Scientific reports

Unité de Virologie Structurale - CNRS URA 3015

Dr. Felix A. Rey

Researchers: BARBA-SPAETH Giovanna PhD (IP),VANEY Marie-Christine PhD (CNRS), DUQUERROY Stéphane PhD (Assistant Professor, Univ Paris-Sud 11), BACKOVIC Marija PhD (IP), KREY Thomas PhD (IP), FLAMAND Marie PhD (IP), BONTEMS
François PhD (CNRS), SAUNIER Bertrand PhD (INSERM).
Engineers: TORTORICI Alejandra PhD (IP), TAMIETTI Carole (IP), MEOLA Annalisa.
Executive secretary: SENLECQUES Danielle (IP).
Laboratory assistant: AJINCA Marthe (IP).
Post-doctoral fellows: JEFFERS Scott Allen PhD, PARK Kyu-Ho PhD, GUARDADO-CALVO Pablo PhD, ROUVINSKY Alexander PhD, CHAUVIAC François-Xavier PhD, MARZA Ester PhD.
M2 students: BOISSIERE Magali, GRAU Nina.

Scientific Report

    Our research is focused in comparative structural studies on proteins with the same or similar function in related and unrelated viruses. Such an approach allows us to identify common themes and their relation to function, as well as specific features for each virus that relate to its particular life cycle. These studies also provide important information about the evolution of viral genes, by identifying homologous proteins in unrelated families. In such cases, sequence analysis alone is not sufficient to identify evolutionary links. In this way, we have first identified viral fusion proteins belonging to class II, which appear to be present in many more viruses than the flaviviruses and alphaviruses for which we have structural studies. Remarkably, we have recently found that cellular genes involved in cell-cell fusion during development of the nematode C. Elegans, are also homologous to the class II viral fusion proteins.

Main axes:
I - Comparative structural studies on viral membrane fusion proteins: The focus is on predicted class II viruses (hepatitis C virus, pestiviruses, rubella virus, members of the Bunyaviridae family). We have also undertaken to study the envelope proteins of the herpesviruses and some class I viruses such as arenaviruses and retroviruses. Because they are exposed at the surface of the virus particles, the viral envelope proteins are the main targets of neutralizing antibodies,a nd we are also studying complexes with antibodies to understand the neutralization mechanism.

II - Viral replication: We focus on the respiratory syncytial virus, a negative-strand RNA virus of high medical relevance, making structural studies of the nucleoprotein (N), the phosphoprotein (P), and the large polymerase protein (L). We are also studying the helicase of positive stranded RNA viruses to understand its role during genome replication, focusing on pestivirus protein NS3 as model system. Comparative structural analysis provides information also valid for NS3 from other members of the Flaviviridae family, including the hepatitis C virus and dengue virus.

III - Capsid assembly: The focus is on the lentiviruses HIV and FIV. We are pursuing our comparative structural analyses to understand the assembly of immature particles and the relevant interactions between gag molecules.

IV - Membrane fusion: A special emphasis in the laboratory is devoted to understanding protein-catalyzed membrane fusion reactions, which are key events not only relevant to viral entry, but to many processes that are fundamental to life. My team has been involved for many years in understanding the structure-function relations of viral membrane fusogenic proteins. Comparison to cellular fusogenic proteins is now revealing that certain proteins controlling cell-cell fusion during organogenesis in multicellular organisms are evolutionary related to the class II viral membrane fusion proteins, and we intend to explore the extent to which other cellular fusogenic proteins are related to viral proteins, for instances those involved in gamete fusion during fertilization, or those involved in muscle formation in vertebrates, or many other processes Obtaining enough amounts of membrane fusogenic proteins for structural studies is intrinsically difficult. In order to make progress in this field, our laboratory has invested heavily in developing adequate systems for production of targeted recombinant proteins. The initial studies that I had carried out, in the early 90s with the flavivirus envelope protein E, and later with the alphavirus protein E1, where done with protein that was released by controlled proteolysis (thus cleaving the ectodomain from the C-terminal membrane anchor) from the surface of purified virus particles, following the pioneering studies by Skehel and Wiley on the influenza virus haemagglutinin. This approach, however, limited the studies to viruses that can be grown to very high titers in order to obtain enough soluble glycoprotein. We also used this approach successfully more recently, in collaboration with Yves Gaudin to crystallize the ectodomain of the vesicular stomatitis virus, directly purified from virus preparations. For the majority of the viruses that we are interested in, it is however not possible to grow the virus to high enough titers for such undertaking. Similarly, an adequate system is necessary for the production of the targeted cell-cell fusion proteins. In particular, the class II fusion proteins are in general notoriously slow to fold, making full use of the folding chaperoning machinery of the transfected cell. We have identified the Drosophila melanogaster Schneider 2 (S2) cells as the optimal cells for production of high amounts of correctly folded class II membrane fusogenic proteins, and we make full use of this systems in most of our projects.

