Interaction of the HCV core protein with GLD-2 in the cytoplasm is required for miR-122 expression regulation.

<p>(A) Huh7 cells were transfected with an empty vector (Ctrl) or an expression vector encoding Flag-tagged core (Core) or NS5B protein. Lysates were immunoprecipitated with anti-Flag (top) or anti-GLD-2 (bottom) antibody, followed by immunoblotting for the indicated proteins. (B) Indirect double-immunofluorescence staining of the core protein and GLD-2 in Huh7 cells transiently expressing the Flag-tagged core protein. Nuclei were visualized by DAPI staining. (C and D) Subcellular localization of GFP-fused full-length HCV core protein and its truncated derivatives transiently expressed in Huh7 cells for 48 h was analyzed by immunofluorescence microscopy (C). Nucl, nucleus; Cyto, cytoplasm. (D) represents the levels of miR-122 and GFP-fused core proteins measured by northern blot (NB) analysis and immunoblotting (IB). The numbers below the northern blot indicate the miR-122 level compared with the GFP-transfected control, quantified as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005714#ppat.1005714.g001" target="_blank">Fig 1C</a>. Total cell lysates resolved by SDS-PAGE were analyzed by immunoblotting using anti-GFP antibody (bottom). (E) RT-PCR quantification of miR-122 levels in Huh7 cells transiently expressing GFP or the indicated GFP-fused core proteins. (F) Analysis of interaction between GLD-2 and the indicated GFP-fused core proteins by co-immunoprecipitation experiments. (G) Quantitative analysis of colocalization between GLD-2 and the indicated GFP-fused core proteins in Huh7 cells. Huh7/HCV RNA, Huh7 cells transfected with HCV (JFH-1) RNA. Scale bar, 10 μm.</p

Dysregulation of miR-122 function by the HCV core protein.

<p>(A) Schematic representation of a reporter plasmid carrying a miR-122 target site [psiCHEK-2_CULT1(WT)] or a miR-122 mismatched target [psiCHEK-2_CULT1(MT)] at its 3′-UTR (top). Huh7 or R-1 cells treated with miR-122 duplexes were transfected with each indicated reporter plasmid together with pcDNA3.1 (Ctrl) or pcDNA3.1-Flag-core (Core). At 48 h post-transfection, cells were harvested, and normalized luciferase activity (Rluc/Fluc) compared with empty vector-transfected control was measured (bottom). *<i>P</i> < 0.005. (B) Intracellular total cholesterol content. The cholesterol content in core-expressing cells (Huh7/core) was 76.5 ± 5.1% of the Huh7/vector control. *<i>P</i> = 0.0013. (C) R-1 cells transfected with the pcDNA3.1-Flag-core (Core) or empty vector (Ctrl) were treated with miR-122 duplex. The HCV subgenomic RNA titer was determined 2 days after transfection by real-time qRT-PCR, normalized with GAPDH, and expressed as a percentage of that in empty vector-transfected control cells. *<i>P</i> = 0.0032. (D) Huh7 cells pre-transfected with pcDNA3.1 (Ctrl) or pcDNA3.1-Flag-core (Core) plasmids were infected with HCV prior to the quantification of the HCV RNA titer by qRT-PCR 2 days later. *<i>P</i> = 0.0011. (E) Huh7 cells were transfected with increasing amounts of pcDNA3.1-Flag-core (Core) or empty vector (6 μg; Ctrl) together with a dual luciferase reporter expressing <i>Renilla</i> luciferase (Rluc) and firefly luciferase (Fluc) by cap- and IRES-dependent translation (top), respectively, before dual luciferase assays at 48 h post-transfection. *<i>P</i> = 0.0022. For all panels, error bars are standard deviations of three independent experiments, each involving triplicate assays. <i>P</i> values were calculated using an unpaired <i>t</i>-test.</p

Isolation of Coralmycins A and B, Potent Anti-Gram Negative Compounds from the Myxobacteria <i>Corallococcus coralloides</i> M23

Two new potent anti-Gram negative compounds, coralmycins A (<b>1</b>) and B (<b>2</b>), were isolated from cultures of the myxobacteria <i>Corallococcus coralloides</i> M23, together with another derivative (<b>3</b>) that was identified as the very recently reported cystobactamid 919-2. Their structures including the relative stereochemistry were elucidated by interpretation of spectroscopic, optical rotation, and CD data. The relative stereochemistry of <b>3</b> was revised to “<i>S*R*</i>” by NMR analysis. The antibacterial activity of <b>1</b> was most potent against Gram-negative pathogens, including <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i>, <i>Acinetobacter baumanii</i>, and <i>Klebsiella pneumoniae</i>, with MICs of 0.1–4 μg/mL; these MICs were 4–10 and 40–100 times stronger than the antibacterial activities of <b>3</b> and <b>2</b>, respectively. Thus, these data indicated that the β-methoxyasparagine unit and the hydroxy group of the benzoic acid unit were critical for antibacterial activity

Non-templated nucleotide addition to the 3′ end of miR-122 is suppressed in liver tissue from patients with HCV and in Huh7 cells expressing HCV core protein or infected with HCV.

