Structure/function study of the respiratory syncytial virus polymerase and search for inhibitors – BRONCHIOLITEASER

The different components of the RdRp will be expressed as recombinant proteins. The functional domains will be characterized in vitro and in vivo. The atomic structure of these proteins will be analyzed using either full-length or truncated proteins by chrystallography and NMR. Critical residues involved in these interactions will be characterized by site-directed mutagenesis using in vitro and in vivo assays.

The NMR structure of the core domain of the M2-1 transcription factor has been solved. The residues involved in the interactions between M2-1 and the phosphoprotein (the main RdRp cofactor) have been characterized by NMR and by using a functional assay. The domain of the nucleoprotein which interact with P has been characterized and the residues involved in this interaction identified. We have started co-crystallization of this domain together with P.

Co-chrystallization of the N domain with P will allow the identification of pockets that can be targeted for drug design. RdRp inhibitor candidates will be tested using a functional assay. Structural studies of the P and M2-1 domains, alone or in complex with their molecular partners, will be performed by NMR.

The molecular characterization of the P-binding domain of the RSV nucleoprotein has been accepted for publication in the Journal of Virology.
The NMR structure of the core domain of M2-1 has been accepted for publication in PLoS Pathogens

Context

RSV is the chief worldwide viral cause of severe acute respiratory tract illness in two species, humans and cattle. Human RSV (HRSV) is the major cause of severe pediatric respiratory tract disease worldwide and is the leading cause of viral death in infants. Like its human counterpart, Bovine Respiratory Syncytial Virus (BRSV) is also the most important viral respiratory pathogen of calves and is responsible for large economic losses in European dairy and beef farming. After fifty years of research, there is still no vaccine available for humans. Some vaccines are commercialized for cattle, but their efficiency has still to be improved. In such context, the development of antiviral drugs with a wide spectrum is an attractive and economical alternative to vaccination. So far, no specific inhibitors are efficient against these viruses, ribavirin being only exceptionally used because of its toxicity and poor efficiency. A humanized monoclonal antibody directed against the surface F glycoprotein (palivizumab Synagis®) is available, with an efficiency of about 50%, but its high cost limits its use. RSV belongs to the Mononegavirales (MNV) order and is a member of the Paramyxoviridae family. It is an enveloped virus and its negative strand RNA genome encapsidated by N is forming a helical ribonucleoprotein complex (RNP). The RNP template is recognized by the RNA-dependent RNA polymerase (RdRp) composed of L, P (which is an essential cofactor of L), N and two small proteins, M2-1 (22K) and M2-2, that act as transcription and replication co-factors, respectively. This viral RdRp has no counterpart in cells and constitutes a good target for drug design.

Proposal

This proposal is based on the collaborative work of 3 groups with complementary competences and knowledge. Our common goal is to investigate the structure and functionning of the RSV RNA-polymerase complex responsible for genome transcription and replication and to define molecular targets for the development of antivirals. The atomic structure of M2-1 and P-N complexes will be investigated by NMR (Team 1+2) and X-ray crystallography (Teams 1+3), respectively. These structural informations will be used for functional analysis by Team 1 using reverse genetics (minireplicon) and mutagenesis. We have previously identified five molecules capable of blocking P-N interaction by screening a chemical library with Team 2. Preliminary experiments have shown that these molecules inhibit RSV replication in cell culture. We have also determined that these molecules do not interact with P by NMR (Team 1+2). Among them, 3 molecules belong totwo natural chemical families, extracted from plants. Other molecules from the same families will be tested for their capacity to block P-N interaction, and their site of binding on N will be determined by crystallography (Teams 1+3+2).
Expected results
Precious structural and functional informations on the RNA-dependant RNA polymerase of RSV will be collected. These data will allow a better understanding of the functioning of the viral RNA polymerase, and will also be useful for drug design and development. P-N inhibitors already identified will be better characterized, in particuluar their site of binding on N, and constitute good candidates for antivirals.