Progress report:
     The main results that have been published in the last four years include the picobirnavirus particle structure (1), the aquabirnavirus sub-viral particle structure(2), the structure of a nucleoprotein/RNA ring complex from the respiratory syncytial virus (3), of a core fragment of the pseudorabies herpesvirus glycoprotein gH in complex with antibody (4), of the chickungunya virus p62/E1 and E3/E2/E1 surface glycoprotein complexes (5), of the lymphocytic choriomeningitis arenavirus (LCMV) glycoprotein GP2 in its post-fusion conformation (6), as well as the structures of the dengue virus envelope protein in complex with a serotype-specific neutralizing antibody (7) and with a dengue-group broad neutralizing antibody in complex with domain III from the envelope protein of each serotype (8). We have also determined the disulfide connectivity of the hepatitis C virus envelope glycoprotein E2, which allowed us to proposed a model for its tertiary organization (9). Finally, we obtained a low-resolution cryo-EM structure of the dengue virus secreted glycoprotein NS1, which is abundant in the blood stream in patients with dengue hemorrhagic fever. The barrel-shaped structure stimulated further studies that allowed us to discover that it is a non-conventional lipoprotein carrying tryglycerides and other lipids in patients (10). These findings on NS1 could have important implications to understand the pathogenicity of dengue virus, and it therefore open the way to further studies, which will be carried out by Marie Flamand in the laboratory.
     On the methodological side, we have set up efficient production systems for antibody fragments to use as crystallization chaperones for proteins that are difficult to crystallize, as outlined in the flow chart above (11, 12).

Keywords : X-ray crystallography; virus structure; viral antigens; icosaedral symmetry; envelope glycoproteins; membrane fusion, viral enzymes; ribonucleoprotein complexes; alphavirus; flavivirus; arbovirus, herpesvirus.

The chikungunya icosahedral surface glycoproteins shell (zoom view).

