Porcine epidemic diarrhea virus (PEDV) is the globally distributed alphacoronavirus that can cause lethal watery diarrhea in piglets, causing substantial economic damage. However, the current commercial vaccines cannot effectively the existing diseases. Thus, it is of great necessity to identify the host antiviral factors and the mechanism by which the host immune system responds against PEDV infection required to be explored. The current work demonstrated that the host protein, the far upstream element-binding protein 3 (FUBP3), could be controlled by the transcription factor TCFL5, which could suppress PEDV replication through targeting and degrading the nucleocapsid (N) protein of the virus based on selective autophagy. For the ubiquitination of the N protein, FUBP3 was found to recruit the E3 ubiquitin ligase MARCH8/MARCHF8, which was then identified, transported to, and degraded in autolysosomes via NDP52/CALCOCO2 (cargo receptors), resulting in impaired viral proliferation. Additionally, FUBP3 was found to positively regulate type-I interferon (IFN-I) signaling and activate the IFN-I signaling pathway by interacting and increasing the expression of tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3). Collectively, this study showed a novel mechanism of FUBP3-mediated virus restriction, where FUBP3 was found to degrade the viral N protein and induce IFN-I production, aiming to hinder the replication of PEDV. IMPORTANCE PEDV refers to the alphacoronavirus that is found globally and has re-emerged recently, causing severe financial losses. In PEDV infection, the host activates various host restriction factors to maintain innate antiviral responses to suppress virus replication. Here, FUBP3 was detected as a new host restriction factor. FUBP3 was found to suppress PEDV replication via the degradation of the PEDV-encoded nucleocapsid (N) protein via E3 ubiquitin ligase MARCH8 as well as the cargo receptor NDP52/CALCOCO2. Additionally, FUBP3 upregulated the IFN-I signaling pathway by interacting with and increasing tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) expression. This study further demonstrated that another layer of complexity could be added to the selective autophagy and innate immune response against PEDV infection are complicated.

p53, the guardian of the genome, is a short-lived protein that is tightly controlled at low levels by constant ubiquitination and proteasomal degradation in higher organisms. p53 stabilization and activation are early crucial events to cope with external stimuli in cells. However, the role of p53 ubiquitination and its relevant molecular mechanisms have not been addressed in invertebrates. In this study, our findings revealed that both HUWE1 (HECT, UBA, and WWE domain-containing E3 ubiquitin-protein ligase 1) and TRAF6 (tumor necrosis factor receptor-associated factor 6) could serve as E3 ubiquitin ligases for p53 in mud crabs (Scylla paramamosain). Moreover, the expression of HUWE1 and TRAF6 was significantly downregulated during white spot syndrome virus (WSSV) infection, and therefore the ubiquitination of p53 was interrupted, leading to the activation of apoptosis and reactive oxygen species (ROS) signals through p53 accumulation, which eventually suppressed viral invasion in the mud crabs. To the best of our knowledge, this is the first study to reveal the p53 ubiquitination simultaneously induced by two E3 ligases in arthropods, which provides a novel molecular mechanism of invertebrates for resistance to viral infection. IMPORTANCE p53, which is a well-known tumor suppressor that has been widely studied in higher animals, has been reported to be tightly controlled at low levels by ubiquitin-dependent proteasomal degradation. However, recent p53 ubiquitination-relevant research mainly involved an individual E3 ubiquitin ligase, but not whether there exist other mechanisms that need to be explored. The results of this study show that HUWE1 and TRAF6 could serve as p53 E3 ubiquitin ligases and synchronously mediate p53 ubiquitination in mud crabs (Scylla paramamosain), which confirmed the diversity of the p53 ubiquitination regulatory pathway. In addition, the effects of p53 ubiquitination are mainly focused on tumorigenesis, but a few are focused on the host immune defense in invertebrates. Our findings reveal that p53 ubiquitination could affect ROS and apoptosis signals to cope with WSSV infection in mud crabs, which is the first clarification of the immunologic functions and mechanisms of p53 ubiquitination in invertebrates.