Biochemical Characterization of Enzyme Fidelity of Influenza A Virus RNA Polymerase Complex Shilpa Aggarwal, Birgit Bradel-Tretheway, Toru Takimoto, Stephen Dewhurst, Baek Kim* Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, United States of America Abstract Background: It is widely accepted that the highly error prone replication process of influenza A virus (IAV), together with viral genome assortment, facilitates the efficient evolutionary capacity of IAV. Therefore, it has been logically assumed that the enzyme responsible for viral RNA replication process, influenza virus type A RNA polymerase (IAV Pol), is a highly error- prone polymerase which provides the genomic mutations necessary for viral evolution and host adaptation. Importantly, however, the actual enzyme fidelity of IAV RNA polymerase has never been characterized. Principal Findings: Here we established new biochemical assay conditions that enabled us to assess both polymerase activity with physiological NTP pools and enzyme fidelity of IAV Pol. We report that IAV Pol displays highly active RNA- dependent RNA polymerase activity at unbiased physiological NTP substrate concentrations. With this robust enzyme activity, for the first time, we were able to compare the enzyme fidelity of IAV Pol complex with that of bacterial phage T7 RNA polymerase and the reverse transcriptases (RT) of human immunodeficiency virus (HIV-1) and murine leukemia virus (MuLV), which are known to be low and high fidelity enzymes, respectively. We observed that IAV Pol displayed significantly higher fidelity than HIV-1 RT and T7 RNA polymerase and equivalent or higher fidelity than MuLV RT. In addition, the IAV Pol complex showed increased fidelity at lower temperatures. Moreover, upon replacement of Mg++ with Mn++, IAV Pol displayed increased polymerase activity, but with significantly reduced processivity, and misincorporation was slightly elevated in the presence of Mn++. Finally, when the IAV nucleoprotein (NP) was included in the reactions, the IAV Pol complex exhibited enhanced polymerase activity with increased fidelity. Significance: Our study indicates that IAV Pol is a high fidelity enzyme. We envision that the high fidelity nature of IAV Pol may be important to counter-balance the multiple rounds of IAV genome amplification per infection cycle, which provides IAV Pol with ample opportunities to generate and amplify genomic founder mutations, and thus achieve optimal viral mutagenesis for its evolution. Citation: Aggarwal S, Bradel-Tretheway B, Takimoto T, Dewhurst S, Kim B (2010) Biochemical Characterization of Enzyme Fidelity of Influenza A Virus RNA Polymerase Complex. PLoS ONE 5(4): e10372. doi:10.1371/journal.pone.0010372 Editor: Dong-Yan Jin, University of Hong Kong, Hong Kong Received March 4, 2010; Accepted April 7, 2010; Published April 29, 2010 Copyright: 2010 Aggarwal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a National Institutes of Health (NIH) contract granted to the New York Influenza Center of Excellence (NIH/NIAID HHSN266200700008C). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: baek_kim@urmc.rochester.edu Introduction The high genetic diversity of influenza A virus (IAV), together with gene reassortment, forms viral quasispecies which enable IAV to acquire its efficient host adaptability. It is widely accepted that the efficient evolutionary capacity of IAV is facilitated by its highly error-prone replication process [1,2]. Every genome copy packaged in newly produced viral progenies is synthesized exclusively by the IAV RNA polymerase (Pol), and thus it has been logically assumed that IAV Pol is a highly error-prone polymerase which provides the genomic mutations necessary for viral evolution and host adaptation. Importantly, however, the actual enzyme fidelity of IAV RNA polymerase, which is the main driving force for viral mutation synthesis, has never been characterized. On the other hand, human immunodeficiency virus type 1 (HIV-1) is also well known for its genomic hypermutability [3,4], and indeed HIV-1 DNA polymerase, called reverse transcriptase (RT), is the most error prone DNA polymerase among those involved in genome replication [5]. However, the replication strategy of these polymerases is very different. HIV-1 RT replicates viral genomes only twice per infection cycle, and every RNA genome in the HIV-1progeny virions is actually produced by host RNA polymerase II. In contrast, IAV Pol not only is responsible for synthesizing every viral RNA genome but also amplifies viral RNAs multiple times per infection cycle, which gives IAV Pol ample chances to generate mutations in every infection event. IAV Pol has been qualitatively studied for its RNA-dependent RNA polymerase activity using various biochemical settings [6,7,8]. However, the mechanistic studies of IAV Pol complex, which determine basic kinetic parameters of the polymerases such as the nucleotide binding affinity, conformational change and chemical catalysis, have not been explored. This is because of several technical difficulties that have hindered this type of biochemical analysis for IAV Pol. First, the expression and purification of IAV Pol has been less than optimal for producing a large quantity of the active enzyme, simply because it is technically more challenging to express and purify the IAV Pol complex PLoS ONE | www.plosone.org 1 April 2010 | Volume 5 | Issue 4 | e10372