Antiviral strategies

  MEMBERSCASTEL Guillaume / CHTEOUI Mohamed / Dr HEYD Bernadette

  Annual Report

Created in October 2004, our Unit develops strategies ultimately aimed at designing new antiviral drugs that will specifically interfere with the replication of Negative Strand RNA Viruses (NSRV). NSRV are a central issue in public health. They cause severe pathologies of the respiratory track (Influenza-, Respiratory Syncytial- virus RSV), encephalitis (Rabies-, Nipah- virus), hemorrhagic fevers (Ebola-, Lassa-, Crimea Congo Hemorrhagic Fever-, Rift Valley Fever virus). Many NSRV are zoonotic agents with emerging potential, several are assigned to biosafety levels 3 or 4, few are potential concern for bioterrorism. Globally, there is a lack of prophylactic/therapeutic tools against NSRV. In particular, no good inhibitors of their replicase function have been found so far.

We study the different components of the transcription/replication complex: the N-RNA template (RNA genome tightly associated with N protein), and the polymerase module (L polymerase, P cofactor). Targets and specific inhibitors are identified by two complementary approaches followed in parallel.

A cognitive approach to elucidate the structure/function relationships and interactions within the transcription/replication complex (and with host factors) in the perspective of destabilizing the functional domains. Yeast 2-hybrid, co-immunoprecipitation, protease analysis combined with structural data and reverse genetics are the major tools used. From a complete interacting map of the rabies virus P protein, the NH2-terminus (50 to 60 amino acids) was found to bind both L and N proteins and thus to be a promising target for inhibition. Two peptides mimicking this NH2–terminus were shown very effective in inhibiting transcription, replication as well as rabies infection in vitro.

A random approach taking advantage of high throughput technologies to screen combinatorial libraries of short peptides (<20 amino acids; >107) and to isolate peptides with inhibitory potential on viral replication. The library peptides can be constrained so as to limit the number of potential conformers. The discovery strategy is progressive: (1) exhaustive screening of the libraries by yeast 2-hybrid or phage display to select peptides with high binding activity to replication-related targets; (2) identification of the destabilized interaction (protein chip mass spectrometry); (3) functional validation of the peptide inhibitory effect on transcription/replication (replicon assay); (4) test for inhibition of viral growth in cell cultures.

Yeast two-hybrid screening has been successfully applied to the rabies virus P protein: from two libraries mimicking naturally constrained bioactive peptides (toxins, defensins), 4 candidates with strong antiviral activity have been identified. Similarly, 5 promising peptides have been selected from large libraries by phage display for their high affinity to the rabies virus N-RNA template. Two of them also show affinity to the N-RNA template of RSV. We currently apply this approach in parallel to various NSRV separated by variable phylogenetic distance, in the perspective of identifying specific antiviral molecules with the widest spectrum of activity and no cellular toxicity. The best inhibitory peptides will also serve as bases to design pharmacologically active peptido-mimetic molecules.


Publications 2006 of the unit on Pasteur's references database

Activity Reports 2006 - Institut Pasteur
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