Interferon-induced BST2 (bone marrow stromal cell antigen 2) inhibits viral replication by tethering enveloped virions to the cell surface to restrict viral release and by inducing the NFKB-dependent antiviral immune response. However, the mechanism by which BST2 uses the selective autophagy pathway to inhibit viral replication is poorly understood. In this study, we showed that BST2 expression was significantly increased during porcine epidemic diarrhea virus (PEDV) infection of Vero cells by IRF1 targeting its promoter. We also showed that BST2 suppressed PEDV replication by binding and degrading the PEDV-encoded nucleocapsid (N) protein. The downregulation of N protein was blocked by macroautophagy/autophagy inhibitors but not a proteasome inhibitor, implying that the N protein was degraded via the selective autophagy pathway. Both the BST2 and N protein interacted with the E3 ubiquitin ligase MARCHF8/MARCH8 and the cargo receptor CALCOCO2/NDP52, and the ubiquitination of N protein was necessary for the degradation of N mediated by the BST2-MARCHF8 axis. The knockdown of MARCHF8 or ATG5 with small interfering RNAs blocked the selective autophagy pathway, rescued the protein abundance of PEDV N in 293T cells, and prevented the inhibition of PEDV replication by BST2 in Vero cells. Together, our data demonstrate the novel mechanism of BST2-mediated virus restriction, in which BST2 recruits MARCHF8 to catalyze the ubiquitination of the PEDV N protein. The ubiquitinated N protein is then recognized by CALCOCO2/NDP52, which delivers it to autolysosome for degradation through the selective autophagy pathway.

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.

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.

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.

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Background IRAV (interferon-regulated antiviral gene) was identified with antiviral activity as a novel interferon-stimulated gene. IRAV is upregulated in response to type I and type II IFNs and a number of virus. However, the antiviral activity of IRAV to virus infection is poorly understood. Methods To obtain the antibody against IRAV, we cloned the full-length IRAV complementary DNA (cDNA) from porcine kidney cells firstly. And then, the porcine IRAV protein was expressed in Escherichia coli BL21 (DE3), purified and immunized to female BALB/c mice. Finally, we determined the specificity of the MAbs by indirect ELISA, Western blotting and IFA. Results Five strains of hybridoma cells named 2B10, 2G12, 2H1,5A8 and 2C5 secreting anti-IRAV MAbs were obtained. By western blot analysis and indirect immunofluorescence assay, the MAbs were identified with the specific reaction with the overexpressed porcine IRAV protein in PK15 cells. Conclusions The MAbs against porcine IRAV, identified by western blot and IFA, provid a valuable tool to study the biological function of IRAV in the future.