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

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

Competitive cell growth (CCG) assay.

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

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

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

Differential Binding of Tenofovir and Adefovir to Reverse Transcriptase of Hepatitis B Virus

<div><p>Introduction</p><p>Resistance of the reverse transcriptase (RT) of hepatitis B virus (HBV) to the tenofovir nucleotide drug has not been observed since its introduction for treatment of hepatitis B virus (HBV) infection in 2008. In contrast, frequent viral breakthrough and resistance has been documented for adefovir. Our computational study addresses an inventory of the structural differences between these two nucleotide analogues and their binding sites and affinities to wildtype (wt) and mutant RT enzyme structures based on <i>in silico</i> modeling, in comparison with the natural nucleotide substrates.</p><p>Results</p><p>Tenofovir and adefovir only differ by an extra CH₃-moiety in tenofovir, introducing a center of chirality at the carbon atom linking the purine group with the phosphates. (R)-Tenofovir (and not (S)-tenofovir) binds significantly better to HBV-RT than adefovir. “Single hit” mutations in HBV-RT associated with adefovir resistance may affect the affinity for tenofovir, but to a level that is insufficient for tenofovir resistance. The RT-Surface protein gene overlap in the HBV genome provides an additional genetic constraint that limits the mutational freedom required to generate drug-resistance. Different pockets near the nucleotide binding motif (YMDD) in HBV-RT can bind nucleotides and nucleotide analogues with different affinities and specificities.</p><p>Conclusion</p><p>The difference in binding affinity of tenofovir (more than two orders of magnitude in terms of local concentration), a 30x higher dosage of the (R)-tenofovir enantiomer as compared to conformational isomeric or rotameric adefovir, and the constrained mutational space due to gene overlap in HBV may explain the absence of resistance mutations after 6 years of tenofovir monotherapy. In addition, the computational methodology applied here may guide the development of antiviral drugs with better resistance profiles.</p></div

Clustering analysis of <i>in silico</i> modeled HBV RT with crystal models of polymerase.

<p>The 3D-structure of HBV-RT and the PDB coordinate files of the other polymerases were aligned and the pairwise RMSD values were put into a distance matrix for tree construction. HBV-RT is indicated in bold-face. The scale bar represents a RMSD value of 1. The date is added to anticipate future additions to the PDB database.</p