Functional characterization of the Alphaviruses Macro domain: Application as a new target for the development of anti-viral agents.

Alphaviruses possess a single positive-stranded genomic RNA coding for structural and non-structural proteins (nsP 1-4). Among alphaviruses nsPs, nsP3 plays a critical role in determining insect vector tropism, neurovirulence, viral vs host protein- protein interaction specificity and viral evasion (1)(2). It contains thee functional modules among which the Macro domain (XD) (3). This Macro domain can bind to mono-ADP-ribose (MAR) and poly-ADP-ribose (PAR) in their free form or conjugated to a protein or RNA substrates (4). Macro domains also carry hydrolase activities such as ADP-ribose 1" phosphate, but also de-MARylation and de-PARylation activities (5)(6). ADP-ribosylation is a crucial regulatory modification implicated in the inflammation process and the regulation of innate immunity. ADP-ribosylation contributes to the establishment of an anti-viral response, by inducing type I interferons (IFN) and inhibiting viral translation and replication (7)(8). Mutagenesis studies on viral Macro domains showed that de-MARylation and de-PARylation activities harbored by this domain might counter ADP-ribosylation in host cells, thereby promoting viral evasion (2)(9). The aim of the present proposal is to design and test specific Macro domain ligands and inhibitors, determine their mechanism of action (ADP-ribose binding and /or hydrolysis) through a functional and biochemical characterization of Chikungunya and Venezuelan equine encephalitis viruses Macro domains, and assay candidate molecules in virus infected cells. The development of this project will rely on the following goals: (i) Conduct a mutagenesis study targeting specifically the divergent residues between Alphavirus members, in order to pinpoint differences in their catalytic function in terms of ligand binding and substrate hydrolysis. (ii) Assess the effect of these mutations on type I IFN susceptibility and ISGs activation in cellula. (iii) Based on the available structural data, test a selected set of candidate inhibitors on recombinant Macro domains. (iv) Validate the active candidates in cell infection experiments. Finally, this study will contribute to the understanding and the functional characterization of viral activities implicated in the shut-off of host innate immune responses, a prerequisite for the development of Alphavirus specific antiviral drugs.