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UMR 1332 - Biologie du Fruit et Pathologie

UMR 1332 Biologie du Fruit et Pathologie

Thierry Michon (Dr)

Thierry Michon
Research director, Institut National de la Recherche Agronomique

Ph.D., Enzymology, University of Aix-Marseille 2 France, 1992.

Research :

Molecular evolution of virus proteins; virus-inspired nanotechnology

Contacts :

Address : Equipe de Virologie, UMR 1332 Biologie du Fruit et Pathologie - INRA et Université de Bordeaux - CS 20032 - 33882 Villenave d'Ornon Cedex France
Phone : +33 (0) 557 12 23 91
Fax : +33 (0) 557 12 23 84

Biography :

Thierry Michon was awarded the PhD in enzymology at the University of Aix-Marseille 2 France in 1992. In 1993, he accepted a position of researcher at the "Institut de la Recherche Agronomique" in Nantes where he worked on peroxidases catalysis in heterogeneous media. Between 1998 and 2001 he was appointed by the California Institute of Technology as an invited scientist and worked in close collaboration with Pr. D. Tirrell (Chemical Engineering Dep’t). During this period he contributed to the development of protein based bio inspired materials. Since 2001 T. Michon has been project leader at the French Institute of Agronomic research (INRA) in Bordeaux France. He studies possible correlations between virus evolution and the intrinsic disorder found in viral proteins. In parallel his team develops virus based Enzymes NanCarriers (ENCs) for nanotechnology applications.

Molecular evolution of intrinsic disorder in viral proteins.

Fischer’s lock-and-key model, 100 years ago, opened up the concept of structure-function relationship whereby the folding of a polypeptide chain into an ordered 3D structure conditions the specificity of its function.

Figure 1 . Two modes of mutational impact on organisms’ fitness (adapted from Tokuriki et al. 2009).

Figure 1 . Two modes of mutational impact on organisms’ fitness
(adapted from Tokuriki et al. 2009).

However, many biologically functional proteins possess Intrinsically Disordered Regions (IDRs). IDRs are regions devoid of stable secondary and tertiary structures under physiological conditions and rather exist as dynamic ensembles of inter-converting conformers. This lack of a stable 3D structure in many cases is relieved only when the protein binds to its target molecule. The ability to exert specific biological functions and to interact with various partners in spite of the lack of a precise 3D scaffold, challenges the classic paradigm according to which specificity in interacting domains can only be achieved through precise surface complementation between structured and conserved domains. The genome of RNA viruses codes for proteins containing an important proportion of IDRs. Within viral proteins, IDRs often contain binding domains for protein-protein interactions that are crucial to the virus replication cycle. Because of the low steric constraints within IDDs, IDDs are expected to be more tolerant to mutations than structured proteins and thus have the potential to evolve faster. Our objective aims at assessing experimentally if ID in viral proteins favors an exploration of a broader sequence space without serious functional consequences, which could afford a faster adaptation to various hosts. It will in turn be examined whether there is a selective pressure for the conservation of intrinsic disorder (ID) in domains of viral proteins interacting with host factors. Potato virus Y (PVY), a phytovirus within the genus Potyvirus, provides an excellent model system. Indeed, it has been demonstrated that pepper infection by PVY requires the interaction between eIF4E, a factor involved in the host translational machinery, and an IDR within the Virus Protein linked to the genome (VPg). In addition, this IDR contains several amino acid positions that are subject to positive selection, i.e. presumably involved in virus adaptation. We also experimentally explore whether a correlation exists between ID and mutational robustness (relaxed constraint) or adaptive evolution (positive selection) from one hand (Figure 1) and between the structural flexibility in the VPgs IDRs and a faster adaptability to the host on the other hand. Our analysis combine, in silico, in vitro and in vivo studies using complementary skills through bioinformatics, biological, biochemical and biophysical approaches[1-7] We performed an in silico analysis of the content in ID of the Potyvirus proteome both at inter- and intra-species scales. This work unveiled a high content in ID, which is conserved during Potyvirus evolution, suggesting a functional advantage of IDRs [8]. In collaboration with the Benoit Moury team (INRA, Avignon), we obtained the first in vivo experimental data supporting the hypothesis that ID could directly modulate virus adaptability to the host, possibly by enabling a faster exploration of the mutational space thereby allowing the virus to bypass the plant resistance. These original data, which strongly suggest that IDRs contribute to adaptive mechanisms of plant viruses, were published in a landmark paper [9]. These results offer an appropriate starting point to explore more systematically correlations between adaptation and ID.       

