Newcastle disease virus forms inclusion bodies with features of liquid-liquid phase separation.

Formation of inclusion bodies (IBs) is a hallmark of infections with negative-strand RNA viruses. Although the Newcastle disease virus (NDV) IBs had been observed in the 1950s, the characteristics of NDV IBs remained largely unknown. Here, we show that NDV infection triggers the formation of IBs that contain newly synthesized viral RNA. The structures of NDV IBs, observed by electron microscopy, were not membrane-bound. Fluorescence recovery after photobleaching a region of NDV IBs occurred rapidly, and IBs were dissolved by 1,6-hexanediol treatment, demonstrating they exhibited properties consistent with liquid-liquid phase separation (LLPS). We find the nucleoprotein (NP) and phosphoprotein (P) are sufficient to generate IB-like puncta, with the N arm domain and N core region of NP and the C terminus of P playing important roles in this process. In summary, our findings suggest that NDV forms IBs containing viral RNA, and provide insights into the formation of NDV IBs.