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  Director : Michel VERON (mveron@pasteur.fr)


  abstract

 

The main projects of the laboratory are :

Study of the phosphorylation of nucleoside analogs used in anti-viral therapies.

Biochemical characterization of the protein NEMO, an essential element of the NF-kB signal transduction pathway involved in several human genetical deseases.

Mechanistic studies on recombination in retrovirus



  report

cale

In vitro study of the phoshorylation of antiviral nucleotide analogs by NDP kinase. Sarah gallois-montbrun and Dominique deville-bonne

Nucleoside analogs like AZT and d4T are used in current therapies against AIDS in association with other drugs in multi-therapy protocoles. However they are delivered to the patients as uncharged nucleosides in order to allow cell penetration. Thus they need to be phosphorylated into triphospho-derivatives by cellular kinases before they can act as chain terminators blocking the viral reverse transcriptase. The last step in this activation pathway is thought to be catalyzed by NDP kinase.

We have studied the reactivity of recombinant human NDP kinase with several analogs currently used in clinics (ddI, ddC, AZT, d4T), both at the biochemical and at the structural level (collaboration J. Janin, LEBS, Gif-sur-Yvette, France). We showed that phosphorylation of the analogs is much slower than for their natural counterpart, indicating that NDP kinase may be a limiting step in the activation process. Both kinetic and binding constants were determined. Combining biochemichal and structural data, we have proposed a precise mechanistic model for the phosphorylation of anti-AIDS nucleotide analogs by NDP kinase.

3TC is a L-derivative of thiacytidine recently introduced in therapeutic protocols for AIDS and hepatitis B and ß-L-deoxynucleosides (ß-L-thymidine and ß-L-2'-deoxycytidine) have recently been recognized as putative anti-hepatitis B agents. We have shown that both nucleotides and desoxynucleotides in L-configuration and in particular 3TC are very poor substrates for NDP kinase. In contrast, another kinase, phosphoglycerate kinase, reacts with L-nculeotides and is probably responsible for the formation of L-dXTP within cells (collaboration A. Faraj and J.P. Sommadossi, Novirio-CNRS and Université Montpellier II).

Ribavirin, a guanosine analog which is the only nucleoside analog used in clinics against RNA virus, is a good drug candidate against dengue, a viral disease responsible for hemorragic fevers. Ribavirin is active in the cell as a triphospho-derivative. We have screened new derivatives of ribavirin modified on the sugar for increased drug efficiency that would allow the use of lower doses and decrease toxicity. The 2'-deoxy derivative of ribavirin showed a phosphorylation rate by NDP kinase and inhibition of C hepatitis polymerase similar to ribavirin and seems an interesting alternative to ribavirin (collaboration L. Mulard, Unité de Chimie Organique, Institut Pasteur et B. Canard, ESIL,CNRS, Marseille).

We have started to modify the active site of human NDP kinase by protein engineering in order to increase its activity for nucleoside analogs. Several mutant proteins were obtained with an increase activity to phosphorylate d4T and AZT as compared to the wild-type human enzyme. The best candidate, combining two mutations in the active site, shows a change in the "specificity factor" of more than 300 as determined on the purified recombinant protein. Current studies are aimed at determining whether the expression of this mutant protein in cultured cells could change their capacity to phosphorylate nucleoside analogs and correspondingly increase their sensitivity to these drugs, in the perspective of cell therapy protocols.

We are also studying the kinases ensuring the addition of the second phosphate. The NMP kinases belong to a family where each kinase is base specific. Human UMP-CMP kinase has been cloned an the enzyme recognize L-3'-TCMP as an excellent substrate. The cloning of all human kinases is underway and will allow us to study the reactivity of phosphonate derivatives which are interesting antiviral drugs.

Biochemical studies of the NEMO protein, an essential component of the NF-kB signalling pathway. Stephane Goffinont, Emilie Vinolo and Fabrice Agou

In response to a wide variety of stimuli like the pro-inflammatory cytokines (TNF-alpha, IL-1) or endotoxines (LPS), cells activate a series of genes involved in the inflammatory and immune responses as well as in oncogenesis and apoptosis. The majority of these genes are under the control of the NF-kB transcription factor whose activation is modulated by a high molecular weight specific protein complex. This multiprotein complex called IKK contains at least three protein components. Two of them, IKK-alpha and IKK-beta, have a protein kinase activity. The third one, NEMO (NF-kB Essential MOdulator) also called IKK-gamma, is a regulatory protein which takes part in the activation of the kinases. The importance of NEMO is underlined by the fact that fibroblast and lymphocyte cells deleted for the NEMO gene loose their ability to activate NF-kB signaling induced by LPS and cytokines. After cell stimulation by pro-inflammatory cytokines or by endotoxines, IKK-kinases are activated without dissociating from NEMO and the inflammatory and immune responses are induced. We investigate the molecular mecanism by which the NEMO protein promotes the activation of the kinases. A better understanding of this mecanism will help us to design new molecules acting as anti-inflammatory and anti-cancer drugs (in collaboration with G. Courtois and A. Israël from the Unité de Biologie Moléculaire de l'Expression Génique, Institut Pasteur).