A controlled positioning of enzymes on virus nano-scaffolds.
A
Michon-fig2A
B
Michon-Fig2B
C
Michon-Fig2C

Figure 2 A. Use of Z33 as a multipurpose “molecular sticker” for interfacing proteins with various supports. Using the antibody-binding peptide Z33 as a fusion tag, virtually any proteins can be bound to any antigenic support by using the appropriate specific antibody. In the picture, a Z33-yellow fluorescent protein fusion is adsorbed on a virus surface by mean of an antibody recognizing the virus major coat protein [12]. B. High resolution AFM-SECM imaging of a single immune complex between a ferrocene functionalized immunoglobuline and the virus protein linked to the genome (VPg) at the very end of a Lettuce mosaic virus (LMV) particle [13]. C.In situ immunoredox Mt/AFM-SECM imaging of a eIF4E-labeled LMV particle  immobilized on a gold surface. A, Topography image. B, simultaneously acquired current image. C, Overlay of the topography and current images showing the precise location of the current spot at one of the extremities of the virus particle. (Lower part) Cross sections of the topography (red trace) and current (blue trace) images taken along the dotted line shown. Both topological and current signals whose position correlate, are showed by a green arrow [15].

 Functional imaging of single biomolecules at work (and especially enzymes) has become the Grail of scientists and technologists. It will not only help to understand in real time molecular mechanisms, for instance enzyme cascades, at the molecular level but will also have high impact for the design of nano-transducers and lab-on-a-chip applications. However, there are very few methods available to systematically evaluate how spatial factors (e.g., position, orientation) influence enzymatic activity. This limitation notably comes from the fact that their small size makes it extremely difficult to organize biomolecules onto surfaces in order to form fully active supramolecular complexes amenable to experimental studies. We have developped a strategy to spatially organize enzymes on a solid substrate making use of robust Virus NanoCarriers (VNC’s) as positioning helpers (Figure 2A). Viruses are attractive building blocks for a large panel of biotechnology applications such as enzyme chips, protein selection, molecular therapy, or to study more fundamental problems raised by modern enzymology Viruses are here highly interesting as natural nano-carriers since their ordered protein backbones afford a high degree of positional control of functional molecules which can be attached selectively to their surface. These VNCs are then used as intermediate building blocks or scaffolds carrying correctly exposed proteins on their surface, and subsequently immobilized onto a solid substrate. Filamentous viruses such as the potyvirus PVA (700 nm long, 14 nm diameter), or M13 Bacteriophage (800 nm, 8 nm diameter), a virus that has shown its potential for biotechnological applications are our work-horse systems. Knowing the structure of these viruses, genetic engineering can be used to optimize the desired enzyme's topology on the virus nanocarrier [10-11]. In the frame of a strong collaboration between the laboratory of molecular electro-chemistry (CNRS-Univ. Paris VII) and our team, high resolution imaging of virus particles were obtained by AFM-SECM (figure 2B). Work is in progress to follow the activity of redox enzymes at the surface of virus nanoparticles [14]. Recently, we bridged the gap between the two main research themes of the team by  using AFM-SECM to identify eIF4E, a plant translation factor interacting specifically to the viral protein VPg exposed at the surface of a potyvirus nanoparticle [15].        

The team
Michon-team2012

June 2012 - Jan, Amandine, Deborah, Mamadou, Thierry, Noëlle, Daniela

Michon-Team2015

April 2015 - Justine, Geneviève, Amandine, Thierry, Jocelyne, Laure

Publications :

1. Tavert-Roudet, G., Abdul-Razzak, A., Doublet, B., Walter, J., Delaunay, T., German-Retana, S., Michon, T., Le Gall, O. and Candresse, T. (2012) The C terminus of lettuce mosaic potyvirus cylindrical inclusion helicase interacts with the viral VPg and with lettuce translation eukaryotic initiation factor 4E. J Gen Virol. 93, 184-193

