In vitro assays to measure the protease activity of Chikungunya virus nsP2

SANTIAGO E. FARAJ; DÉBORA A. GONZÁLEZ; ROLANDO C. ROSSI; MÓNICA R. MONTES; CLAUDIA V. FILOMATORI

Rosario, Santa Fe

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

Chikungunya virus (CHIKV) is an important human pathogen transmitted by mosquitoes that belong to the family Togaviridae, genus Alphavirus, which has recently caused explosive epidemics in the Indian Ocean and the Americas. CHIKV infection causes a febrile syndrome typically accompanied by debilitating arthralgia that can persist for years. Despite its high prevalence, there are no vaccines or specific therapies against CHIKV. CHIKV nsP2 protease is crucial for the processing of the viral non-structural polypeptide and the release of viral enzymes, making it a promising drug target.In vitro assays are valuable tools to test the activity of protease inhibitors and to define the molecular determinants for enzymatic activity. In this work, we expressed the protease domain of CHIKV (nsP2-pro) fused to an N-terminal His6-tag, and purified it by metal-chelate chromatography.In order to determine the protease activity, we developed two in vitro cell-free assays. In one of them, we use as substrate a large polyprotein composed of two independently folded domains linked by the biological cleavage site of nsP2; protease activity was visualized by the mobility shift of the polyprotein on polyacrylamide gels. The alternative method is based on the proteolysis of a small peptide that contained the viral cleavage site flanked by two FRET tags. We measured the increase of fluorescence as a function of time to calculate the velocity of proteolysis, which allowed the kinetic characterization of nsP2-pro. We used both assays to test the effect of small compounds such as flavonoids, which have been shown to inhibit the protease activity of RNA viruses from other families. Also, to evaluate the determinants for activity, we compared the nsP2-pro sequences of different alphaviruses and identified key amino acids that were not present in other members of the genus but appeared to be important for CHIKV nsP2 catalysis. As a proof of concept, we used in our assays the nsP2 pro of Venezuelan Encephalitis Virus, a phylogenetically distant alphavirus, and found that it was incapable to process a substrate bearing the CHIKV cleavage site. In summary, we developed two simple techniques to test nsP2-pro activity, which are exploitable both for the rational search of inhibitory compounds and for the study of the molecular mechanism of nsP2-pro.