which consists of three different subunits, PA, PB1 and PB2, as compared to other single subunit polymerases such as HIV-1 RT. Second, polymerase studies which determine the basic kinetic parameters of DNA/RNA polymerases employ pre-steady state conditions instead of the steady state condition [9,10]. However, since the pre-steady state condition requires the saturation of the template/primer complex used (100–200 nM) by the polymerase, a large amount of the purified polymerase is necessary for this type of analysis. Third, the IAV Pol assays use very high concentrations (mM) of primers such as ApG and cellular mRNAs, while other polymerase assays (i.e. HIV-1 RT) employ much lower concen- trations of the primers (10–100 nM). Therefore, under the current IAV Pol reaction conditions available, only a fraction of the total primer is extended in each polymerase reaction, and thus the use of the 59 end labeled primers, which are routinely used in kinetic assays of other polymerases, are not easily applicable to the IAV Pol assay. Most frequently used polymerization assays of IAV Pol in current studies are gel-based standing start primer extension reactions that incorporate NTP substrates using ApG or cellular mRNAs as primers. In typical IAV Pol assays, one of the four NTPs is an a-32P-labeled radioactive NTP (i.e. a-32P-GTP), which aids in the visualization of the extended products, and the other three NTPs are nonradioactive (i.e. ATP, CTP and UTP), [6,7,8,11,12]. However, while the three nonradioactive NTPs are normally used at mM ranges, the concentration of the radioactive NTP (i.e. a-32P-GTP) is relatively very low (0.08 to 50 mM), simply because the highly concentrated radioactive NTP is not readily available, and use of a large quantity of radioactivity is technically challenging. Indeed, many recent reports that studied the steady state kinetic NTP incorporation by IAV Pol still used approximately 1/100 to 1/1000 biased nucleotide pools for the radioactive NTP (i.e. GTP) to the other three nonradioactive NTPs (i.e. ATP, CTP and UTP) [8,13,14]. These unbalanced NTP pools in the reaction raise several concerns. First, it is highly likely that the use of 100- or 1000-fold lower concentration of the radioactive GTP, as compared to the three other nonradioactive NTPs, induces a serious polymerase kinetic bias: the entire IAV Pol reaction rate is simply dependent on the limited availability of radioactive GTP in the reaction, ultimately leading to the underestimation of the kinetic activity of IAV Pol. Second, biased NTP pools are a well known factor for mutagenesis [15], implying that some of the extension products, which were used for assessing polymerase activity, are actually the products of mutation synthesis. Third, it is well established that cellular NTP concentrations range between 0.25–3 mM (mM, ATP, 3,152 +/2 1,698; GTP, 468 +/2 224; UTP, 567 +/2 460 and CTP, 278 +/2 242) [16]. In this report, we established new assay conditions that use unbiased and biologically relevant NTP substrate concentrations and avoid the kinetic bias found in the currently available gel- based assays. Importantly, this adjustment of the assay conditions not only revealed the robust polymerase activity of IAV Pol complex, but also enabled us to investigate the enzyme fidelity of IAV Pol. Also, with this new assay condition, we were able to conduct a series of comprehensive biochemical analysis to study the impact of various reactions parameters such as temperature, metals and viral NP on the IAV Pol fidelity. Overall, our results suggest that IAV Pol is least error-prone when compared to two reverse-transcriptases, HIV-1 RT and MuLV RT and also to bacterial phage T7 RNA polymerase. Importantly, the biochem- ical finding of the high fidelity nature of IAV Pol introduces a new concept of the mechanistic relationship between IAV Pol fidelity and the IAV replication strategy which uniquely provides IAV Pol Influenza Polymerase Fidelity with ample opportunities to generate genomic mutations during the multiple rounds of cRNA and vRNA amplification in a single infection cycle. Materials and Methods Expression and purification of influenza A virus RNA polymerase complex and other polymerases For IAV polymerase, the PA, PB1 and PB2 genes (codon- optimized, Geneart) were amplified by reverse-transcription PCR from the H3N2 IAV strain (A/chicken/Nanchang/3-120/01), obtained from Dr. R. Webster, St. Jude Children’s Research Hospital, Memphis, TN, USA. They were then cloned to the baculovirus expression vector pVL1392 (Invitrogen) between the BglII and XbaI sites. The PA gene was tagged at the C-terminus with the tandem affinity purification (TAP) tag, which consisted of a thrombin cleavage site followed by 6X His tag, tobacco etch virus (TEV) cleavage site and finally an IgG-binding domain. Each of the three pVL1392 plasmids were individually transfected into Sf9 insect cells for the production of recombinant viruses in SF900II SFM media (GIBCO), following the protocols provided by the vendor (BD Biosciences Pharmingen). To prepare the purified trimeric IAV polymerase, the three recombinant viruses were used for coinfecting Tni insect cells grown in Express Five SFM media supplemented with L-Glutamine (GIBCO). Cells were harvested 72 hours post infection and the lysates obtained were purified by the TAP purification technique as described previously [12,17]. Typically, 1l expression culture produced 0.7 mg of the purified complex with approximately 95% purity, which was qualitatively compared with the band intensity of BSA with 95% purity (Sigma-Aldrich).The approximate molar ratio of PA:PB1:PB2, which was estimated by the protein band intensity in SDS-PAGE, was 1.8:1:1. T7 RNA polymerase was purchased from Ambion, and HIV-1 and MuLV RT proteins were isolated in our previous study [18]. Overexpression and purification of H3N2 NP in E. coli The H3N2 Nanchang NP gene (A/Chicken/Nanchang/3-120/ 01) was cloned to pET28a (Novagene), expressing NP protein fused to the six histidine tag at its N-terminal end. NP was purified using Ni++ column chromatography following the protocol provided by the vendor (Novagen). Fractions containing NP were pooled and dialyzed to a low salt buffer (50 mM Tris-Cl (pH 7.5), 1 mM EDTA, 200 mM NaCl, 10% glycerol, 1 mM DTT). The dialyzed protein was further purified using a second DEAE- Sephacel column to minimize any RNase contamination. The RNA binding of the purified NP was examined by a filter binding assay employing both protein and nucleic acid binding filters (data not shown). Template preparation 59 vRNA (59 AGUAGAAACAAGGCC 39) and the 14- nucleotide (nt) 39 vRNA which serves as a template (59 GGCCUGCUUUUGCU 39) from the PA gene of influenza A virus were purchased from Dharmacon. The other two templates (50-nt and 137-nt) used encode the first 50-nt and 137-nt sequences from the 39 end of the Nanchang PA gene. The 50-nt template was purchased from Integrated DNA Technologies. The 137-nt RNA template was prepared by in vitro transcription using a double stranded (ds) PCR product amplified by a 37-mer forward primer encoding T7 promoter sequence and 19-nt of the viral sequence and a 16-mer reverse primer encoding 39 sequence of the PA gene. The PCR product was used for synthesizing the 137-mer PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e10372