We have succeeded in obtaining a functional recombinant NEMO purified in E. coli in the presence of non-ionic detergents. We have studied its quaternary structure by analytical centrifugation and gel filtration and evaluated its secondary structures by circular dichroism. The cloning and the purification of the fragment corresponding to the C-terminal domain showed that NEMO is able to associate into dimers or trimers. A specific sequence forming coiled-coils structures is responsible for the oligomerization of the protein. The presence of dimeric and trimeric forms of NEMO has also been shown in living cells using a cell permeable bifunctional crosslinking reagent. In addition, the association of recombinant NEMO with the E. coli DnaK suggests a role for chaperonins in the formation of oligomeric complexes of NEMO in vivo. Current experiments are aimed at determining the precise mechanism by which the oligomerization of NEMO controls the activation of the kinases.

Recently, it was shown by Israël's group in collaboration with teams at Necker Hospital that two human pathologies, Incontinentia Pigmenti and the ectodemal displasia with immunodeficiency, were essentially due to mutations within the coding sequence for the NEMO protein. In this context, we are studying in vitro recombinant NEMO proteins bearing mutations found in patients in order to establish the effect of these mutations on the biochemical properties of the protein and in particular on its state of oligomerization.

Mechanisms of genetic recombination in retroviruses. Abdeladim Moumen, Véronique Giacomoni-Fernandes, Roman Galetto and Matteo Negroni

Homologous recombination is a major source of genetic variability in retroviruses. During their extracellular life retroviruses store genetic information as a single RNA molecule, which is present in two copies within each viral particle. Genetic recombination occurs in retroviruses mostly through template switching during reverse transcription between these two copies of genomic RNA, a process known as "copy choice" or "strand transfer". The impact of recombination on the dynamics of retroviral infections has been dramatically illustrated for the spreading of the AIDS pandemic. In this case at least 10% of the infectious strains of HIV originate through recombination among different viral subtypes. We study the mechanism of retroviral recombination in a reconstituted system, using purified nucleic acids and proteins. We have focused our attention on the role of the structures of the genomic RNA and of a major co-factor of the reverse transcription process, the nucleocapsid protein (NC), an RNA chaperone known to enhance strand transfer in vitro. Our results indicate that the structure of the genomic RNA is the main determinant in the recombination process, in contrast to the current opinion of a copy choice process governed by pausing of reverse transcription. In particular we have shown that template switching in vitro preferentially occurs within hairpin regions of the genome. We have also assessed that the folding of the acceptor RNA (the one onto which DNA synthesis is continued after strand transfer) constitutes the crucial parameter for the efficiency of template switching. These observations have led to the proposal of a possible mechanism accounting for strand transfer within hairpin regions of genomic RNAs. According to this model the transfer process would proceed through a mechanism reminiscent of branch migration taking place during DNA-DNA recombination. Our current efforts are now put on the one hand in the testing of this model, and on the other in the development of a system to study retroviral recombination in infected cells in culture (collaboration with P. Charneau, Groupe de Virologie Moléculaire et Vectorologie, in our institute). In this system the viral infection is limited to one cycle and the analysis of the recombinant virions is performed in the absence of selective pressure. This system is developed in such a way as to parallel as closely as possible the one employed for the in vitro study and will allow to compare the results issued from these two different experimental approaches.

Keywords: NDP kinase, Nucleotides analogs, Anti-viral therapy, HIV, Recombination, Retrovirus, NEMO, NF-kB



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  personnel

  Office staff Researchers Scientific trainees Other personnel
  Tran Catherine (cathtran@pasteur.fr) Agou Fabrice, CR IP (fagou@pasteur.fr)

Deville-Bonne Dominique, MC P6 (ddeville@pasteur.fr)

Negroni Matteo, CR IP (matteo@pasteur.fr)

Veron Michel, DR1 CNRS (mveron@pasteur.fr)

Galetto Roman, Post-doc (rgaletto@pasteur.fr)

Gallois-Montbrun Sarah, Thèse (sarahgm@pasteur.fr)

Moumen Abdeladim, Thèse

Pasti Claudia, Thèse

Vinolo Emilie, DEA (vinolo@pasteur.fr)

Traincard François, Ing. Rech. IP (traincar@pasteur.fr)

Giacomoni-Fernandes Véronique, Techn. Sup. IP (verofern@pasteur.fr)

Goffinont Stéphane, Ingénieur AI CNRS

Cortes Marie-Thérèse, Agent de labo IP


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