Inhibition of recombinant IAVpol using a 50-mer RNA template.

<p>(A) Principle of the reaction. Recombinant IAVpol (PA/PB1/PB2) was incubated in the presence of a 50-mer RNA template sequence derived from the 3′-end of the PA gene of the NanChang strain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068347#pone.0068347-Aggarwal1" target="_blank">[12]</a>. The 15-nt 5′vRNA oligo that is partially complementary to the 3′vRNA is needed as promoter for the enzyme. The 5′-pApG dinucleotide primer is extended and allows for multiple incorporation events of α-³³P-GMP used as tracer (star). (B) Representative curves of inhibition potency of 3′dGTP and T-705 RTP against IAVpol RNA synthesis activity. IC₅₀s were determined by adding increasing concentrations of each inhibitor, and quantitative analysis of the amount of remaining full length RNA product is expressed as % inhibition (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068347#s2" target="_blank">Materials and Methods</a>). Each experiment was conducted at least twice to calculate the average value and standard deviation. (C) The inhibition percentage was measured in the presence of a saturating concentration of T-705 RTP (100 µM), and either low (3 µM) or high (300 µM) concentration of one of the two purines GTP or ATP. (D) The same experiment as (C) instead with either low (3 µM) or high (300 µM) of pyrimidine UTP or CTP.</p

GMP and T-705 RMP incorporation opposite C on template.

<p>(A) A 14-mer RNA template sequence (t14-1) was designed to favor the extension of the 5′-pApG dinucleotide primer (AG) to a 9-mer in the presence of CTP and UTP, or a 14-mer full-length product (FL) in the presence of the additional GTP that can be specifically incorporated at position +10. (B) Example of a polyacrylamide gel electrophoresis result showing the products of primer extension. In addition to 25 µM CTP and UTP, 100 µM of GTP (lane 4), T-705 RTP (lane 5), ATP (lane 6), or 3′dGTP (lane 7) were added to the enzymatic reaction. The 9-mer and 14-mer product sequences were chemically synthesized, radiolabeled, and used as molecular size markers during the gel migration (lane 1 and 2). (C) Natural GTP was added to the primer extension reaction at increasing concentrations up to 10 µM in the presence of 25 µM CTP and UTP. (D) Quantitative analysis of GMP incorporation, based on the extension of the 9-mer RNA product obtained in Fig. 3C. The percentage of the extended products from 9-mer was plotted against GTP concentration and the data was fitted to a hyperbolic equation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068347#s2" target="_blank">Materials and Methods</a>) to derive the <i>K</i>_app for GTP incorporation. The inset shows the same plot on semi-log scale. Each experiment was conducted at least twice to calculate the average value and standard deviation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068347#pone-0068347-t002" target="_blank">Table 2</a>).</p

Inhibition of the influenza virus RNP complex by 3′dGTP and T-705 RTP.

<p>(A) Chemical structures of the obligate chain terminator 3′dGTP and the base-modified T-705 ribofuranosyl 5′-triphosphate (T-705 RTP). (B) Polyacrylamide gel electrophoresis (6%) showing the decrease in radiolabeled viral RNA product from the enzymatic reaction in the presence of increasing concentrations of 3′dGTP. Concentrations of inhibitor are as follows: lane 1 (0), lane 2 (0.023), lane 3 (0.069), lane 4 (0.21), lane 5 (0.62), lane 6 (1.9), lane 7 (5.6), lane 8 (16.7), lane 9 (50), and lane 10 (150 µM). (C) same as (B), with the same concentration range of T-705 RTP as inhibitor.</p

Enzymatic efficiency of single NMP incorporation opposite uridine (N:U).

*<p>calculated as <i>K</i>_app,NTP/<i>K</i>_app,ATP.</p

Schematic of ambiguous base pairing of T-705 opposite cytidine and uridine on RNA template.

<p>Enzyme kinetics of single nucleotide incorporation combined with competitive inhibition experiments demonstrated that T-705 RTP is able to base pair with a cytidine and a uridine prior to being incorporated by influenza virus polymerase with low discrimination against natural GTP or ATP.</p

AMP and T-705 RMP incorporation opposite U on the template.

<p>(A) A 14-mer RNA template sequence (t14-2) was designed to favor the extension of the 5′-pApG dinucleotide primer (AG) to a 9-mer in the presence of CTP and UTP, or a 14-mer full-length product (FL) in the presence of additional ATP that can be specifically incorporated at position +10. (B) Polyacrylamide gel electrophoresis result showing the product of primer extension in the presence of 100 µM of either ATP (lane 4), T-705 RTP (lane 5), or GTP (lane 6). The 9-mer and 14-mer product sequences were chemically synthesized, radiolabeled, and used as molecular size markers (lane 1 and 2). (C) Natural ATP was added to the reaction at increasing concentrations up to 10 µM. (D) Quantitative analysis of AMP incorporation, based on the extension of the 9-mer RNA product obtained in (C). The percentage of the extended products from 9-mer was plotted against ATP concentration, and the data was fit to a hyperbolic equation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068347#s2" target="_blank">Materials and Methods</a>) to derive the <i>K</i>_app for ATP incorporation. The inset shows the same plot on semi-log scale. Each experiment was conducted at least twice to calculate the average value and standard deviation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068347#pone-0068347-t003" target="_blank">Table 3</a>).</p