Single-stranded nucleic acid binding enhances the in vitro catalytic activity of Chikungunya virus nsP2 protease

Chikungunya virus (CHIKV) is an emerging arbovirus whose replication relies on the multifunctional nonstructural protein 2 (nsP2), particularly its viral protease (nsP2^pro), which is essential for polyprotein processing. In this study, we investigated how interactions with nucleic acids influence nsP2^pro activity. Using High-Throughput Sequencing-Fluorescent Ligand Interaction Profiling (HiTS-FLIP), we identified specific single-stranded DNA aptamers that enhanced nsP2^pro activity. Additionally, both random single-stranded DNA and single-stranded RNA were found to stimulate protease activity, whereas double-stranded DNA showed no such effect. Circular dichroism spectroscopy and secondary structure predictions confirmed that the identified aptamers adopt stable folded conformations. Similarly, structured RNA sequences were also capable of promoting protease activity. The observed stimulatory effect depended on the nucleic acid strand type, length, and buffer conditions, suggesting the involvement of electrostatic interactions. NMR experiments demonstrated that nsP2^pro interacts with single-stranded DNA, accompanied by changes in its conformational dynamics upon binding. Molecular dynamic simulations of nsP2^pro-RNA and -DNA complexes revealed that these nucleic acids interact specifically with the nsP2^pro methyltransferase domain and induce an opening process in the protease active site. Our findings provide novel insights into the regulation of nsP2^pro, enhancing our understanding of CHIKV replication mechanisms, and may guide future antiviral development strategies.