRNA or control siRNA or cells that were mock treated (SIV infection only). (B) SIVmac-251 replicated in CD34^– cells of the same donor. SIV antigen p27 was detected in CD34^– cells infected with virus (SIV) but not in mock-infected CD34^– cells (M^–). Assays were run in duplicate. Values represent means ± SD (0.01–0.66).