Figure 8

Effects of endogenous HIV-1 antisense RNA on HIV-1 expression. (A) Targeted regions of shRNA#1 and #2. Lower panel depicts secondary structures of expressed shRNAs predicted by m-fold. Lower case letters indicate mutated sites. Boxed sequences are guide strand for HIV-1 asRNAs. (B) Reporter-based confirmation of strand-specific knockdown. Luciferase reporter plasmids that contain sense HIV-1 sequence or antisense HIV-1 sequence were transfected into the shRNA-expressing Molt-4 cells. Relative changes of luciferase activities were calculated by pMIR-sense or AS ASP-L/pMIR-report. The mean ± S.D. of triplicate experiments are shown. Asterisks indicate statistical significance (p < 0.01). (C) Levels of endogenous HIV-1 asRNAs in HIV-1-infected shRNA expressing cells at 24 h of infection evaluated by strand-specific qRT-PCR at R7. Asterisks indicate statistical significance (p < 0.009). (D) HIV-1 sense RNA levels at 4 dpi measured by strand-specific qRT-PCR at R7 (env region). (E) Levels of viral particle production. At the indicated time points, cultured supernatant were collected and subjected to RT-assay. Asterisks indicate statistical significance.