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Projets de recherche

Interactions between avian and influenza virus proteins

Project funded by a Marie Curie Career Integration Grant (2013-2017) to Mariette Ducatez

 

Influenza virus is a segmented single stranded negative sense RNA virus of the Orthomyxoviridae family, which includes three Genera: influenza A, B, and C [1]. We will focus on influenza A virus and study host/pathogen interactions. Influenza A strains are classified into subgroups (or serotypes) based on their surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). Today we know 16 HA and 9 NA types. Aquatic birds are the natural reservoir of influenza A viruses and many mammals can also be infected by the virus either sporadically or with well adapted lineages. Human, swine and horse are for instance natural hosts of H1N1 and H3N2; H1N1, H1N2, and H3N2; H3N8 and H7N7 strains, respectively [1].

 

On rare occasions, swine influenza viruses can infect humans (and human viruses infect swine), generally resulting in a mild disease [2, 3]. Avian influenza viruses (AIVs) can also sporadically infect humans. It occurred in 1999 and 2003 in Hong Kong following H9N2 virus cases in poultry [4-6], in 2003 in the Netherlands after a large outbreak of highly pathogenic avian influenza (HPAI) H7N7 virus in poultry [7], or since 2003 in Asia and Africa with HPAI H5N1 virus.

Avian influenza interspecies transmissions are rare but remain a major concern for public health.

Avian influenza was indeed involved in all influenza pandemics identified so far. The 1918 H1N1 “Spanish flu” pandemic origin is still debated and was caused either by a direct zoonotic transmission from avian to human or by a re-assortment between avian, swine and human gene segments in human. Similarly Asian H2N2 (1957-58) and Hong Kong H3N2 (1968-69) pandemics  also emerged after the introduction of avian influenza gene segments [8].

 

State of the art

The systematic study of interactions between the viral protein and host cells is a powerful approach for characterizing the determinants of viral cellular infection.

In influenza both the non structural protein 1 (NS1) and the ribonucleoproteic complex proteins have been shown to widely interact with host cellular proteins on a mammal model.

 

Interactions between NS1 and cellular proteins

NS1 has been identified as an important virulence factor of the influenza virus. Multiple functions have been attributed to NS1 as reviewed by Hale et al. in 2008 [9]. NS1 mainly impacts the host immune response: it reduces the induction of IFN-β [10], inhibits the function of intracytoplasmic pathogen sensor human retinoic acid-inducible gene product I (RIG-I) [11], and acts on the host protein synthesis by suppressing the protein kinase (PKR) mediated inhibition of replication [12] and limiting the activation of the 2′-5′-oligo (A) synthetase/RNase L pathway [13]. NS1 together with phosphatidylinositol-3-kinase (PI3K) can prevent apoptosis during infection [14].

In the last couple of years, several research groups have worked on the interactions between NS1 and cellular proteins. In mammal cells (293T, A549, and/or HeLa cells), they have shown that NS1 interacts with Scribble, MAGI-1,2,3, Dgl1 [15, 16], Akt(-pH) [17], Pdlim2 [18], Hsp90 [19], hnRNP-F [20], hStau-1 [21], RIL, regulating cellular tyrosine kinase Src (Bavagnoli et al., 2011), and PSD-95 (Zhang et al., 2011) as shown by pull-down, two-hybrid and immunofluorescence assays and sometimes confirmed by interference RNA experiments. Thanks to RNAseq and microarray technologies, NS1 has also been shown to bind to hPAF1C (provided NS1 possessed a histone-like sequence), resulting in the suppression of hPAF1C-mediated transcriptional elongation and with consequences on the antiviral response (Marazzi et al., 2012). The role of the discovered interactions is not always well understood yet. MAGI proteins play a role in the membrane permeability [15], and Dgl1 and Scribble associated with NS1 disrupt tight junctions (Gobiewski et al., 2011).  HnRNP-F and hStau-1 with NS1 seem to increase viral replication [20, 21]. Several interactions moreover suggest a regulation (or even often an inhibition) of virus-induced apoptosis such as Scribble, Akt, Hsp90 [15, 17, 19].

Avian and mammal NS1 C-terminal motifs differ with ESEV and RSKV on avian and mammal NS1, respectively. These C-terminal motifs have also been associated with interspecies transmission as well as with species-specific virulence [22, 23]. The anti-apoptotic effect of NS1-Scribble interaction was shown with the avian NS1 ESEV motif [15]. The nuclear localization of NS1 in mammal cells (but not avian cells) seem to also be dependent on the amino acid (aa) sequence of the C-terminal part of NS1 [24].

Objectives of the proposal and expected results

To date most of the knowledge on influenza virus and host cell interactions was gained using mammal models. Very little is known on avian cell/influenza virus interactions. Both the influenza non structural protein 1 (NS1) and the ribonucleoproteic complex proteins have been shown to play an important role during the host cell infection process. However, there are well established differences between avian and mammal NS1 and avian and mammal hosts (cells) also display obvious specificities.

We therefore propose to study the interactions between influenza virus and host proteins in an avian model and to compare them with what is known on the mammalian model.