1. Duquerroy S, Da Costa B, Henry C, Vigouroux A, Libersou S, Lepault J, Navaza J, Delmas B, Rey FA. 2009. The EMBO journal 28: 1655-1665.
2. Coulibaly F, Chevalier C, Delmas B, Rey FA. 2010. Journal of Virology 84: 1792-9.
3. Tawar RG, Duquerroy S, Vonrhein C, Varela PF, Damier-Piolle L, Castagné N, MacLellan K, Bedouelle H, Bricogne G, Bhella D, Eléouët JF, Rey FA. 2009. Science 326: 1279-83.
4. Backovic M, DuBois RM, Cockburn JJ, Sharff AJ, Vaney MC, Granzow H, Klupp BG, Bricogne G, Mettenleiter TC, Rey FA. 2010. Proc Natl Acad Sci USA 107: 22635-22640.
5. Voss JE, Vaney MC, Duquerroy S, Vonrhein C, Girard-Blanc C, Crublet E, Thompson A, Bricogne G, Rey FA. 2010. Nature 468: 709-12.
6. Igonet S, Vaney MC, Vonhrein C, Bricogne G, Stura EA, Hengartner H, Eschli B, Rey FA. 2011. Proc Natl Acad Sci USA 108: 19967-19972.
7. Cockburn JJ, Navarro Sanchez ME, Goncalvez AP, Zaitseva E, Stura EA, Kikuti CM, Duquerroy S, Dussart P, Chernomordik LV, Lai CJ, Rey FA. 2011. The EMBO journal 31: 767-779.
8. Cockburn JJ, Navarro-Sanchez ME, Fretes N, Urvoas A, Staropoli I, Kikuti CM, Coffey LL, Arenzana Seisdedos F, Bedouelle H, Rey FA. 2012. Structure 20:303-314.
9. Krey T, d'Alayer J, Kikuti CM, Saulnier A, Damier-Piolle L, et al. 2010. PLoS pathogens 6: e1000762.
10. Gutsche I, Coulibaly F, Voss JE, Salmon J, d'Alayer J, Ermonval M, Larquet E, Charneau P, Krey T, Mégret F, Guittet E, Rey FA, Flamand M. 2011. Proc. Natl. Acad Sci USA 108: 8003-8008.
11. Gilmartin AA, Lamp B, Rumenapf T, Persson MA, Rey FA, Krey T. 2012. Protein Eng Des Sel. 25:59-66.
12. Backovic M, Johansson DX, Klupp BG, Mettenleiter TC, Persson MA, Rey FA. 2010. Protein Eng Des Sel. 23:169-174.

Selected Publications

  1. Functional and evolutionary insight from the crystal structure of rubella virus protein E1. DuBois RM, Vaney MC, Tortorici MA, Al Kurdi R, Barba-Spaeth G, Krey T, Rey FA. Nature 2013. Jan 6.

  2. Mechanism of Dengue Virus Broad Cross-Neutralization by a Monoclonal Antibody. Cockburn JJ, Navarro-Sanchez ME, Fretes N, Urvoas A, Staropoli I, Kikuti CM, Coffey LL, Arenzana Seisdedos F, Bedouelle H, Rey FA. Structure. 2012 Feb 8;20(2):303-14.

  3. Structural insight into the meutralization mechanism of a higher primate antibody against dengue virus. Cockburn JJ, Navarro-Sanchez E, Goncalvez A, Zaitseva E, Stura EA, Kikuti CM, Duquerroy S, Chernomordik L, Lai CJ, Dussart P, Rey FA. EMBO J. 2011 Dec 2;31(3):767-779.

  4. Structure of a core fragment of glycoprotein H from Pseudorabies virus in complex with antibody. Backovic M, Dubois R, Cockburn JJ, Sharff AJ, Vaney MC, Granzow H, Klupp BG, Bricogne G, Mettenleiter TC, Rey FA. Proc Natl Acad Sci U S A. 2010 Dec 13.

  5. Glycoprotein organization of chikungunya virus particles revealed by X-ray crystallography. Voss JE, Vaney M-C, Duquerroy S, Vonrhein C, Girard-Blanc C, Crublet E, Thompson A, Bricogne G, Rey FA. Nature. 2010 Dec 2;468:709-712. [News and Views: ’Structural biology: An alphavirus puzzle solved’ by Margaret Kielian. Nature. 2010 Dec 2;468:645-646.]

  6. The disulfide bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of the molecule. Krey T, d’Alayer J, Kikuti CM, Saulnier A, Damier-Piolle L, Petitpas I, Johansson DX, Tawar RG, Baron B, Robert B, England P, Persson MA, Martin A, Rey FA. PLoS Pathog. 2010 Feb 19;6(2):e1000762.

  7. Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus. Tawar RG, Duquerroy S, Vonrhein C, Varela PF, Damier-Piolle L, Castagné N, MacLellan K, Bedouelle H, Bricogne G, Bhella D, Eléouët JF, Rey FA. Science. 2009 Nov 27;326(5957):1279-83.

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