Influenza Polymerase Fidelity Figure 4. Effect of manganese on polymerase activity and misincorporation efficiency of IAV Pol complex. (A) IAV polymerization with 5 mM Mg(OAC)2 and varying concentration of MnCl2 (lanes 1 to 4, 0.5, 1, 5, 7.5 mM). LC: loading control (B) Fully extended products for the IAV Pol polymerase activity with different metal concentrations were compared after being normalized with the value obtained with 5 mM Mg(OAC)2 (100%). (C) The misincorporation efficiency of IAV Pol at 0.5 mM MnCl2 was calculated as described in Figure 2F, and compared with the value obtained with 5 mM Mg(OAC)2 (100%). C: no polymerase control. doi:10.1371/journal.pone.0010372.g004 activity of the IAV Pol increases with increasing NP concentration, which has been previously observed [39]. However, upon the normalization with the total activity, the misincorporation efficiency decreased slightly in reactions with NP. Virological implications of the high fidelity nature of IAV Pol with respect to other RNA viruses It has been well established that the enzyme fidelity of viral polymerases is a main determinant for viral genetic hyper- mutability and evolution; and in the case of HIV-1, the highly error-prone HIV-1 RT is considered as a key contributor for viral genomic hypervariability [5,40]. This concept has been supported because alterations in the HIV-1 RT fidelity directly affect HIV-1 mutation rate [40]. Considering the high viral capacity of influenza viruses to evolve both within and between host species as well, the observation of the relatively high enzyme fidelity of the IAV Pol complex was rather unexpected. Interestingly, however, several primer extension based biochemical studies also reported PLoS ONE | www.plosone.org 9 April 2010 | Volume 5 | Issue 4 | e10372