Article

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

ABSTRACT

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.

  • Source
    • "The National Institute of Allergy and Infectious Disease (NIAID) created the Systems Biology for Infectious Diseases Research program specifically to investigate the interactions between viruses and the host immune system [28]. Transcriptomic and proteomic analyses done at two of the program's centers, the Systems Virology Center and the Center for Systems Influenza, have identified transcriptome changes in response to infection with influenza and SARS-CoV, as well as novel interactions between the H5N1 influenza polymerase and a number of host proteins [29, 30, 31]. Efforts to identify the complete set of mechanisms by which viruses interact with host immune systems will provide numerous benefits for vaccine development. "
    [Show abstract] [Hide abstract] ABSTRACT: Numerous challenges have been identified in vaccine development, including variable efficacy as a function of population demographics and a lack of characterization and mechanistic understanding of immune correlates of protection able to guide delivery and dosing. There is tremendous opportunity in recent technological and computational advances to elucidate systems level understanding of pathogen-host interactions and correlates of immunity. A systems biology approach to vaccinology provides a new paradigm for rational vaccine design in a ‘precision medicine’ context.
    Full-text · Article · Oct 2013 · Current opinion in immunology
  • Source
    • "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. "
    [Show abstract] [Hide abstract] 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.
    Preview · Article · Apr 2012 · PLoS ONE
  • Source
    • "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]. "
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Apr 2010 · PLoS ONE
Show more