In vitro translation of ANDV-like SmRNAs in the presence of partially purified recombinant ANDV GST-N protein.
(A) Schematic representation of the capped and polyadenylated Glo-FLuc RNA and capped ANDV like SmRNA, N-RNA-3’UTR [27], used in this study. In the ANDV like SmRNA RNAs, the FLuc reporter gene is in-frame with the ANDV-N protein initiation codon (depicted by the arrow) [27]. (B-E) In vitro translation (90 min at 30°C) in nuclease-treated Flexi-rabbit reticulocyte lysate (RRL; 35% v/v) programmed with in vitro transcribed cap-Glo-FLuc-poly(A) (B-C), or the cap-N-RNA-3’UTR (D-E) RNAs (0.01 pmol) was conducted in the presence of increasing concentrations of the ANDV GST-N protein (protein/RNA molar ratio from 0.25:1 to 250:1). GST alone at a protein/RNA molar ratio of 1:1 was used as a control. Data are shown as ratios, the number of ANDV GST-N protein molecules added to each reaction, relative to the estimated number of cap-N-RNA-3’UTR molecules added to the reaction (6.02 x109 molecules/μL). The number of RNA molecules present in 0.01 pmol (6.02 x109 molecules) was estimated considering an RNA length of 2286 bp for cap-N-RNA-3’UTR, and 2086 bp for the cap-Glo-FLuc-poly(A) RNAs, using the free online calculator [http://www.molbiol.ru/eng/scripts/01_07.html]. The number of molecules of GST (35kDa) or the recombinant ANDV GST-N (77.5 kDa) added to the reaction was estimated using the free online calculator [https://www.bioline.com/us/media/calculator/01_04.html]. (C and E) After the in vitro translation assay was performed 1μL of the reaction mix was taken from the indicated experimental points and diluted (1/100) and directly used as a template for a real-time RT-qPCR reaction to determine the relative levels of cap-Glo-FLuc-poly(A) RNA (C) and cap-N-RNA-3’UTR (E) RNA. The RNA abundance was expressed relative to the value obtained for the RRL supplemented only with GST at a 1:1 RNA to protein ratio set to 1. Values are the mean (+/- SEM) of at least three independent experiments, each conducted in duplicate. Statistical analysis was performed by a one-way ANOVA with Dunnett’s multiple comparisons test (P<0.05).
