PEDV nucleocapsid (N) protein induces liquid-liquid phase separation (LLPS) to selectively aggregate the viral genome

As an important structural protein, porcine epidemic diarrhea virus (PEDV) nucleocapsid (N) plays critical roles in the viral life cycle. Liquid-liquid phase separation (LLPS) is required for multiple biological functions for coronaviridae infection. However, whether PEDV has the ability to cause LLPS and its roles in this process remain unknown. In this study, we carried out bioinformatic analysis to show that PEDV N protein had the strongest potential to induce LLPS among all PEDV proteins. PEDV N protein caused both endoplasmic reticulum stress (ERS) and protein translation stall in vivo, which has been associated with biomolecular condensates mediated by LLPS. At the same time, we purified the N protein and found that either RNA or molecular crowding agent can induce LLPS of N protein in a concentration-dependent manner in vitro. Subsequently, the different truncated domains of N protein were further investigated, of which results showed that the dimerization domain (DD) was responsible for the self-aggregation of N protein, and that the intrinsically disordered region (IDR) played decisive roles in LLPS formation with mRNA. In addition, we showed that the RNA sequence and structural differences in genomic RNA (gRNA) such as 5' end contribute to LLPS triggered by N protein, as revealed by larger, more liquid-like condensates of 5'-UTR combined with N protein. Disruption of LLPS attenuated ERS and interfered with the binding of N protein with RNA, as well as N gene transcription and protein expression. These findings not only provide new insights into the novel functions of PEDV N protein, but also provide a foundation for understanding the mechanism of genome aggregation and its role in the virus life cycle.