The human H5N1 influenza A virus polymerase complex is active in vitro over a broad range of temperatures, in contrast to the WSN complex, and this property can be attributed to the PB2 subunit

Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
Journal of General Virology (Impact Factor: 3.18). 01/2009; 89(Pt 12):2923-32. DOI: 10.1099/vir.0.2008/006254-0
Source: PubMed


Influenza A virus (IAV) replicates in the upper respiratory tract of humans at 33 degrees C and in the intestinal tract of birds at close to 41 degrees C. The viral RNA polymerase complex comprises three subunits (PA, PB1 and PB2) and plays an important role in host adaptation. We therefore developed an in vitro system to examine the temperature sensitivity of IAV RNA polymerase complexes from different origins. Complexes were prepared from human lung epithelial cells (A549) using a novel adenoviral expression system. Affinity-purified complexes were generated that contained either all three subunits (PA/PB1/PB2) from the A/Viet/1203/04 H5N1 virus (H/H/H) or the A/WSN/33 H1N1 strain (W/W/W). We also prepared chimeric complexes in which the PB2 subunit was exchanged (H/H/W, W/W/H) or substituted with an avian PB2 from the A/chicken/Nanchang/3-120/01 H3N2 strain (W/W/N). All complexes were functional in transcription, cap-binding and endonucleolytic activity. Complexes containing the H5N1 or Nanchang PB2 protein retained transcriptional activity over a broad temperature range (30-42 degrees C). In contrast, complexes containing the WSN PB2 protein lost activity at elevated temperatures (39 degrees C or higher). The E627K mutation in the avian PB2 was not required for this effect. Finally, the avian PB2 subunit was shown to confer enhanced stability to the WSN 3P complex. These results show that PB2 plays an important role in regulating the temperature optimum for IAV RNA polymerase activity, possibly due to effects on the functional stability of the 3P complex.

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    • "A purified H5N1polymerase shows significantly higher polymerase activity in vitro when compared to human strain A/WSN/33 (H1N1) [14], [46], [47]. The polymerase activity of WSN was increased by the introduction of the PB2 or PA subunit of H5N1. "
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    ABSTRACT: Genetic reassortment plays a critical role in the generation of pandemic strains of influenza virus. The influenza virus RNA polymerase, composed of PB1, PB2 and PA subunits, has been suggested to influence the efficiency of genetic reassortment. However, the role of the RNA polymerase in the genetic reassortment is not well understood. Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes, and demonstrated that the PB2 subunit of A/HongKong/156/1997 (H5N1) [HK PB2] dramatically reduced the synthesis of mRNA, cRNA and vRNA when introduced into the polymerase of other influenza strains of H1N1 or H3N2. The HK PB2 had no significant effect on the assembly of the polymerase trimeric complex, or on promoter binding activity or replication initiation activity in vitro. However, the HK PB2 was found to remarkably impair the accumulation of RNP. This impaired accumulation and activity of RNP was fully restored when four amino acids at position 108, 508, 524 and 627 of the HK PB2 were mutated. Overall, we suggest that the PB2 subunit of influenza polymerase might play an important role for the replication of reassortant ribonucleoprotein complexes.
    PLoS ONE 04/2012; 7(2):e32634. DOI:10.1371/journal.pone.0032634 · 3.23 Impact Factor
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    • "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 55°C [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]. "
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    ABSTRACT: 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. 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. 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.
    PLoS ONE 04/2010; 5(4):e10372. DOI:10.1371/journal.pone.0010372 · 3.23 Impact Factor
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    ABSTRACT: The declaration of pandemic alert Phase 6 for human influenza A (H1N1) by the WHO and the measures taken by individual countries in June 2009 has shown the world how fragile today's resources in pandemic and prepandemic, but also seasonal, vaccines are. Conventionally, human influenza vaccines are produced in embryonated chicken eggs. However, significant efforts of authorities and vaccine manufacturers over the last decade have led to the establishment of cell culture-derived vaccines. Currently, vaccines produced in three different host cell lines (Madin-Darby Canine Kidney, Vero and PER.C6) are in clinical trials, and the first licenses for seasonal as well as pandemic H5N1 vaccines have been granted. These encouraging developments for cell culture-based influenza virus production are summarized and an overview on potential cell substrates is given.
    Expert Review of Vaccines 12/2009; 8(12):1681-92. DOI:10.1586/erv.09.128 · 4.21 Impact Factor
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