at the initiation sites may be minimal. However, it is still possible that some fraction of the pauses observed in our study may result from the pausing induced during the initiation of RNA synthesis. In addition, there are a number of other factors known to affect the processivity of polymerases such as template sequences, template RNA secondary structures, substrate availability, salt, and temperature. In this study, we employed identical template sequences for all four enzymes to minimize any template-induced pausing. Quantitative comparison of the misincorporation efficiency of IAV Pol complex with T7 RNA polymerase, HIV-1 RT and MuLV RT Finally, we estimated the fidelity difference between IAV Pol and three other enzymes analyzed in Figure 2. Since in the assay described in Figure 2, the misincorporation level is proportional to the total polymerase activity used, we could simply calculate the misincorporation efficiency in the (2) UTP (or TTP) reactions with each of the four polymerase reactions by normalizing the band intensity of the total misincorporated product (‘‘]’’) of the (2) UTP (or TTP) reaction with the band intensity of the total polymerase activities used which is represented by the full length products in the all four nucleotide reactions. First, we examined if the misincorporation efficiency remains similar in the reactions with three different activities of IAV Pol (1x, 2x and 3x: Fig. 2B). As shown in Figure 2F, the misincorporation efficiency of IAV Pol was relatively constant regardless of the IAV Pol activity used, and this validates that the polymerase activity and reaction conditions employed in Figure 2 are proper for fidelity comparisons. Second, HIV-1 RT showed 3 times higher misincorporation efficiency than MuLV RT in this gel based assay. In fact, HIV-1 RT previously showed 4 to 10 times lower fidelity than MuLV RT in various fidelity assays (1, 29, 31) and this further validates our gel based assay system. With this validation, next, we calculated and compared the misincorporation efficiency of IAV Pol with the three other polymerases. As summarized in Figure 2G, indeed, IAV Pol is 7 and 2.3 times less error-prone than HIV-1 and MuLV RTs, respectively, and 6.1 times less error-prone than T7 RNA polymerase. Importantly, since all of the four polymerases used in Figure 2 lack 39 to 59 proof-reading activity, the mutation synthesis observed in the misincorporation assay must be catalyzed only by their polymerase active sites. Effect of temperature, Mn++, and influenza nucleoprotein (NP) on the misincorporation efficiency of IAV Pol complex Previous studies with various DNA and RNA polymerases demonstrated that the enzyme fidelity of DNA polymerases is highly sensitive to reaction conditions such as metals [30,31] and reaction temperature [32,33]. Next, by using the physiological NTP condition showing robust activity of IAV Pol and the misincorporation assay established in Figure 2, we investigated a series of comprehensive biochemical characterizations for the effects of the temperature, Mn++ and viral RNA binding protein, NP, on the polymerase activity and fidelity of IAV Pol. First, we examined the effect of the temperature on the IAV Pol fidelity. The ApG-initiated RNA synthesis was conducted in the presence of all four NTPs or (2) UTP mix by the IAV Pol complex at 30, 37, 39 and 42uC for 1 hr. 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 (‘‘LC’’ Figure 3). The fully extended products in the Influenza Polymerase Fidelity reactions with all four NTPs were used for estimating the temperature dependence of IAV RNA polymerase after being normalized with the loading control (LC) band intensity. As shown in Figure 3A and 3B, the H3N2 IAV Pol complex showed the highest polymerase activity at 37uC and its activity largely decreased at 42uC. However, while the polymerase activity significantly reduced at 42uC, the level of the misincorporation beyond the 1st stop site at 42uC remained similar with the reactions at lower temperatures. Since the misincorporation capability was defined as the ratio of the level of the primer extended beyond the first stop site with the biased NTP pool to the total polymerase activity measured in the reaction with all four NTPs, the misincorporation capability of IAV Pol actually increased at 42uC, compared to that at the lower temperatures. Indeed, when the misincorporation efficiency at each temperature was calculated (Fig. 3C), the IAV Pol complex was more error prone at 42uC compared to the low temperatures. In fact, this temperature effect of the IAV Pol fidelity is novel because our previous study with RT proteins showed elevated fidelity at high temperatures such as 55uC [32]. In addition, the optimal temperature for the IAV Pol activity varies among viral strains as we recently reported [12], and avian strain Pol complexes appear to display its optimal activity at higher temperature in general [34]. Mg++ is a common metal ion used by DNA and RNA polymerases which essentially facilitates the nucleophilic attack of the 39 OH at the primer to the a-phosphate of the incoming nucleoside triphosphate to form a phosphodiester bond. It has been shown that replacing Mg++ with Mn++ promotes mutation synthesis in some DNA polymerases [35]. First, we examined the effect of Mn++ on the polymerase activity of H3N2 IAV Pol. 5 mM Mg++, which is commonly used in the IAV Pol assay, was used a control (Figure 4A), and the same reaction was repeated with varying Mn++ concentrations. As shown in Figure 4A and 4B, the H3N2 IAV Pol showed much lower polymerase activity with 0.5mM Mn++ than the reaction with 5 mM Mg++. Interestingly, at 1, 5 and 7.5 mM Mn++ the IAV Pol actually showed increased total NTP incorporation than with 5 mM Mg++. However, the primer extension at 1, 5 and 7.5 mM Mn++ became highly distributive as shown by the accumulation of abortive products, suggesting that the processivity of IAV Pol significantly reduced at high Mn++ concentrations. A recent report which employed biased NTP pool conditions, also showed increased activity by increasing Mn++ concentration although the activity with Mn++ is much lower than Mg++ [36] Next, we calculated the misincorporation efficiency using the equation described above. For this, we used the concentration of Mn++ which showed comparable activity to 5 mM Mg++ i.e., 0.5 mM Mn++ without any processivity defect. Even after normalizing these two reactions for activity differences, the reaction with Mn++ shows higher misincorporation. Thus, this data supports numerous earlier reports in literature stating that Mn++ promotes mutation synthesis by inserting a higher ratio of incorrect to correct substrate. Also, it leads to decreased hydrolysis of the mismatched product which explains the abortive products in the figure [37]. Lastly, we investigated the effect of influenza virus NP which is known to both bind viral RNA template as well as interact with the IAV polymerase [38]. For this, we expressed and purified the H3N2 NP protein in E. coli. Since one single NP molecule binds to RNA every 24 nucleotides [38], the 30-mer RNA template used in the previous figures is too short for this test. Thus we used the 50- nt long RNA template which was used in Figure 1B. The IAV Pol reactions were conducted in the absence and presence of varying amounts of NP which can coat 100, 150 and 200% of the 50-nt RNA template. As shown in Figure 5A and 5B, the polymerase PLoS ONE | www.plosone.org 8 April 2010 | Volume 5 | Issue 4 | e10372