State-of-the-art and preparatory work

Influenza viruses (IV) are a major threat for humans and can lead to severe disease and cause pandemic outbreaks, such as the actual H1N1 strain. Highly pathogenic avian influenza viruses (HPAIV) of the H7‐ and H5‐subtypes not only affect birds but also sometimes humans with high mortality rates. Their widespread occurrence and possibility to infect humans increase the danger of reassortment between human IV and HPAIV that could create new human pathogenic viruses.

The IV genome is composed of eight ssRNA‐segments (1, 2). They encode different viral proteins and can only be replicated and transcribed by the viral RNA‐dependent‐RNA‐polymerase (RdRp) within the nucleus of the infected cell. The RdRp consists of three subunits (PB1, PB2, PA) and together with the viral RNA (vRNA) and the nucleocapsid protein (NP) forms the replication/transcription active ribonucleoprotein complex (RNP). The NS segment encodes both, the viral Nonstructural Protein 1 (NS1) as well as the Nuclear Export Protein (NEP) (3). NEP was shown to be involved in the nuclear/cytoplasmatic transport of the viral RNPs. NS1 is a multifunctional regulatory factor that counteracts the cellular innate immunity reaction. NS1 binds to dsRNA, a viral replication product, and blocks the activation of the dsRNA‐activated protein kinase (PKR) and of transcription factors, which induce the expression of type I interferon (IFN) and thereby activate the cellular immune response. Furthermore, NS1 inhibits splicing and polyadenylation of cellular pre‐mRNAs, leading to preferential expression of viral proteins (4). NS1 can therefore directly or indirectly affect the replication efficiency of the viral RNPs.

Previous own work in the IRTG has shown that different NS segments of the HPAIV strains A/Goose/Guangdong/1/1996 (GD, H5N1) (5), A/Mallard/NL/12/2000 (Ma, H7N3) and A/Vietnam/1203/2004 (VN, H5N1) (Wang et al., unpublished) reassorted with the strain A/chicken/FPV/Rostock/34, (FPV, H7N1), resulting in altered viral replication characteristics including alteration of the polymerase activity. Furthermore, the reassortant viruses show different growth rates and effects on the innate immunity in mammalian cell culture systems. In avian cell culture systems all replicate similar except the FPV NS VN reassortant. These results suggest that the effect of the NS segments may depend on host specific factors. The NS segment of the different strains GD, Ma and VN are well distinguished from the FPV strain, and the effect of specific amino acids on viral growth and RdRp activity can therefore be analyzed by mutational analysis and generation of recombinant reassortant viruses with mutated NS segments.

References

1. Wright, P. F., Naumann, G., & Kawaoka, Y. (2007) in Fields ‐ Virology, eds. Knipe, D. M. & Howley, P. M. (Lippincott Williams & Wilkins, Philadelphia), pp. 1691‐1740.

2. Palese, P. & Shaw, M. (2007) in Fields ‐ Virology, eds. Knipe, D. M. & Howley, P. M. (Lippincott Williams & Wilkins, Philadelphia), pp. 1646‐1689.

3. O'Neill, R. E., Talon, J., & Palese, P. (1998) The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J 17, 288‐296.

4. Hale, B. G., Randall, R. E., Ortin, J., & Jackson, D. (2008) The multifunctional NS1 protein of influenza A viruses. J Gen Virol 89, 2359‐2376.

5. Ma, W., Brenner, D., Wang, Z., Dauber, B., Ehrhardt, C., Hogner, K., Herold, S., Ludwig, S., Wolff, T., Yu, K., et al. (2010) The NS segment of an H5N1 highly pathogenic avian influenza virus (HPAIV) is sufficient to alter replication efficiency, cell tropism, and host range of an H7N1 HPAIV. J. Virol. 84, 2122‐2133.

Aims

The overall aim of this project is to gain a broader understanding of how reassortment of different HPAIV NS segments influences the strictly avian HPAIV strain FPV and to provide a better understanding of how this affects the host‐range and pathogenicity. The four different NS segments of the strains FPV, GD, Ma and VN belong to two different alleles (A: FPV, VN; B: GD, Ma). They show variations within their group as well as between their groups. For example the GD‐ and the Ma‐NS segment are distinguished only by eight amino acids in the NS1 protein sequence and two amino acids in the NS2 protein sequence. We therefore aim to analyze which amino acids or which combination of amino acids are implicated in

I. an effect on the viral polymerase regarding replication, transcription and the export of viral ribonucleoproteins (RNP).

II. viral propagation (growth rate) and transmission.

III. the effects of the NS proteins on the innate immunity and cell‐mediated apoptosis.

Work programme and methods

In order to investigate the effect of variations in the NS1/NS2 amino acid sequence between the FPV/VN and Ma/GD segments we will (i) create different mutants of the VN‐ and Ma‐NS segments to approach their sequence to either the FPV‐ or GD‐NS segment sequence by site directed mutagenesis of the cloned segments. The mutated NS segment encoding plasmids together with seven plasmids encoding for the other viral segments of the FPV wild type virus strain will be transfected into cells to (ii) rescue reassorted viruses by reverse genetics (required methods will be transfection methods, titration and plaque assay). The effect on replication of reassorted FPV NS Ma and FPV NS VN strains carrying the mutated respective NS segments will be investigated (iii) in avian and mammalian cell cultures. In case that the FPV reassortants with the mutated NS segments show an effect in the replication characteristics, the RdRp activity will be assessed by determination of the amounts of vRNA using (iv) a special primer extension protocol. Furthermore, the viral polymerase genes will be sequenced to exclude that the effect in replication depends on a mutation in these genes. To elucidate the effect of different mutant NS proteins in RNP export (v) indirect immunofluorescence methods are available. The influence of NS proteins to suppress IFN‐β will be investigated by using (vi) ELISA and VSV bio‐assay. Another aspect is the NS1 protein dependent effect on cell‐mediated apoptosis, which will be methodical elucidated by (vii) in situ tunel assay combined with a FACS assay.

Titles for dissertations (prospective)

• Role of specific amino acids of the NS1 of highly pathogenic avian influenza viruses for the activity of the viral polymerase

Relationships/connections within the research training group

Bindereif, Niepmann, Friedhoff, Shatsky, Ziebuhr: analysis of RNA/protein complexes exploiting the group specific expertise

Kubareva, Oretskaya: Protein/protein‐interactions regulating transcription

Bujnicki: Molecular modelling of RNA and RNA‐protein complexes

Benefits of the scientific exchange

The project will benefit from the exchange of expertise in specialized techniques and approaches in nucleic acid‐protein interactions, RNA biochemistry, and bioinformatics. The IRTG has significantly intensified the exchange of methods and know‐how between the RNA groups and also made methodical expertise of other groups available for our projects. In future the doctoral students involved in the project will get familiar with molecular modelling and bioorganic chemistry and learn how the interdisciplinary cooperation will benefit the progress of the project. The project itself will be fostered by the supportive interaction of partners with different expertise.