Journal: Journal of Virology

Article Title: The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components

doi: 10.1128/jvi.01954-23

Figure Lengend Snippet: Identification of ZIKV sfRNA-binding proteins in hNPCs. (A) hNPCs and BHK-21 cells were infected with ZIKV FSS13025 or ZIKV GZ01. Thirty-six hours post-infection, cell-associated RNA was harvested and levels of gRNA and sfRNA were analyzed by Northern Blot. (B) Workflow of RNA pulldown and LC-MS/MS analysis. Biotinylated RNAs containing FSS13025 sfRNA, GZ01sfRNA, or a size-matched control transcript were incubated with hNPC lysates. Proteins that co-purified with the RNAs on streptavidin beads were identified by chromatography mass spectropmetry. (C) Volcano plots of eluted proteins. The x axis shows the mean 2Log difference in protein abundance between the FSS13025 sfRNA (left) or GZ01 sfRNA (right) and control samples from three independent biological replicates. The y axis shows the 10Log of the P value by Student’s t test from the comparison of the protein abundance in the FSS13025 sfRNA (left) or GZ01 sfRNA (right) with the control samples. Significantly enriched proteins are shown as red. The 12 proteins that bond by both FSS13025 sfRNA and GZ01 sfRNA are shown as red diamonds, and their names are labeled aside. The names of proteins associated with RISC assembly are shown in blue. (D) Go enrichment classification of the 12 proteins that bond by both FSS13025 sfRNA and GZ01 sfRNA. (E) Biotinylated RNAs containing FSS13025 sfRNA, GZ01 sfRNA, or a size-matched control transcript were incubated with hNPC lysates. Associated proteins were eluted and analyzed by Western blot.

Article Snippet: Human neural progenitor cell (hNPC) line 15167 derived from fetal brains (Lonza) ( 66 ) was cultured as neurospheres in neurocult-XF basal medium (STEMCELL technologies) supplemented with neurocult-XF proliferation supplement (STEMCELL technologies), basic fibroblast growth factor (bFGF, 10 ng/mL, STEMCELL technologies), and epidermal growth factor (EGF, 20 ng/mL, STEMCELL technologies), Heparin solution (2 μg/mL, STEMCELL technologies).

Techniques: Binding Assay, Infection, Northern Blot, Liquid Chromatography with Mass Spectroscopy, Control, Incubation, Purification, Chromatography, Quantitative Proteomics, Comparison, Labeling, Western Blot

Journal: Journal of Virology

Article Title: The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components

doi: 10.1128/jvi.01954-23

Figure Lengend Snippet: Mapping the region(s) of sfRNA responsible for RHA or PACT binding and RNAi repression activity. (A) Schematic representation of the truncate RNAs. (B) Biotinylated truncate RNAs were incubated with hNPC lysates. Associated proteins were eluted, separated on a 10% SDS-PAGE and probed with the antibodies indicated on the left by Western blot. (C) hNPCs were cotransfected with plasmids pEGFP-N1(encoding eGFP) (100 ng), shGFP, or shNC (300 ng) and 3′SL RNA or control RNA (300 ng). The expression of eGFP was analyzed 48 h after cotransfection. The intensity of eGFP was observed under fluorescence microscopy. (D) Cell lysates from (C) were harvested and analyzed by Western Blot. (E) The levels of GFP expression in panel D of three independent experiments were quantified by densitometry and normalized to that of actin. The expression level of GFP in the first column was set at 1.0. Scale bar, 200 μm. Data are expressed as mean ± SD from three independent experiments. **P ≤ 0.01, as determined by Student’s t test.

Article Snippet: Human neural progenitor cell (hNPC) line 15167 derived from fetal brains (Lonza) ( 66 ) was cultured as neurospheres in neurocult-XF basal medium (STEMCELL technologies) supplemented with neurocult-XF proliferation supplement (STEMCELL technologies), basic fibroblast growth factor (bFGF, 10 ng/mL, STEMCELL technologies), and epidermal growth factor (EGF, 20 ng/mL, STEMCELL technologies), Heparin solution (2 μg/mL, STEMCELL technologies).

Techniques: Binding Assay, Activity Assay, Incubation, SDS Page, Western Blot, Control, Expressing, Cotransfection, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components

doi: 10.1128/jvi.01954-23

Figure Lengend Snippet: Multiple flaviviral 3′SLs can bind to RISC components and exhibit higher affinities than siRNA. (A) Biotinylated 3′SL RNAs from ZIKV, DENV, WNV, JEV, YFV, or siRNA were incubated with hNPC lysates. Associated proteins were eluted, separated on a 10% SDS-PAGE and probed with the antibodies indicated on the left by Western blot. (B) BLI assay. Biotinylated RNAs were captured onto streptavidin (SA) biosensors and assayed for binding to RHA or PACT at the indicated concentrations. The data collected were processed on the Gator software. (C) The binding affinities (KD) of (B) were compared.

Article Snippet: Human neural progenitor cell (hNPC) line 15167 derived from fetal brains (Lonza) ( 66 ) was cultured as neurospheres in neurocult-XF basal medium (STEMCELL technologies) supplemented with neurocult-XF proliferation supplement (STEMCELL technologies), basic fibroblast growth factor (bFGF, 10 ng/mL, STEMCELL technologies), and epidermal growth factor (EGF, 20 ng/mL, STEMCELL technologies), Heparin solution (2 μg/mL, STEMCELL technologies).

Techniques: Incubation, SDS Page, Western Blot, Binding Assay, Software