RNA-protein and Protein-protein interactions within the replication complex of Influenza A Virus – RNAP-IAV

With a complementary, pluridisciplinary and multiscale (cellular-, molecular- and atomic-scale) approach combining cellular, biochemical, structural and computational studies, the 2 partners combine their expertise to address important questions on the structure and function of IAV RNPs, based on solid experimental data both on the nucleoprotein and the polymerase subunits.
Biochemistry of protein and nucleic acids, biophysics, structural biology (X-Ray crystallography, SAXS, electron microscopy, ...) and molecular modeling are the main technical components used for the project.

The results obtained over the first 18 months of the project are:
- specificity of the viral polymerase for the conserved ends of the vRNA.
- Characterization of PA-PB1-RanBP5 complex.
- X-Ray structure of influenza D virus nucleoprotein.
- Collection and the deep characterization of thermosensible mutants (engineered by nucleotide substitutions or codons deletions) in the PA subunit linker. Collection and characterization of their corresponding revertants.
- Identification of a peptide stabilizing the NP-RNA complex
- Identification in silico of a new polymerase inhibitor
- Quantification of the attenuation of substitution and deletion on the RNP proteins mutants in mice.

For the continuation of the project, we will focuse on :
- the molecular basis of the interaction between the viral PA-PB1 heterodimer and the human karyopherin-ß RanBP5,
- the molecular dynamic of NP, its oligomerization and the interaction with RNA,
- the molecular basis of the interaction between NP and RNA
- the effects of identified molecules on the different components of the RNP.
At the end, the objective is to combine all the data obtained in the course of the project to detail and to understand the assembly process of the RNP.

Publications:
1 - Monod A, Swale C, Tarus B, Tissot A, Delmas B, Ruigrok RW, Crépin T, Slama-Schwok A (2015) Learning from structure-based drug design and new antivirals targeting the ribonucleoprotein complex for the treatment of influenza. Expert Opin Drug Discov. doi: 10.1517/17460441.2015. 1019859. PMID : 25792362

2 - Crépin T, Swale C, Monod A, Garzoni F, Chaillet M, Berger I (2015) Polyproteins in structural biology. Curr Opin Struct Biol. doi: 10.1016/j.sbi.2015.04.007. PMID : 25996897

3 - Da Costa B, Sausset A, Munier S, Ghounaris A, Naffakh N, Le Goffic R, Delmas B. (2015) Temperature-Sensitive Mutants in the Influenza A Virus RNA Polymerase: Alterations in the PA Linker Reduce Nuclear Targeting of the PB1-PA Dimer and Result in Viral Attenuation. J Virol, 89 (12):6376-90. PMID : 25855727

4 - Swale C, Monod A, Tengo L, Labaronne A, Garzoni F, Bourhis JM, Cusack S, Schoehn G, Berger I, Ruigrok RW, Crépin T (2016) Structural characterization of recombinant IAV polymerase reveals a stable complex between viral PA-PB1 heterodimer and RanBP5. Sci Rep. doi: 10.1038/srep24727. PMID : 27095520

5 - Meyer L, Sausset A, Sedano L, Da Costa B, Le Goffic R, Delmas B (2016) Codon Deletions in the Influenza A Virus PA Gene Generate Temperature-Sensitive Viruses. J Virol, 90 (7): 3684-3693. PMID : 26792748

Patent:
1 - Extension application 20140163107 USA patent by INSERM-transfert ANTIVIRAL COMPOSITIONS DIRECTED AGAINST THE INFLUENZA VIRUS NUCLEOPROTEIN. Anny Slama-Schwok et al.

The 2009 A/H1N1 pandemic has highlighted the unpredictability and potential public health danger posed by influenza viruses. Despite numerous studies, detailed replication mechanism, virus escape mechanism from the immune system, the mechanisms by which species barriers crossing takes place remained unsolved issues. Fundamental answers to these questions are required for improving vaccines, anti-viral drugs and diagnostics and protecting from a new severe pandemic provoked by a pathogenic virus.

Influenza A virus (IAV) is a negative-sense single-stranded RNA virus. Its genome consists of eight segments individually encapsidated into ribonucleoprotein (RNP) complexes that are central to the viral life cycle. Each RNP is comprised of a single heterotrimeric RNA polymerase bound to the complementary vRNA termini and multiple copies of the viral nucleoprotein (NP) thus forming an intricate RNA-protein architecture. Crystal structures of isolated fragments of the IAV RNA polymerase subunits as well as NP have been solved, but how the subunits form a biologically functional complex, interact with NP and the viral genome, or modulate interactions with host factors is still unknown.

The objective of the collaborative RNAP-IAV project is to understand the molecular mechanisms of human IAV RNP assembly by dissecting the protein-protein and RNA-protein interactions inherent to this intricate architecture. With a complementary, pluridisciplinary and multiscale (cellular-, molecular- and atomic-scale) approach combining cellular, biochemical, structural and computational studies, the 2 partners combine their expertise to address important questions on the structure and function of IAV RNPs, based on solid experimental data both on the nucleoprotein and the polymerase subunits.

A new strategy developed by Partner-1 allows the production of the heterotrimeric human IAV RNA polymerase in quantities that are large enough for structural (i.e. crystallography and SAXS) and functional studies, especially to detail the molecular basis of the specific vRNA-polymerase recognition. Recent advances by Partner-1 in generating high quality complexes of partly truncated PA-PB1 heterodimers are especially promising for solving PB1 structure with and without RNA. The skill of Partner-1 in electronic microscopy with a high resolution 5Å resolution will be also extremely valuable and complementary to crystallography and SAXS techniques. Furthermore, the two partners have gained an invaluable skill to control the dynamic oligomeric state of NP that represents another important prerequisite for the achievement of RNAP-IAV project. The structural data obtained by Partner-1 will be exploited by Partner-2 through the design new antivirals candidates targeting protein-protein and RNA-protein interactions based on ongoing work with naproxen, a generic drug targeting NP with novel antiviral properties. Reversed genetics developed by Partner-2 will “translate” the structural results at the cellular scale. In addition, Partner-2 will focus on the nuclear import of neo-synthesized viral PBA-PB1 proteins and their specific interactions with importins nuclear receptors. These cellular studies will take advantage from temperature-sensitive (ts) mutants and revertants on polymerase sub-units, which modulate viral replication and have clear biological significance in terms of adaptation of avian viruses to human hosts.

The skills of both partners will be used to combine the different components in order to reconstitute and assemble in vitro a fully functional RNP. This interdisciplinary project will provide new insights into IAV transcription, replication, and how these activities are modified by genetic mutation during host species adaptation and will illuminate fundamental aspects of the viral cycle. The current project also provides new approaches in the field of anti-influenza drug design and attenuated live vaccines.