<i>De novo</i> RNA synthesis by NiV L-P and analysis of promoter sequence requirements.

<p>(a) The 12-mer RNA template from the leader promoter region of the NiV genome was used in a <i>de novo</i> RdRp assay and products were analyzed using a 22.5% polyacrylamide urea sequencing gel. NiV L(wild-type [wt])-P, abbreviated as WT L, and template were incubated in the presence of α³³P-GTP tracer (lane 1) or with the addition of ATP (A) and CTP (C) (lane 2). NiV L(D832A-N833A)-P mutant, abbreviated as D832A-N833A, and template were incubated in the presence of α³³P-GTP tracer (lane 3) or with the addition of ATP and CTP (lane 4). NiV L(H1347A)-P, abbreviated as H1347A, and template were incubated in the presence of α³³P-GTP tracer (lane 5) or with the addition of ATP and CTP (lane 6). Product sizes are indicated using a kinase-labeled set of four combined oligonucleotides: 4-, 5-, 6-, and 12-mer primer (lane M). (b) A nanoluciferase-based NiV minigenome assay comparing luciferase activity for NiV L(wt), NiV L(H1347A), NiV L(R1348A), and a control sample with no L. Sample size was n = 2, error bars represent standard deviation, and sample luciferase activity was normalized to NiV L(wt). (c) Product formation was quantified and the Michaelis constant (<i>K</i>_<i>m</i>) for ATP and CTP was calculated. Sample size was n = 2 and error bars represent standard deviation. (d) Using systematically introduced adenine at the first nine bases of the promoter sequence, RNA synthesis activity was quantified, and normalized to the 12-mer RNA template from the leader promoter. For these experiments, nucleotide substrates included ATP+ CTP+UTP at 1 mM each, and α³³P-GTP. Samples size was n = 2 and error bars represent standard deviation.