Figure 4

Stable TRN-SR2 depletion inhibits multiple round infection by HIV-1 WT and N74D CA mutant virus. (A) Western blot showing TRN-SR2 levels in HeLaP4 cells stably depleted of TRN-SR2 by shRNA expressing vectors (shTR3 and shTR4) and control cells expressing a scrambled (shSCR) shRNA. β-tubulin was detected as loading control. (B) TRN-SR2 knockdown in the shTR3 and shTR4 HeLaP4 cells measured by QPCR. TRN-SR2 mRNA levels are normalized for β-actin. Shown are mean values ± SD of triplicate measurements. (C) Analysis of CD4 expression levels of the shSCR, shTR3 and shTR4 HeLaP4 cells by anti-CD4 immunostaining and flow cytometry. 293T cells were analyzed in parallel as control for non-specific staining. Averages of triplicate samples ± SD are shown. (D) Stably TRN-SR2 depleted cells and control cells were infected with 6 × 10⁴ pg p24 of infectious HIV NL4-3 (WT) or HIV-1 NL4-3 N74D CA mutant virus (N74D). From four days post infection on supernatants were sampled daily for p24 measurements. One of three independent experiments each performed in duplicate is shown. (E) shTR3, shTR4 and shSCR cells were transfected with 1 μg of pNL4-3 molecular clone plasmid. 24 hours post transfection supernatants were analyzed for p24 production. 5 μM of ritonavir was used as a positive control of inhibition of p24 production. Shown are mean values ± SD of one experiment out of two performed in triplicate.