Porcine epidemic diarrhea virus (PEDV) is a globally distributed alphacoronavirus that has re-emerged lately, resulting in large economic losses. During viral infection, interferon (IFN-I) plays a vital role in the antiviral innate immunity. However, PEDV has evolved strategies to limit IFN-I production. To suppress virus replication, the host must activate the IFN-stimulated genes and some host restriction factors to circumvent viral replication. This study observed that PEDV infection-induced early growth response gene 1 (EGR1) expression in PEDV-permissive cells. EGR1 overexpression remarkably suppressed PEDV replication. In contrast, depletion of EGR1 led to a significant increase in viral replication. EGR1 suppressed PEDV replication by directly binding to the IFN-regulated antiviral (IRAV) promoter and upregulating IRAV expression. A detailed analysis revealed that IRAV interacts and colocalizes with the PEDV nucleocapsid (N) protein, inducing N protein degradation via E3 ubiquitin ligase MARCH8 to catalyze N protein ubiquitination. Knockdown of endogenous MARCH8 significantly reversed IRAV-mediated N protein degradation. The collective findings demonstrate a new mechanism of EGR1-mediated viral restriction, in which EGR1 upregulates the expression of IRAV to degrade PEDV N protein through MARCH8. IMPORTANCE PEDV is a highly contagious enteric coronavirus that has rapidly emerged worldwide and caused severe economic losses. No currently available drugs or vaccines could effectively control PEDV. PEDV has evolved many strategies to limit IFN-1 production. We identified EGR1 as a novel host restriction factor and demonstrated that EGR1 suppresses PEDV replication by directly binding to the IRAV promoter and upregulating the expression of IRAV, which interacts and degrades the PEDV N protein via E3 ubiquitin ligase MARCH8 to catalyze nucleocapsid protein ubiquitination, which adds another layer of complexity to innate antiviral immunity of this newly identified restriction factor. A better understanding of the innate immune response to PEDV infection will aid the development of novel therapeutic targets and more effective vaccines against virus infection.

Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, but none of the currently available drugs or vaccines can effectively control these diseases. During viral infection, the host will activate the interferon (IFN) signaling pathways and host restriction factors in maintaining the innate antiviral responses and suppressing viral replication.

Background: Porcine epidemic diarrhea virus (PEDV) infection causes an acute enteric tract infectious disease characterized by vomiting, anorexia, dehydration, weight loss and high mortality in neonatal piglets. During PEDV infection, the spike protein (S) is a major virion structural protein interacting with receptors and inducing neutralizing antibodies. However, the neutralizing B-cell epitopes within PEDV S protein have not been well studied.Methods: To accurately identify the important immunodominant region of S1, the purified truncated S1 proteins (SA, SB, SC, SD and SE) were used to immunize BALB/c mice to prepare polyclonal antibodies. The antisera titers were determined by indirect ELISA, western blot and IFA after four immunizations to find the important immunodominant region of S1, and then purified the immunodominant region of S1 protein and immunized mice to generate the special antibodies, and then used recombinant peptides to determine the B-cell epitopes of monoclonal antibodies.Results: Five antisera of recombinant proteins of the spike protein region of PEDV were generated and we found that only the polyclonal antibody against part of the S1 region (signed as SE protein, residues 666-789) could recognize the native PEDV. Purified SE protein was used to immunize BALB/c mice and generate mAb 2E10. Pepscan of the SE protein demonstrated that SE16 ( 722 SSTFNSTREL 731 ) is the minimal linear epitope required for reactivity with the mAb 2E10. Further investigation indicated that the epitope SE16 was localized on the surface of PEDV S protein in the 3D structure.Conclusions: A mAb 2E10 that is specifically bound to PEDV was generated and identified a specific linear B-cell epitope (SE16, 722 SSTFNSTREL 731 ) of the mAb. The epitope region of PEDV S1 localized in the different regions in comparison with the earlier identified epitopes. These findings enhance the understanding of the PEDV spike protein structure for vaccine design and provide a potential use for developing diagnostic methods to detect PEDV.