pppAG-elongation on the correct antigenome 3′-end and on variants with an incorrect last nucleotide.

<p>pppAG-elongation by NS5Pol_DV on DV₁₀3′- templates (ACUAACAA-CN) varying the last nucleotide (correct -CU versus -CC, -CA and -CG). Control reactions were included without template. (<b>A</b>) pppAG elongation in the presence of Mn²⁺. pppAG (100 µM) and UTP (100 µM, containing αUTP) were used as substrates. Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 2 mM MnCl₂, 500 nM NS5Pol_DV, and 1 µM template. Reactions were started by addition of MnCl₂ and UTP. Samples were taken at given time points and analyzed by PAGE and autoradiography. OligoG marker is shown on the left, the identity of product bands is given on the right. (<b>B</b>) pppAG-elongation in the presence of Mg²⁺. pppAG (100 µM) and UTP (100 µM, containing αUTP) were used as substrates. Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 5 mM MgCl₂, 5 µM NS5Pol_DV, and 1 µM template. Reactions were started by addition of MgCl₂ and UTP. Samples were taken at given time points and analyzed by PAGE and autoradiography. OligoG marker is shown on the right; the identity of product bands is given on the left.