In recent years, an emerging porcine epidemic diarrhea virus (PEDV) variant has gained attention because of serious outbreaks of piglet diarrhea in China and the United States. Coronavirus nonstructural protein 7 (nsp7) has been proposed to act with nsp8 as part of an RNA primase to generate RNA primers for viral RNA synthesis. ABSTRACT Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus that causes high mortality in piglets. Interferon (IFN) responses are the primary defense mechanism against viral infection; however, viruses always evolve elaborate strategies to antagonize the antiviral action of IFN. Previous study showed that PEDV nonstructural protein 7 (nsp7), a component of the viral replicase polyprotein, can antagonize ploy(I:C)-induced type I IFN production. Here, we found that PEDV nsp7 also antagonized IFN-α-induced JAK-STAT signaling and the production of IFN-stimulated genes. PEDV nsp7 did not affect the protein and phosphorylation levels of JAK1, Tyk2, STAT1, and STAT2 or the formation of the interferon-stimulated gene factor 3 (ISGF3) complex. However, PEDV nsp7 prevented the nuclear translocation of STAT1 and STAT2. Mechanistically, PEDV nsp7 interacted with the DNA binding domain of STAT1/STAT2, which sequestered the interaction between karyopherin α1 (KPNA1) and STAT1, thereby blocking the nuclear transport of ISGF3. Collectively, these data reveal a new mechanism developed by PEDV to inhibit type I IFN signaling pathway. IMPORTANCE In recent years, an emerging porcine epidemic diarrhea virus (PEDV) variant has gained attention because of serious outbreaks of piglet diarrhea in China and the United States. Coronavirus nonstructural protein 7 (nsp7) has been proposed to act with nsp8 as part of an RNA primase to generate RNA primers for viral RNA synthesis. However, accumulating evidence indicates that coronavirus nsp7 can also antagonize type I IFN production. Our present study extends previous findings and demonstrates that PEDV nsp7 also antagonizes IFN-α-induced IFN signaling by competing with KPNA1 for binding to STAT1, thereby enriching the immune regulation function of coronavirus nsp7.

Porcine Epidemic Diarrhea Virus nsp7 Inhibits Interferon-Induced JAK-STAT Signaling through Sequestering the Interaction between KPNA1 and STAT1

Porcine epidemic diarrhea virus (PEDV) is an enteric swine coronavirus. Recent PEDV eruption in East Asian and North American countries made it notorious and caused huge economic losses to the porcine industry. Nonstructural protein 13 (nsp13) is a nucleic acid helicase/NTPase that plays a critical role in viral gene transcription and viral replication. To investigate the function of nsp13 in the context of PEDV infection, here, PEDV nsp13 gene was amplified and cloned into pET28a/pET30a/pGEX-6P-1 expression vectors. The recombinant his-tagged nsp13 and GST-tagged nsp13 were expressed. Purified his-tagged nsp13 from pET28a-nsp13 vectors was chosen for immunization. Three monoclonal antibodies (mAbs) named 5A9, 5C7, and 5G7 were identified from the hybridoma cells, and were characterized by Western blot analysis and immunofluorescent assay, which demonstrated high specificity of these three mAbs with pCAGGS-HA-nsp13. All three mAbs belong to IgG1+ kappa subclass. However, only mAb 5A9 could effectively and specifically recognize PEDV expressing nsp13. Furthermore, the generated antibody against nsp13 could be applied to investigate nsp13 function during PEDV replication.

Generation and Characterization of Monoclonal Antibody against Porcine Epidemic Diarrhea Virus Nonstructural Protein 13

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Generation and Characterization of Monoclonal Antibody against Porcine Epidemic Diarrhea Virus Nonstructural Protein 13