Fig 1.
Schematic of a regular shRNA (top) and AgoshRNA molecule (bottom).
In the canonical pathway the stem of the shRNA is cleaved by Dicer into an siRNA duplex of ~21 bp with a 3’ UU overhang that is loaded into RISC. One strand (the passenger, white arrow) is cleaved and degraded, the other acts as guide (black arrow) in RNAi-silencing. Alternatively, AgoshRNA is recognized directly by Ago2, triggering cleavage on the 3’ stem of the duplex between bp 10 and 11, counted from the 3’-end, yielding a single guide RNA molecule of ~30 nt (grey arrow). The predicted Dicer and Ago2 cleavage sites are marked with black and grey arrows, respectively. AgoshRNA subsequently may instruct Ago2 for RNAi-silencing or may be trimmed by PARN to create an unpaired ~24 nt guide named AgoshTRIM. Base pairs: bp, nucleotides: nt.
Fig 2.
AgoshRNA molecules and CCR5 target sequences.
13 AgoshRNAs against human CCR5 were designed. The target sequences in CCR5 mRNA (GenBank: AY874120) are shown with the position in subscript. The predicted structure of the AgoshRNA molecules by MFold is shown in the third column with the guide sequence boxed in grey and the bottom mismatch A C boxed in black. The potent shRNA sh1005 was included as positive control.
Fig 3.
Knockdown activity of the AgoshRNAs against luciferase-CCR5.
(A) Luciferase knockdown by the AgoshRNA was determined by co-transfection with the AgoshRNA constructs. HEK293T cells were co-transfected with 100 ng of the firefly luciferase reporter plasmid, 1 ng of renilla luciferase plasmid, and 25 ng of the corresponding AgoshRNA constructs. pBluescript (pBS) plasmid and an unrelated shRNA (shNef) served as negative control. The luciferase activity scored with shNef activity was set at 100%. (B) Luciferase knockdown by the most potent AgoshRNA constructs is dose-dependent when HEK293T cells were co-transfected with firefly luciferase reporter plasmid, renilla luciferase plasmid and increasing amount of AgoshRNA constructs (1, 5 and 25 ng). The mean values and standard deviation are based on six independent transfections.
Fig 4.
Identification of potent AgoshRNAs against CCR5.
(A) PM1 T cells were transduced with lentiviral vectors expressing AgoshRNAs against CCR5, cultured for four days and analyzed by flow cytometry for CCR5 expression in GFP-expressing cells. (B) Percentage of CCR5+ cells in the GFP+ versus GFP- population in transduced PM1 T cells at day 4. The mean values and standard deviation are based on three independent experiments.
Fig 5.
Stable AgoshRNA-mediated CCR5 silencing in PM1 T cells.
PM1 T cells were transduced with lentiviral vectors expressing AgoshRNAs against CCR5, cultured for 22 days and analyzed by flow cytometry once a week for CCR5 expression in GFP-expressing cells.
Table 1.
Competitive cell growth (CCG) assay.
Fig 6.
Reduction of CCR5 surface expression on human PBMC transduced by anti-CCR5 AgoshRNA lentiviral vectors.
PHA and IL2-stimulated PBMC were transduced with the indicated lentiviral vector. The transduced cells were cultured in IL2-containing medium for 7 days before FACS analysis for CCR5 expression on the cell surface. (A) Representative FACS analyses are shown. (B) Percentage of CCR5+ cells in the GFP+ versus GFP- population was calculated. Three independent experiments were performed. The mean values and standard deviation are indicated.
Fig 7.
The impact of anti-CCR5 AgoshRNAs in a spreading HIV-1 infection.
Stably transduced PM1 T cells expressing the AgoshRNAs variants or the sh1005 control were challenged with (A) the R5-tropic BaL isolate or (B) the X4-tropic LAI isolate at different moi: 0.01 (left panel) and 0.1 (right panel). Cells transduced with the empty lentiviral vector JS1 served as control. Virus replication was monitored by measuring CA-p24 in the supernatant for 25 days.