(A) SLBP depletion in HeLa-T4, CEM, and primary CD4⁺ T lymphocytes led to higher (2.7- to 4.4-fold) levels of HIV-1 integration at 48 hours after infection as quantified by Alu-PCR. (B and C) Among the 8 critical cellular factors (BANF1, EED, HMGA1, SMARCB1, IPO7, TNPO3, LEDGF, and UNG2) involved in HIV-1 integration, SLBP depletion led to increases in HMGA1 mRNA (B) and protein (C) expression. The fold changes in protein levels were quantified by densitometric analysis and are noted on the right of gels. (D) The impact of SLBP and HMGA1 on viral integration was further validated by simultaneous depletion of both proteins. When both SLBP and HMGA1 were depleted, HIV-1 integration levels were reduced and approached control conditions. (E) SLBP depletion in HeLa-T4, CEM, and primary CD4⁺ T lymphocytes led to significantly higher (1.7- to 2.3-fold) levels of unspliced HIV-1 mRNA expression at 48 hours after infection as quantified by real-time PCR. (F) The specificity of SLBP depletion on viral RNA levels was validated by a siRNA-resistant SLBP cDNA variant [SLBP(res.)]. Levels of HIV-1 unspliced RNA increased (~2.4-fold) upon SLBP depletion but returned toward control levels when SLBP siRNA–treated cells expressed SLBP(res.). (G) SLBP depletion in HeLa-T4, CEM, and primary CD4⁺ T lymphocytes led to significantly higher (~1.6- to 2.1-fold) levels of p24 in culture supernatant at 48 hours after infection as quantified by ELISA. Western blots were used to measure and verify HMGA1 and SLBP protein levels for each experimental condition. (n = 3 biologic replicates.) Student’s t test was performed for A, B, E, and G. One-way ANOVA with post hoc Tukey’s test was used for D and F. *P < 0.05, **P < 0.01; error bars represent ± SD. Si(h), short interfering (hairpin) RNA.