Structure and function of the transcription et replication machinery of the non-segmented negative strand RNA viruses – NNViPol

By a multidisciplinary approach that combines in vitro and in vivo studies, we propose to pursue our structural and functional characterization of the VSV polymerase complex. By reconstituting the transcription and replication processes in vitro and in vivo, we propose to answer specific questions about the mechanisms of these reactions. Finally, we will also seek to validate molecular targets for the development of antiviral inhibitors.

Currently, no atomic structure of a non-segmented negative sense RNA is known. Among the expected results, the determination of the structure of VSV polymerase will provide a major advance in the understanding of the catalytic mechanisms specific of this polymerase and will open the door for the development of specific inhibitors.

The fulfillment of each of the three major goals of this project will have major benefits for understanding the replication of non-segmenté negative sense RNA viruses and for developing inhibitors in our fight against known and emerging virus of this order.

To come

Understanding the molecular mechanisms of viral replication and of the interactions between viruses and their host-cells is a major undertaking that will shed light on the evolution of viruses, on host-virus adaptations and that will give us new tools to fight against current and emerging pathogens. In this project, we focus on the non-segmented negative strand RNA viruses (NNV). Some of these viruses are highly prevalent in humans (measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, metapneumovirus) while others continue to emerge and cause serious diseases (rabies virus, Ebola virus, Nipah virus …). Although vaccines are available against some NNV, we are still lacking an efficient drug to fight these pathogens. Because all NNV share a common organization of their genome and similar mechanisms of transcription and replication, we believe that deciphering the structure and the molecular mechanisms involved in the replication of a model virus such as vesicular stomatitis virus (VSV) will help in understanding the replication machine of other NNV and will point to Achilles’ heels that might then be specifically targeted for structural studies and the development of antiviral therapies.

NNV carry their own machinery for transcribing the viral genome into mRNAs and for replication of this genome. This machinery is specific and thus represents a target for developing inhibitors of viral replication. It consists of a large ribonucleoprotein complex made of the RNA genome coated by the nucleoprotein (N), the phosphoprotein (P) and the large subunit (L) of the RNA-dependent RNA polymerase. The mechanism of gene expression of NNV has been studied for more than 40 years, but many details of action and regulation of this viral machine remain the subject of speculation and controversy. In particular, we are still lacking detailed structural information about the polymerase and we still do not know whether a unique complex catalyzes both types of RNA synthesis or whether complexes of different composition are involved. Also, we lack a clear description of the mechanisms by which the polymerase switches between its two activities, by which it gains access to the N-coated RNA and by which it moves along this template.

With this project we aim to understand the fundamental mechanisms of the viral machine that transcribes and replicates the RNA genome of a model NNV, the vesicular stomatitis virus (VSV). The project relies on structures of several essential components of this machine that we solved recently, in particular of the N0-P complex. These structures brought new insights and raised new hypotheses about the functioning of these complex processes. It also relies on preliminary results obtained in our laboratories. In particular, we are now able to produce all highly purified components, including the L protein, and we have been able to reconstitute transcription activity by mixing them together. Also, for the first time, we have obtained a preliminary 3D reconstruction of the L protein by electron microscopy.

By a multidisciplinary approach that combines in vitro and in vivo studies, we propose to pursue our structural and functional characterization of the VSV polymerase complex. We propose to determine the structure of the L protein at the highest possible resolution and to characterize the organization and structure of the polymerase machine in complex with its partners. By reconstituting the transcription and replication processes in vitro and in vivo, we propose to answer specific questions about the mechanisms of these reactions. Finally, we will also seek to validate molecular targets for the development of antiviral inhibitors and to determine their structures at high resolution.