2. Dielen, A. S., Sassaki, F. T., Walter, J., Michon, T., Menard, G., Pagny, G., Krause-Sakate, R., Maia, I. D., Badaoui, S., O, L. E. G., Candresse, T. and German-Retana, S. (2011) The 20S proteasome alpha(5) subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the Lettuce mosaic potyvirus HcPro protein. Mol Plant Pathol. 12, 137-150

3. Chroboczek, J., Hebrard, E., Makinen, C., Michon, T. and Rantalainen, K. (2012) Intrinsic disorder in genome-linked viral proteins VPgs of potyviruses. In Flexible viruses : structural disorder within viral proteins (Uversky, V. N. and Longhi, S., eds.). pp. 277-312, John Wiley and Sons

4. German-Retana, S., Walter, J., Doublet, B., Roudet-Tavert, G., Nicaise, V., Lecampion, C., Houvenaghel, M. C., Robaglia, C., Michon, T. and Le Gall, O. (2008) Mutational analysis of plant cap-binding protein eIF4E reveals key amino acids involved in biochemical functions and potyvirus infection. J Virol. 82, 7601-7612

5.  Hebrard, E., Bessin, Y., Michon, T., Longhi, S., Uversky, V. N., Delalande, F., Dorsselaer, A. V., Romero, P., Walter, J., Declerk, N. and Fargette, D. (2009) Intrinsic disorder in Viral Proteins Genome-Linked: experimental and predictive analyses. Virol J. 6, 23

6.  Michon, T., Estevez, Y., Walter, J., German-Retana, S. and Le Gall, O. (2006) The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue. Febs J. 273, 1312-1322

7. Roudet-Tavert, G., Michon, T., Walter, J., Delaunay, T., Redondo, E. and Le Gall, O. (2007) Central domain of a potyvirus VPg is involved in the interaction with the host translation initiation factor eIF4E and the viral protein HcPro. J Gen Virol. 88, 1029-1033.

8. Charon J, Theil S, Nicaise V, Michon T. 2016. Protein intrinsic disorder within the Potyvirus genus: from proteome-wide analysis to functional annotation. Mol. BioSyst. 12:634–652.

9. Charon J, Barra A, Walter J, Millot P, Hébrard E, Moury B, Michon T. 2017. First experimental assessment of protein intrinsic disorder involvement in an RNA virus natural adaptive process. Mol. Biol. Evol. 2017 Sep 18. doi: 10.1093/molbev/msx249. [Epub ahead of print].

10. Cardinale, D., Carette, N. and Michon, T. (2012) Virus scaffolds as enzyme nano-carriers. Trends Biotechnol. 30, 369-376

11. Carette, N., Engelkamp, H., Akpa, E., Pierre, S. J., Cameron, N. R., Christianen, P. C., Maan, J. C., Thies, J. C., Weberskirch, R., Rowan, A. E., Nolte, R. J., Michon, T. and Van Hest, J. C. (2007) A virus-based biocatalyst. Nat Nanotechnol. 2, 226-229

12. Pille, J., Cardinale, D., Carette, N., Di Primo, C., J., B.-N., Walter, J., Lecoq, H., van Eldijk, M. B., Smits, F. C. M., Schoffelen, S., van Hest, J. C. M., Mäkinen, K. and Michon, T. (2013) A General Strategy for Ordered Non-covalent Protein Assembly on Well-defined Nano-scaffolds. Biomacromolecules, 14(12), 4351-4359.

13. Nault, L., Taofifenua, C., Anne, A., Chovin, A., Demaille, C., Besong-Ndika, J., Cardinale, D., Carette, N., Michon, T. and , Walter, J. (2015). Electrochemical Atomic Force Microscopy Imaging of Redox-Immunomarked Proteins on Native Potyviruses: From Subparticle to Single-Protein Resolution. ACS Nano. doi:10.1021/acsnano.5b00952

14. Patel AN, Anne A, Chovin A, Demaille C, Grelet E, Michon T, Taofifenua C. 2017. Scaffolding of Enzymes on Virus Nanoarrays: Effects of Confinement and Virus Organization on Biocatalysis. Small:doi: 10.1002/smll.201603163.

15. Tavert-Roudet G, Anne A, Barra A, Chovin A, Demaille C, Michon T. 2017. The potyvirus particle recruits the plant translation initiation factor eIF4E by means of the VPg covalently linked to the viral RNA. Mol. Plant-Microbe Interact. 30:754–762.