Virology

HOST/PATHOGENE INTERACTION

Subjects

• Adenovirus penton base protein, viral capsomer responsible for the virus internalisation. The structural studies of the protein and the identification of its cellular partners. Study of the adenovirus dodecahedron, the nano-particule built of 12 penon bases, a remarkably efficient vector of the intracellular protein delivery.
• Enteric adenovirus serotype 41. Structural characterization of the virus with the unique tropism and the identification of its cellular partners.
• Terminal protein VPg of the plant virus Potato Virus Y. Characterization of its interaction with the host eukaryotic initiation factor eIF4E.

Technical approaches

Methods of molecular and cell biology. Expression library screening. Recombinant protein expression systems (bacteria, baculovirus). Electron microscopy (negative stain and cryoelectronmicroscopy, in collaboration). Confocal microscopy. Protein analysis. Crystallogenesis.

Collaborations

Several groups at IBS, EMBL Grenoble Ouststation, Institute of Biochimistry and Biophysics of Polish Academy of Sciences in Warsaw, Institute of Molecular Biology and Genetics of Ukrainian Academy of Sciences in Kiev.

Main results

1. Study on the penton base protein of adenovirus

We identified 6 novel partners of this viral protein responsible for virus internalisation, which opens the new vista in our understanding of its multiple intracellular interactions. In collaboration with EMBL, we resolved the atomic structure of this pentameric protein, which permits in a more precise way to understand its stability, its structural role in the viral capsid and the mechanism of its intracellular interactions. In the study concerning the dodecahedron we have shown that the remarkable efficacy of the intracellular entry of this nano-particle stems from its ability to interact with the heparan sulfates proteoglycanes, the omnipresent components of the eukaryotic membranes, which are not recognized by the virus of origin. We have shown that the dodecahedron is able to transduce 100% of human cells in culture. The amount of dodecahedra in one cell is 300-400 000 and they can deliver 10 to 20 millions of foreign proteins (manuscript in preparation).

Publications:

Adenovirus dodecahedron allows large multimeric protein transduction in human cells. (2003) Fender P, Schoehn G, Foucaud-Gamen J, Gout E, Garcel A, Drouet E, Chroboczek J. J Virol. 77:4960-4.

Adenovirus protein involved in virus internalization recruits ubiquitin-protein ligases. (2002) Galinier R, Gout E, Lortat-Jacob H, Wood J, Chroboczek J. Biochemistry 41:14299-305.

Heparan sulfate proteoglycan mediates the selective attachment and internalization of serotype 3 human adenovirus dodecahedron. (2004) Vives RR, Lortat-Jacob H, Chroboczek J, Fender P. Virology 321:332-40.

The structure of human adenovirus 2 penton C. Zubieta, G. Schoehn, J. Chroboczek, and S Cusack. Molecular Cell, accepted

2. Enteric adenovirus of serotype 41 (Ad41).

The virus with enteric tropism has to survive in the particular conditions of the human gastrointestinal system. We demonstrated that the infectivity of enteric Ad41 is not inhibited by the exposure to acid whereas the infectivity of respiratory Ad2 is abolished under these conditions. We showed that this phenomenon depends on the global basic charge of the Ad41 capsid and on the unusual stability of the fiber protein responsible for the virus attachment at the beginning of infection.

Publications :

Structural studies of human enteric adenovirus type 41. (2002) Favier A-L, Schoehn G, Jaquinod M, Harsi C and Chroboczek J. Virology 293:75-85.

Unique physicochemical properties of human enteric Ad41 responsible for its survival and replication in the gastrointestinal tract. (2004) Favier AL, Burmeister WP, Chroboczek J. Virology 322:93-104.

3. Terminal protein VPg of the plant virus Potato Virus Y (ms in preparation)

Potyvirus RNA contains at the 5’ end a covalently linked virus-encoded protein VPg, which is required for virus infectivity. This role has been attributed to VPg interaction with the eukaryotic initiation translation factor eIF4E (E4), a cap-binding protein. We characterized the dissociation constants for the interaction of the potato virus Y VPg with different plant E4s and mapped the E4 attachment region on VPg. VPg/E4 interaction results in the inhibition of cell-free protein synthesis, and we show that it stems from the liberation of the cap moiety from the complex with E4. Since VPg does not attach the cap, we infer that VPg induces changes in the E4 structure, diminishing its affinity to the cap. We show here for the first time that the initiation complex scaffold protein eIF4G increases VPg interaction with E4. These data suggest similar cap and VPg interactions with E4 and characterize VPg as a novel eIF4E-binding protein, which inhibits host protein synthesis at a very early stage of the initiation complex formation through the inhibition of cap attachment to the initiation factor E4

Other publications

Deubiquitinating function of adenovirus proteinase. (2002) Balakirev MY, Jaquinod M, Haas AL, Chroboczek J. J Virol. 76:6323-31.

Otubains: a new family of cysteine proteases in the ubiquitin pathway. (2003) Balakirev MY, Tcherniuk SO, Jaquinod M, Chroboczek J. EMBO Rep. 4:517-22.

Gene transfer by adenovirus-mimetic peptides in the presence of a cationic lipid and/or adenovirus. (2003) Moritz S, Colin M, Keller M, Klonjkowski B, Capeau J, Coutelle C, Chroboczek J, Brahimi-Horn MC. Analysis of the contribution of the viral and nonviral components. Arch Virol. 148:1-18.

Binding of adenovirus capsid to dipalmitoyl phosphatidylcholine provides a novel pathway for virus entry. (2003) Balakireva L, Schoehn G, Thouvenin E, Chroboczek J. J Virol. 77:4858-66.

Crystal structure of human otubain 2. (2004) Nanao MH, Tcherniuk SO, Chroboczek J, Dideberg O, Dessen A, Balakirev MY. EMBO Rep. 5:783-8

Construction of tumor specific toxins using ubiquitin fusion technique S. Tcherniuk, J. Chroboczek and M. Balakirev. Molecular Therapy 2004, accepted.