Journal: International Journal of Molecular Sciences

Article Title: Hepatitis C Virus 5′UTR Sequences That Bind eIF3 and Ribosomal 40S Subunits Confer Stimulation of Minus-Strand RNA Synthesis

doi: 10.3390/ijms27073234

Figure Lengend Snippet: Detection of HCV replicon minus-strand RNA synthesis. In vitro transcribed RNAs of three replicon constructs were transfected into Huh-7.5 cells. The “hp” construct has only a protective stem-loop at its 5′end (“Gene Cluster I”, see B), while the “5′UTR” constructs have the complete HCV 5′UTR. The latter come with either wild-type NS5B polymerase activity (WT; Pol +), or with the NGND mutation in the NS5B active center (Pol −). At 4, 24, 48 and 72 h after transfection (hpt), cells were either lysed and the cell extracts were used for RNA isolation and detection ( A , C , D ), or separate cell lysates were prepared for firefly luciferase (Fluc) measurement ( B ). The same construct color code is used through all panels. ( A ) Detection of HCV replicon RNA plus strands by specific strand-specific RT-qPCR. Values show means and standard deviations (SDs), dots show values of individual experiments. ( B ) Firefly luciferase (Fluc) activity expressed from Gene Cluster II as an indirect measure for replicon RNA plus-strand abundance. ( C ) Detection of HCV replicon minus strands by strand-specific RT-qPCR after 5EU labeling, biotin linkage and streptavidin bead purification of RNA newly synthesized in the Huh-7.5 cells after replicon RNA transfection (see improved protocol in ). ( D ) 40-fold magnification (“blowup”) of Y-axis from ( C ) to show low-value details. At 72 hpt, the 5′UTR-NGND (Pol-) replicon showed 0.025% of the 5′UTR WT (Pol +), with an SD of 0.014%. Statistical significance for pair-wise comparisons is shown with ** p < 0.01; *** p < 0.001; and p > 0.05 not significant (n.s.).

Article Snippet: Data processing: The Ct values obtained from RT-qPCR curves (using the Bio-Rad CFX Maestro Software ( www.bio-rad.com/de-de/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments ; Version 1.0, accessed on 26 February 2018) for Real-Time PCR) were first normalized to the corresponding expression value obtained with GAPDH mRNA (∆Ct = Ct [sample] − Ct [GAPDH]).

Techniques: In Vitro, Construct, Transfection, Activity Assay, Mutagenesis, Isolation, Luciferase, Quantitative RT-PCR, Labeling, Purification, Synthesized

Journal: International Journal of Molecular Sciences

Article Title: Hepatitis C Virus 5′UTR Sequences That Bind eIF3 and Ribosomal 40S Subunits Confer Stimulation of Minus-Strand RNA Synthesis

doi: 10.3390/ijms27073234

Figure Lengend Snippet: Minus-strand synthesis proficiency of HCV replicon RNAs with different lengths of 5′UTR sequences. Replicon constructs contain either the hairpin (hp) or HCV 5′UTR sequences as shown in B, lower panel, with the NS5B replicase active (Pol +) or inactive (Pol −). The experiment was performed as shown in , and the same construct color code is used through all panels. ( A ) Strand-specific RT-qPCR detection of plus strands. ( B ) Strand-specific RT-qPCR detection of 5EU labeled minus strands. ( C ) 30-fold magnification of Y-axis from ( B ) to show low-value details. At 72 hpt, the SLI-III-NGND (Pol −) replicon showed 0.032% of the SLI-III WT (Pol +), with an SD of 0.016%. Statistical significance for pair-wise comparisons is shown with ** p < 0.01.

Article Snippet: Data processing: The Ct values obtained from RT-qPCR curves (using the Bio-Rad CFX Maestro Software ( www.bio-rad.com/de-de/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments ; Version 1.0, accessed on 26 February 2018) for Real-Time PCR) were first normalized to the corresponding expression value obtained with GAPDH mRNA (∆Ct = Ct [sample] − Ct [GAPDH]).

Techniques: Construct, Quantitative RT-PCR, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Hepatitis C Virus 5′UTR Sequences That Bind eIF3 and Ribosomal 40S Subunits Confer Stimulation of Minus-Strand RNA Synthesis

doi: 10.3390/ijms27073234

Figure Lengend Snippet: Effect of mutations affecting binding of eIF3 and the ribosomal 40S subunit on minus-strand synthesis. ( A ) Strand-specific RT-qPCR detection of 5EU labeled minus strands. In constructs “Δb” the SLIIIb was deleted (compare B, lower panel), which severely affects eIF3 binding. In mutant constructs “de” the SLIIId and SLIIIe were mutated, which severely affects 40S subunit binding. ( B ) Expression of HCV core protein with C-terminal triple HiBiT tags from of the above constructs. This expression is a direct measure for the ability of the respective HCV IRES in Gene Cluster I to mediate IRES-dependent translation. ( C ) 10-fold magnification of Y-axis from ( B ) to show low-value details. Statistical significance for pair-wise comparisons is shown with ** p < 0.01.

Article Snippet: Data processing: The Ct values obtained from RT-qPCR curves (using the Bio-Rad CFX Maestro Software ( www.bio-rad.com/de-de/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments ; Version 1.0, accessed on 26 February 2018) for Real-Time PCR) were first normalized to the corresponding expression value obtained with GAPDH mRNA (∆Ct = Ct [sample] − Ct [GAPDH]).

Techniques: Binding Assay, Quantitative RT-PCR, Labeling, Construct, Mutagenesis, Expressing