Influenza Polymerase Fidelity Figure 5. Effect of H3N2 NP on polymerase activity and the misincorporation efficiency of the IAV Pol complex. (A) IAV polymerization in the presence and absence of varying concentrations of H3N2 NP with the 50-mer template used in Figure 1B. Molar ratios between the RNA template and NP molecule were altered between 0 and 2. The RNA template was preincubated with NP before adding the polymerase complex. LC: loading control; (B) Fully extended products for the IAV Pol activity with different molar ratios of RNA/NP were compared after being normalized with the value obtained with no NP (100%). (C) The misincorporation efficiency of IAV Pol was calculated as described in Figure 2F, and compared using the value obtained with no NP (100%). C: no polymerase control. doi:10.1371/journal.pone.0010372.g005 that the RNA-dependent RNA polymerase of poliovirus, which is a positive strand RNA virus, is a high fidelity enzyme [41]. Thus, we reasoned about the disagreement between high genetic variability of IAV and relatively high enzyme fidelity of IAV Pol complex found in this report. IAV has a replication (RNA synthesis) strategy that amplifies their genomes many times per infection by using exclusively their own polymerase complex without any assistance from host RNA polymerases. This is contrasted to retroviruses (i.e. HIV-1) that replicate their single stranded RNA genomes into double-stranded proviral DNA in two ways. First, HIV-1 replicate their RNA genome to proviral double stranded DNA only twice per infection (conversion of (+) single strand RNA genomes to (2) single strand DNA and then conversion of the synthesized (2) DNA to double stranded proviral DNAs) by using their own polymerases. Second, the RNA genomes of every HIV-1 progenies are synthesized by host RNA polymerase II by transcribing the proviral DNA integrated into the host chromosomes. It is a reasonable assumption that the viruses, which require a high level of genomic mutations for their evolution and escape but have only a few chances to replicate their genomes by their own polymerases (i.e. HIV-1), might have evolved to harbor highly error-prone polymerases (i.e. HIV-1 RT) to generate sufficient mutations in really limited chances. In contrast, due to numerous rounds of the amplification of single viral RNA genomes to viral RNAs only by the viral polymerase, IAV Pol has ample chances to make and accumulate genomic mutations in each viral infection cycle. It has been estimated that the viral burst size in case of influenza, in one cycle of replication, is about 1000 [42], and thus influenza virus should undergo ,1000 rounds of genome replication solely by viral RNA polymerase. If IAV Pol PLoS ONE | www.plosone.org 10 April 2010 | Volume 5 | Issue 4 | e10372