Abstract 1042 Liquid-liquid phase separation induced by interactions between the picornavirus 3C(D) main protease and genomic RNA control elements

The picornaviral 3C(D) protein is a key protease that catalyzes the cleavage of both the viral polyprotein and host cell defense proteins. Additionally, it interacts with cis-acting RNA replication elements (CREs; oriL, oriI, and oriR) in the viral genome to control viral replication and translation processes. Despite these known functions, uncertainty persists regarding the mode of interaction between 3C(D) and RNA, and how such interactions may impact protease function. In this study, we investigated the interactions of poliovirus (PV) 3C and 3CD (a form incorporating both 3C protease and 3D polymerase domains), with a specific focus on RNA elements derived from oriL and oriR. RNA binding led to NMR chemical shift perturbations in the N-terminal α-helix (e.g., Arg13), the active site (His40 and Glu71, part of the catalytic triad), the previously identified RNA binding region (residues Glu81-His89), and nearby Thr142, Met160 residues. Furthermore, RNA binding resulted in decreased peak intensities, suggesting the formation of larger multimeric protein-RNA complexes. To delve deeper into these interactions, we employed multiwavelength analytical ultracentrifugation (AUC), revealing the formation of a 1:1 complex between PV-3C and the RNA derived from oriL, as validated by small-angle X-ray scattering (SAXS). Conversely, the RNA derived from oriR led to a multimeric protein: RNA complex with evidence for 1:1, 1:2, 2:1, 2:2, 3:2 etc complexes. Remarkably, fluorescence microscopy provided evidence of stress granule formation through liquid-liquid phase separation (LLPS) induced by the interaction of 3C with RNA. This phenomenon was not only observed with PV-3C but also with 3CD which is more abundant during the virus life cycle. The formation of LLPS in the presence of various RNAs suggests a potential role for 3C(D) in apoptosis post-viral infection and introduces an intriguing possibility of an additional regulatory mechanism in the picornavirus life cycle. This research was supported by NIHs grant AI104878 (to DDB), S10-OD028589 (to NY for SAXS) and S10-OD032215-01 (to NY for AUC).