Rabies virus RNA capping machinery as antiviral target – RAB-CAP

Mononegavirales such as Ebola, rabies, VSV and measles virus are non-segmented negative strand RNA viruses causing important public health threats. Although prophylactic or post exposure vaccination is available against a limited set of Mononegavirales, we lack antiviral molecules for the treatment of non-vaccinated and/or people infected by rabies. It is therefore essential to identify conserved viral enzymes, playing a key role during the viral replication cycle, in order to design new antiviral strategies. The L protein of Mononegavirales is the main actor of virus replication, catalyzing the replication and transcription of the viral genome. The C-terminal half of L protein contains conserved polyribonucleotidyltransferase (PRNTase) and methyltransferase (MTase) domains involved in the addition of a cap structure at the 5’ end of viral mRNA. Interestingly, Mononegavirales members use an unconventional capping mechanism in which the PRNTase domain forms a covalent link with the 5’ end of nascent RNA. A GDP molecule is subsequently ligated to the viral nascent pRNA forming the cap structure (GpppN-RNA). The cap structure is then methylated on the 2’O of the first nucleotide and on its N7 position (mGpppNm-RNA). This cap-1 structure is essential for viral mRNA translation into protein and blocks the detection of viral mRNA by host cell sensors such as RIG-I and MDA5, as was recently demonstrated for positive strand RNA viruses (Dengue & Coronaviruses). Although these positive strand RNA viruses are distantly related to Mononegavirales, these results demonstrate that the inactivation of N7-MTase blocks viral replication and of 2’O-MTase leads to the clearance of viral infection in small animal models with a strong induction of antiviral response. Thus, the capping pathway can be considered a promising antiviral target. However, although individual steps of the pathway have been observed in vitro in VSV, the full capping process has never been unravelled for a single virus model, from the molecular mechanism description to corresponding phenotypic effects in animal models.
The aim of this project is to decipher the Mononegavirales capping machinery by characterizing the structure and the function of capping enzymes (PRNTase, N7- and 2’O-MTases) and to allow the identification of compounds blocking the MTase activity. We will address this question using the rabies virus (RABV) as a model for two main reasons: First, rabies is an important zoonotic disease provoking lethal encephalitis in humans and animals. Rabies infection is fatal in the absence of post exposure prophylaxis and always fatal after the onset of the symptoms. It is at least responsible for 59 000 human deaths each year. Second, this virus model provides the opportunity to perform a complete study ranging from biochemical and structural analysis, to exploiting reverse genetic tools in cell culture and subsequent in vivo translational applications using a mouse model.
By combining structural and biochemical studies together with the powerful tools of reverse genetics we propose to characterize the effects of mutations blocking these capping activities on RABV replication and on the immune response. We expect these mutations to affect virus replication and/or induce a strong antiviral response clearing the viral infection, therefore providing proof of concept that the cap synthesis pathway is a promising antiviral target and represents a powerful strategy limiting the viral replication. This should make important contributions to understand the Mononegavirales transcription mechanism and potentially lead to the discovery of a viral strategy that couples transcription to immune evasion. The present experience of the consortium in antiviral projects should accelerate the translation of our results and deliverables such as new live attenuated vaccines and drugs to fight rabies and potentially other currently incurable viral diseases caused by Mononegavirales.