CONICET | Buscador de Institutos y Recursos Humanos

Chikungunya virus (CHIKV) is a pathogen transmitted by mosquitoes, which belongs to the family Togaviridae, genus Alphavirus. Its infection causes fever and joint pain that can last for years. Recently, it has caused epidemics in the Indian Ocean and the Americas. Despite being prevalent, there are no therapies or vaccines against it.The CHIKV nsP2 protease is critical for nonstructural polypeptide processing and viral enzyme release, making it an important drug target.The objective of this work is to study the determinants for the interaction between recombinant protease domain (nsP2-Pro) and its substrates. As a first approach, we used the AlphaFold algorithm and identified possible interaction pairs between segments with the cleavage sites and residues located in the C-terminal domain of nsP2pro. Based on these observations, we designed mutations that disrupt the predicted interactions or alter the catalytic site. To assess the effect of the engineered mutations we developed two in vitro cell-free assays that use purified nsP2-Pro. The first one used as substrate a polyprotein composed by GFP and thioredoxin linked by sequences containing the nsP2 biological cleavage site; protease activity was measured by appearance of lower molecular weight species on polyacrylamide gels. The other assay used a peptide containing the cleavage site flanked by FRET tags. In this case, the increase in fluorescence as a function of time was measured to calculate the rate of proteolysis.To complete the study, we have in the laboratory a CHIKV replicon containing reporter genes, which allows us to evaluate the impact of nsP2 mutations on viral translation and replication.These studies will help to understand the enzymatic mechanism of nsP2-Pro and provide useful information for drug design.