Competitive cell growth (CCG) assay.

<p>Competitive cell growth (CCG) assay.</p

Reduction of CCR5 surface expression on human PBMC transduced by anti-CCR5 AgoshRNA lentiviral vectors.

<p>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. <b>(A)</b> Representative FACS analyses are shown. <b>(B)</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.</p

AgoshRNA molecules and CCR5 target sequences.

<p>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.</p

The impact of anti-CCR5 AgoshRNAs in a spreading HIV-1 infection.

<p>Stably transduced PM1 T cells expressing the AgoshRNAs variants or the sh1005 control were challenged with <b>(A)</b> the R5-tropic BaL isolate or <b>(B)</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.</p

Stable AgoshRNA-mediated CCR5 silencing in PM1 T cells.

<p>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.</p

Schematic of a regular shRNA (top) and AgoshRNA molecule (bottom).

<p>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 Agosh^TRIM. Base pairs: bp, nucleotides: nt.</p

Identification of potent AgoshRNAs against CCR5.

<p><b>(A)</b> 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>(B)</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.</p

Influence of the loop size and nucleotide composition on AgoshRNA biogenesis and activity

<p>Short hairpin RNAs (shRNAs) are widely used for gene silencing by the RNA interference (RNAi) mechanism. The shRNA precursor is processed by the Dicer enzyme into active small interfering RNAs (siRNAs) that subsequently target a complementary mRNA for cleavage by the Argonaute 2 (Ago2) complex. Recent evidence indicates that shRNAs with a relatively short basepaired stem bypass Dicer and are instead processed by Ago2. We termed these molecules AgoshRNAs as both processing and silencing steps are mediated by Ago2 and proposed rules for the design of effective AgoshRNA molecules. Active and non-cytotoxic AgoshRNAs against HIV-1 RNA were generated, but their silencing activity was generally reduced compared with the matching shRNAs. Thus, further optimization of the AgoshRNA design is needed. In this study, we evaluated the importance of the single-stranded loop, in particular its size and nucleotide sequence, in AgoshRNA-mediated silencing. We document that the pyrimidine/purine content is important for AgoshRNA-mediated silencing activity.</p

Design of AgoshRNAs with an extended guide.

<p><b>(A)</b> The 19-nt guide strand of the original shGag5 and the derived AgoshGag5 are boxed in grey. The complementarity between the guide and the HIV-1 target sequence was increased in variants E3, E6 and E8 by addition of anti-HIV nt in the loop and into the 3’ strand of the stem. The thermodynamic stability (ΔG in kcal/mole) was determined using the Mfold program. <b>(B)</b> Knockdown activity on the HIV-sense and HIV-antisense reporters (upper and lower graph, respectively). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128618#pone.0128618.g003" target="_blank">Fig 3</a> for further details.</p

Four potent anti-HIV shRNAs and the matching AgoshRNAs.

<p>The four shRNAs shown are potent antivirals that target highly conserved HIV-1 RNA sequences. The Dicer processing sites are indicated (<b>¯</b>I<b>_</b>), with the 3’-guide strand highlighted in grey (<b>◄</b>). Based on these guide strands, four matching Aggo2 molecules were designed against the same HIV-1 targets. The Ago2 processing site is indicated and the 5’-guide is highlighted in grey.</p