<p>(A) Analysis of miR-122 isomers identified by the deep sequencing of small RNA libraries from human liver biopsies. Fold change (log2) of the proportion of each miR-122 isomer (each isomer read count divided by the total count of miR-122 isomers) in the indicated specimens compared with the corresponding value in the normal liver tissue (N-1). (B) Fold-change analysis for the 23-nt isomers as described in (A). Isomers are sorted according to their 3′-end dinucleotide sequences. Presented at the top is the pri-miR-122 sequence showing processing sites (blue arrows) for the 22-nt prototype miR-122 and an aberrant 3′-terminal processing site (black arrow) for the 23-nt isomer bearing a template-derived 3′ GU-tail. (C) Proportions (% of each isomer read count compared with the total read counts of the four major isomers shown at the top) of four major miR-122 isomers (isomers whose relative percentage to the total read counts of miR-122 isomers is >2%) in the indicated liver biopsies. (D) Proportions of four major miR-122 isomers in Huh7 cells transiently expressing HCV core protein (Core) or infected with HCV.</p

Inhibition of GLD-2 by the HCV core protein.

<p>(A) Coomassie blue staining of purified recombinant proteins used in terminal transferase assays. (B and C) A terminal transferase assay with miR-122-5p (22-nt long guide-strand RNA) and GLD-2 or its inactive form GLD-2(D215A) in the presence of [α-³²P]ATP (B) or each of the indicated radiolabeled ribonucleotides (C). Radiolabeled RNA products were resolved by PAGE on a denaturing polyacrylamide gel (15%) and visualized using a PhosphorImager. As size markers, 5′-³²P-labeled miR-122 isomers were used. (D) Inhibition of GLD-2 3′ adenylation activity by the HCV core protein. The amount of the HCV core protein or SARS-CoV capsid protein added to the reactions is shown as a molar ratio to GLD-2. The radioactivity present in the bands was quantified using a PhosphorImager and expressed as a percentage compared with the GLD-2 alone control. Shown below the autoradiogram is ethidium bromide (EtBr) staining of the gel showing the amounts of miR-122 template used in the assays.</p

Inhibition of HCV IRES-mediated translation by systemically delivered LNP-formulated siIE22.

<p>(A and B) Schematic diagram of siIE22 LNP (A) and LNP particle size analysis (B). (C) Experimental schedule and schematic representation of the pDual-IRES plasmid. The pDual-IRES plasmid was hydrodynamically injected through the tail vein of BALB/c mice (n = 4 per group). After 1 h, mice were iv injected with siIE22 LNP at a dose of 1 mg/kg body weight. The Fluc expression level in the liver was determined 16 h after the injection. Luciferase activity is reported as RLU per mg protein. *, <i>P <</i> 0.01. (D) BALB/c mice (n = 4 per group) were iv injected with indicated siRNAs (1 mg/kg body weight) complexed with ND98 or formulated with LNP. Poly(I:C) (1 mg/kg) complexed with ND98, formulated with LNP, or free form (each in 170 μl) was administered. PBS or LNP vesicles alone were used as control treatments. Two hours later, serum IFN-α levels were quantified by ELISA. The dotted line indicates the detection limit of the assay (15 pg/ml). (E) hPMBCs grown in 96-well plates were transfected with indicated siRNA at 10 nM concentration or with 1 μg/well poly(I:C) using the lipidoid ND98 or stimulated by a direct addition of 50 μg/ml poly(I:C) to the medium. After 16 h, cell culture supernatants from stimulated cells were analyzed for IFN-α by ELISA. Data shown are from one of the two independent experiments with similar results. ND, non-detectable. (F) HEK293 cells were transfected with the luciferase expressing plasmids (IFNβ-pGL3 and pRL-TK) for the IFN-β promoter activity assay. After 6 h, cells were transfected with 100 nM siIE22 or scrambled (Sc) siRNA, or 1 μg/ml poly(I:C). After 8 h, cells were harvested for dual luciferase assays. Fluc activity was normalized to Rluc activity from the pRL-TK plasmid. Normalized luciferase activity (Fluc/Rluc) of mock-treated cells was defined as 100. Data are presented as the mean ± SD of six measurements from two independent experiments.</p

Screening for potent HCV IRES-targeting siRNA by siRNA tiling experiments.

