Article Figures & Data
Figures
- FIG. 1.
Schematic representation of a short hairpin siRNA expression cassette introduced within the ITRs of a recombinant AAV-2 DNA vector. The H1 promoter was used to express tat siRNA in the form of a hairpin transcript consisting of a 21-nucleotide (nt) sense and 21-nucleotide antisense sequence separated by a hexaloop (5′-CTG CTT GTA CCA ATT GCT ATT AGG ATC AAT AGC AAT TGG TAC AAG CAG TTT TT-3′). A five-thymidine transcription termination signal was placed downstream of the hairpin sequence. To allow the generation of shRNA-expressing cell lines, an SV40 early promoter-driven neomycin resistance gene was introduced downstream of the hairpin expression cassette.
- FIG. 2.
Cotransfection of various synthetic RNA species including tat-specific sense (s), antisense (as), and short interfering (si) molecules with HIV-1NL4.3 in 293T cells. An siRNA with no sequence homology to HIV-1 was included as a control. Cell-free supernatant was assayed for HIV-1 p24 antigen (Ag) levels on day 4 posttransfection and demonstrated 91% reduction of p24 in cells transfected with tat siRNA compared to controls. Values represent averages of three independent experiments, with the ranges indicated.
- FIG. 3.
To verify the antiviral activity of H1 promoter-driven tat siRNA, 293T cells were cotransfected with HIV-1NL4.3 and pAAV DNA constructs. Quantification of viral p24 antigen levels 48 h posttransfection in cell-free supernatant revealed a 97% decrease in cells expressing tat shRNA (pAAV-tat) compared to cells that were transfected with HIV-1NL4.3 and control mock vector lacking tat-specific sequences (pAAV-Δtat). Values represent averages of three independent experiments, with the ranges indicated.
- FIG. 4.
Northern blot analysis of tat shRNA expression in HIV-1NL4.3-infected H9 cells transduced with AAV-tat or AAV-Δtat. Continuous expression of tat shRNA was observed on days 14 and 35 after HIV-1NL4.3 infection. rRNA expression was used as a loading control.
- FIG. 5.
(A) Inhibition of HIV-1 replication in H9 cells stably expressing tat shRNA. Cells were challenged with 100 TCID50 of HIV-1NL4.3, and p24 antigen levels were assessed over a 2-month period. Viral replication was suppressed by 95% until day 21 compared to control cells. Viral escape started to emerge on day 25, and viral replication was no longer suppressed by day 35, with observed p24 antigen levels being similar to those in control cells. (B) Sequence analysis of HIV-1NL4.3 in culture supernatant at indicated time points. A mutation in position 9 of the tat target sequence emerged at day 25, coincident with the loss of tat shRNA antiviral activity.
- FIG. 6.
(A) Cotransfection of HIV-1NL4.3mut with pAAV-tatwt, pAAV-tatmut, and mock vector (pAAV-Δtat). When the targeted tat sequence and shRNA were homologous (HIV-1NL4.3-mut/tat-mut), virus production was reduced by 92%. When the targeted tat sequence and shRNA were not homologous (HIV-1NL4.3-mut/tat-wt), virus production was suppressed by only 50%. (B) H9 cells stably expressing tat shRNA (H9-tat) or Δtat (H9-mock) were infected with 20 ng of p24 of HIV-1NL4.3 or HIV-1NL4.3mut. On day 10 viral replication was reduced with wild-type virus (H9-tat/NL4.3-wt) but not with mutant virus (H9-tat/NL4.3-mut), demonstrating the importance of complete sequence homology between the viral target sequence and the expressed shRNA.