Our project has three operational objectives:

 

  • Objective 1: identify the avian cellular proteins that interact with influenza virus proteins
  • Objective 2: narrow the identified interaction(s) to the protein domain level and identify molecular determinants of host specificity
  • Objective 3 (longer term): study the mechanism of interaction

 

We are expecting to observe both similarities and differences in the interactions between influenza proteins and avian cells and those reported in mammal cells. This comparison could allow us to identify proteins or protein domains, which play an important role in interspecies transmission.

Our final goal is to highlight parts of the virus genome to closely survey for pandemic preparedness.

 

 

1.            Wright, P.F., G. Neumann, and Y. Kawaoka, Orthomyxoviruses, in Fields Virology, D.M. Knipe, Howley, P.M., Editor. 2007, Lippincott Williams & Wilkins, a Wolters Kluwer business: Philadelphia. p. 1691-1740.

2.            Myers, K.P., C.W. Olsen, and G.C. Gray, Cases of swine influenza in humans: a review of the literature. Clin Infect Dis, 2007. 44(8): p. 1084-8.

3.            Yu, H., et al., Isolation and genetic analysis of human origin H1N1 and H3N2 influenza viruses from pigs in China. Biochem Biophys Res Commun, 2007. 356(1): p. 91-6.

4.            Butt, K.M., et al., Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003. J Clin Microbiol, 2005. 43(11): p. 5760-7.

5.            Peiris, M., et al., Human infection with influenza H9N2. Lancet, 1999. 354(9182): p. 916-7.

6.            Saito, T., et al., Characterization of a human H9N2 influenza virus isolated in Hong Kong. Vaccine, 2001. 20(1-2): p. 125-33.

7.            Koopmans, M., et al., Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands. Lancet, 2004. 363(9409): p. 587-93.

8.            Guan, Y., D. Vijaykrishna, and J. Bahl, Zhu, H., Wang, J., Smith, G. J., The emergence of pandemic influenza viruses. Protein Cell, 2010. 1(1): p. 9-13.

9.            Hale, B.G., et al., The multifunctional NS1 protein of influenza A viruses. J Gen Virol, 2008. 89(Pt 10): p. 2359-76.

10.          Garcia-Sastre, A., et al., Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology, 1998. 252(2): p. 324-30.

11.          Guo, Z., et al., NS1 protein of influenza A virus inhibits the function of intracytoplasmic pathogen sensor, RIG-I. Am J Respir Cell Mol Biol, 2007. 36(3): p. 263-9.

12.          Bergmann, M., et al., Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication. J Virol, 2000. 74(13): p. 6203-6.

13.          Min, J.Y. and R.M. Krug, The primary function of RNA binding by the influenza A virus NS1 protein in infected cells: Inhibiting the 2'-5' oligo (A) synthetase/RNase L pathway. Proc Natl Acad Sci U S A, 2006. 103(18): p. 7100-5.

14.          Hale, B.G., et al., Influenza A virus NS1 protein binds p85beta and activates phosphatidylinositol-3-kinase signaling. Proc Natl Acad Sci U S A, 2006. 103(38): p. 14194-9.

15.          Liu, H., et al., The ESEV PDZ-binding motif of the avian influenza A virus NS1 protein protects infected cells from apoptosis by directly targeting Scribble. J Virol, 2010. 84(21): p. 11164-74.

16.          Thomas, M., et al., Analysis of the PDZ binding specificities of Influenza A virus NS1 proteins. Virol J, 2011. 8: p. 25.

17.          Matsuda, M., et al., Characterization of the interaction of influenza virus NS1 with Akt. Biochem Biophys Res Commun, 2010. 395(3): p. 312-7.

18.          Yu, J., et al., PDlim2 Selectively Interacts with the PDZ Binding Motif of Highly Pathogenic Avian H5N1 Influenza A Virus NS1. PLoS One, 2011. 6(5): p. e19511.

19.          Zhang, C., et al., The NS1 protein of influenza a virus interacts with heat shock protein Hsp90 in human alveolar basal epithelial cells: implication for virus-induced apoptosis. Virol J, 2011. 8: p. 181.

20.          Lee, J.H., et al., Direct interaction of cellular hnRNP-F and NS1 of influenza A virus accelerates viral replication by modulation of viral transcriptional activity and host gene expression. Virology, 2010. 397(1): p. 89-99.

21.          de Lucas, S., et al., Human Staufen1 protein interacts with influenza virus ribonucleoproteins and is required for efficient virus multiplication. J Virol, 2010. 84(15): p. 7603-12.

22.          Soubies, S.M., et al., Species-specific contribution of the four C-terminal amino acids of influenza A virus NS1 protein to virulence. J Virol, 2010. 84(13): p. 6733-47.

23.          Zielecki, F., et al., Virulence determinants of avian H5N1 influenza A virus in mammalian and avian hosts: role of the C-terminal ESEV motif in the viral NS1 protein. J Virol, 2010. 84(20): p. 10708-18.

24.          Volmer, R., et al., Nucleolar localization of influenza A NS1: striking differences between mammalian and avian cells. Virol J. 7: p. 63.