<p>(A) The proposed secondary structure of HCV IRES. The IRES region spanning nts 277–343 (shown in gray) was targeted by siRNAs. The target site of the selected potent anti-HCV siRNA siIE22 is shown in blue. The base-pairings in the proposed pseudoknot (PK) structures (PKs 1 and 2) are shown in green. (B) siRNA sequences tiled over HCV IRES. The underlined sequence represents a mapped druggable region (nts 313–343) where the targets of selected potent siRNAs were enriched. (C) Anti-HCV activity of HCV IRES-targeting siRNAs in Huh7 cells transfected with Rluc-JFH1 (top panel), an HCV replicon encoding the Rluc reporter. The Rluc gene was fused in frame to the DNA sequence encoding 17 N-terminal amino acid residues of the HCV core protein. Huh7 cells were electroporated with the Rluc-JFH1 <i>in vitro</i> RNA transcript and pGL3 plasmid used for normalization of transfection efficiency. After 24 h, the cells were transfected with each of the IRES-specific siRNAs or a scrambled (Sc) siRNA (50 nM). At 48 h post-transfection, luciferase activity was measured. (D) Huh7 cells harboring an HCV subgenomic replicon RNA (R-1) were transfected with each of the IRES-specific siRNAs or Sc siRNA (10 nM each). At 48 h post-transfection, HCV RNA levels were quantified by real-time qRT-PCR. *, <i>P <</i> 0.01. (E and F) Dose-dependent inhibition of HCV-replication by siIE22 was assessed in R-1 cells, as described in (D). HCV genome copy number and HCV proteins (NS5A and NS5B) levels were analyzed by qRT-PCR (E) and western blotting (F), respectively.</p

Anti-HCV efficacy of chemically modified siIE22 derivatives.

<p>(A) Non-modified siIE22 (2 μM) was incubated in 45% human plasma for the indicated time periods. RNA extracted from each sample was resolved by electrophoresis on a denaturing 15% polyacrylamide gel and subjected to northern blotting (NB) analysis for detection of siIE22 guide-strand. The Phosphorimager image shown is from one representative experiment of three independent experiments with similar results. Densitometric analysis of siIE22 guide strand signal was done using a Phosphorimager. Relative intensity of signals was plotted using SigmaPlot to estimate siIE22 guide strand half-life. Relative signal (% of signal at time 0) is shown below a representative blot. (B) Sequences of modified guide and passenger strands of siIE22 derivatives used in this study. Modified residues are shown in green or blue. “s”, phosphorothioate linkage. (C) Plasma stability of a set of the selected siIE22 derivatives was evaluated as in (A). (D) Anti-HCV activity of the selected modified siIE22 (1 nM) was evaluated in R-1 cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146710#pone.0146710.g001" target="_blank">Fig 1D</a>. *, <i>P <</i> 0.01. (E) Analysis of half-life of the gs_PS1 siIE22 as in (A).</p

Anti-HCV efficacy of gs_PS1 siIE22 LNP in a mouse model for HCV replication.

<p>(A) HCV genome in the HCV-replicating Huh7 (+ HCV) or Huh7 (- HCV) sc xenograft was detected by northern blotting. An <i>in vitro</i> transcribed HCV RNA genome was used as a size marker. The 28S rRNA detected by ethidium bromide staining is shown as a loading control. (B) Immunostaining for HCV viral proteins (E2 and NS5B) in the xenograft at 4 weeks post-xenografting. DAPI, nuclear staining. Scale bar, 10 μm. (C) The NOD-SCID mice (n = 3) carrying HCV-replicating Huh7 xenograft were treated with gs_PS1 siIE22 LNP at a dose of 1 mg/kg body weight via tail vein injection. Shown are relative serum HCV RNA titers at the indicated time points. *, <i>P</i> < 0.01. (D) Relative serum HCV RNA titers in the mice treated with LNP-formulated Sc siRNA (Sc LNP) or gs_PS1 siIE22 LNP (1 mg/kg) once in every three days for 4 times. The data were generated from two independent experiments with a total of 6 mice per group. Each differently colored diamond represents an individual mouse. *, <i>P</i> < 0.01.</p

Analysis of anti-HCV potency and RNAi activity of various siRNAs targeting the IRES subdomain IIIf.

<p>(A) Passenger strand sequences of siIE22 and six siRNAs sharing their targets with siIE22 in the IRES subdomain IIIf. (B) Evaluation of anti-HCV efficacy of a set of selected siRNAs (100 pM each) using an HCV replicon expressing an Rluc reporter. Luciferase activity at 48 h post-siRNA treatment is shown. Sc, scrambled siRNA. (C) Antiviral efficacy of gs_PS1 siIE22 and siIE318_27 in R-1 cells harboring an HCV subgenomic replicon. (D) <i>In vitro</i> target cleavage assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146710#pone.0146710.g002" target="_blank">Fig 2A</a>. P, 5′ ³²P-radiolabeled 31-nt long HCV IRES probe; CP, cleaved probe. (E) Comparison of antiviral activity of indicated siRNAs targeting the region shared with siIE22 in R-1 cells. In (B), (C), and (E), *, <i>P <</i> 0.05; **, <i>P <</i> 0.01.</p