RNA template using in vitro transcription kit (Ambion), which was purified as described [19]. For the IAV Pol misincorporation reactions, a 30-nt RNA template (59 GCAUUGUCGCAAUCAGUACCUGCUUUCG- CU 39) encoding the first 30-nt sequence from the 39end of the PA gene was used (Integrated DNA Technologies). The T7 template sequence (59 TAATACGACTCACTATAGGCGAAAGCAGG- TACTGATTGCGACAATGC 39) contains the T7 promoter region which is the first 18 nucleotides and the remaining 29 nucleotides (see the sequence underlined) are the same sequence as that of the flu template. The first A nucleotide is deleted because T7 pol requires G at the +1 site. This also explains why the T7 bands run one nucleotide lower than the IAV Pol reaction bands on the PAGE. For the HIV-1 RT and MuLV RT reactions, a 48- nt RNA template is used. This 48-mer RNA template (59 GCAUUGUCGCAAUCAGUACCUGCUUUCGCUCUGGC- UUAAGGGCGAUCG 39) encodes not only the same first 30-nt sequence of the PA gene as the 30-mer Flu Pol template, but also the 18-nt non-PA gene which is annealed to a 18-mer DNA primer containing the A–G sequence at its 39 end. ApG initiated RNA transcription We tested the transcriptional activity of the purified polymer- ase complex using the ApG transcription assay in which the 39 and 59 vRNA and the dinucleotide primer, ApG (Biosynthesis) are provided. ApG was extended using the 39 vRNA template (14-nt) by the IAV polymerase (200 ng), generating a 14-nt long RNA fragment product. The ApG transcription reactions were performed as described elsewhere [20] at 37uC in 5 ml reactions mixtures containing transcription buffer (25 mM Tris-Cl, pH 7.5, 100 mM KCl, 5 mM Mg(OAC)2, 0.1 mM EDTA, 2 mM DTT, 0.25 ml Nonidet P-40, 12.5% glycerol), IAV Pol complex, 0.25 U/ml RNasin, 0.3 mM ApG, 1.6 mM 14nt 39vRNA, 1.6 mM 59vRNA, 0.08 mM radioactive a-32P-GTP (0.125 ml GTP, 3000 Ci/mmole, Perkin Elemer), and varying concentra- tions of nonradioactive NTPs (ATP, CTP, GTP and UTP) as described in figure legends. Transcription reactions were stopped after 1 h by the addition of 5 ml trichloroacetic acid (TCA) dye, denatured at 95uC for 3 min and transcription products were separated by 8 M urea denaturing 18–20% polyacrylamide gel electrophoresis (PAGE). The dried gels were analyzed by autoradiography using a PhosphorImager (Molecular Dynamics) and the intensity of the fully extended products was quantitated by OptiQuant (Version 3.10, Packard Instrument). In order to validate the linearity of the radioactive signals in this assay, we determined the signal intensity of the samples diluted by known ratios (i.e. 1, 1/2, 1/4, 1/8 and 1/16), and used that for all subsequent analysis of the signals. In the reactions described in Figure 1 which used varying amounts of radioactive and nonradioactive GTP, the ApG-primed reactions were performed in the presence of every 10-fold increasing concentrations of the nonradioactive GTP (0.1, 1, 10, 100 and 1000 mM) with 0.08 mM of radioactive a-32P-GTP and 1 mM of other three nonradioac- tive NTPs (ATP, CTP and UTP). Misincorporation assay In the misincorporation assays with all four polymerases, an equal concentration of all the nucleotides (NTPs or dNTPs) was provided using a mix of four a-32P-NTPs or dNTPs (0.16 mM, PerkinElmer, specific activity, 3000 Ci/mmol) and nonradioactive NTPs or dNTPs (500 mM). For IAV Pol, the ApG-transcription reactions were performed with the 30-nt 39 RNA template as described (Figure 2A), and the reactions were repeated with only three kinds of NTPs after deleting ATP [(2) A] or UTP [(2) U]. Influenza Polymerase Fidelity Similarly, for the T7 RNA polymerase reactions, 2 ml of normalized protein (Ambion) was mixed with 50 nM ds DNA template containing T7 promoter (Figure 2A) in the provided reaction buffer at 37uC for 1 hr. For HIV and MuLV RT, a mixture of 200 nM 48-mer RNA template annealed to 100 nM 18-mer DNA primer (Figure 2A), 500 mM dNTPs, 0.16 mM a-32P- dNTPs (PerkinElmer, 3000 Ci/mmol) were incubated with RT for 1 hour at 37uC under conditions described previously (16). In the (2) A and (2) T reactions, dATP and dTTP were left out, respectively. For further biochemical characterization, misincorporation reactions were carried out at various temperatures (30, 37, 39 and 42uC). For testing the effect of Mn++, Mg(OAC)2 (5 mM) in the buffer was replaced with varying concentrations of MnCl2 (0.5, 1, 5, 7.5 mM). Further, the reactions were performed with and without different amounts of purified NP. For reactions with NP, NP was preincubated with the RNA template prior to the addition of IAV Pol in the reaction mixture. Note that to minimize any loading variations between reactions, which can affect the total activity measurement, we added an equal amount of a 59 end 32P- labeled non specific primer to each reaction, and used this as the loading control (i.e. ‘‘LC’’ Figure 3). The transcription products in all the misincorporation reactions were separated by 8 M urea denaturing 18–20% PAGE. Results and Discussion RNA polymerase activity of IAV Pol complex using biologically relevant NTP concentrations First, in order to address the potential kinetic bias induced by the biased NTP pools that have been typically used in the gel- based IAV Pol assays, we conducted the ApG-initiated IAV RNA polymerase reaction using gradually increasing concentrations of the limited NTP (GTP) in the reaction, which abolishes the NTP pool bias (Figure 1). The ApG-primed reactions were performed in the presence of every 10-fold increasing concentrations of the nonradioactive GTP (0.1, 1, 10, 100 and 1000 mM) with a 0.08 mM of radioactive a-32P-GTP and 1 mM of other three nonradioactive NTPs (ATP, CTP and UTP). The polymerase was expressed and purified from the baculovirus system as we described previously [17]. The reaction mixtures contained the Nanchang H3N2 IAV Pol complex, ApG (0.3 mM), 59 RNA 15- mer (1.6 mM), and one of three different 39 RNA templates, 14-nt (Fig. 1A), 50-nt (Fig. 1B) and 137-nt (Fig. 1C) encoding the 39 end sequence of the Nanchang IAV PA gene. The reactions were performed at 37uC for 60 min, and were analyzed by denaturing 8 M urea-PAGE. The extent of the fully extended products, which is indicative of the polymerase activity in the multiple nucleotide incorporation assays, was determined. Importantly, note that the actual portion of the radioactive a-32P-GTP proportionally decreases upon mixing with every 10- fold increase in concentration of the nonradioactive GTP (0.1, 1, 10, 100 and 1000 mM, lanes 1 to 5 in Fig. 1). Thus, the NTP bias was abolished in the reaction with 1000 mM nonradioactive GTP where the portion of the radioactive GTP (0.08 mM) is minimal in the final GTP concentration (1000.15 mM). If the polymerase activity remains unchanged under this condition, the radioactive GTP incorporation in the primer extension, which can be visualized and quantitated from the radioactive band intensity of the extended products, should proportionally decrease, due to the increasing probability of the nonradioactive GTP incorporation instead of a-32P-GTP. However, as shown in Figure 1, in all of the reactions with the three different RNA templates, we did not observe the predicted proportional 10-fold reduction of the fully PLoS ONE | www.plosone.org 3 April 2010 | Volume 5 | Issue 4 | e10372