Abstract
Transmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus that causes diarrhea in pigs and is associated with high morbidity and mortality in sucking piglets. S1 is one of two protein domains in the spike (S) glycoprotein and is responsible for enteric tropism, sialic acid recognition, and host receptor binding. Although there has been extensive research on the S1 protein of TGEV, little is known about the intracellular role of TGEV-S1. In the present study, we used yeast two-hybrid screening of a cDNA library from porcine intestinal cells to identify proteins that interact with TGEV-S1. Among 120 positive clones from the library, 12 intracellular proteins were identified after sequencing and a BLAST search. These intracellular proteins are involved in protein synthesis and degradation, biological signal transduction, and negative control of signaling pathways. Using a glutathione-S-transferase (GST) pulldown assay and Co-IP, we found that UBXN1 interacts with the S1 protein. Here, we observed that TGEV infection led to increased UBXN1 expression levels during the late phase of infection in IPEC-J2 cells. Inhibition of UBXN1 in IPEC-J2 cells via siRNA interference significantly decreased the viral titer and downregulated the expression of S1. UBXN1 overexpression significantly increased the viral copy number. Additionally, we provided data suggesting that UBXN1 negatively regulates IFN-β expression after TGEV infection. Finally, our research indicated that UBXN1 plays a vital role in the process of TGEV infection, making it a candidate target for the development of a novel antiviral method.
Highlights
Transmissible gastroenteritis virus (TGEV) is a pathogenic agent of porcine transmissible gastroenteritis (TGE), which causes vomiting, diarrhea, and high mortality in suckling piglets, resulting in heavy losses to the pig breeding industry [1]
Identifying host proteins interacting with TGEV‐S1 via a two‐hybrid assay The bait vector pGBKT7 in the yeast two-hybrid system was constructed to screen the interactions between the TGEV-S1 protein and host proteins, and the recombinant plasmid was successfully transformed into Y2HGold yeast cells (Figure 1A)
No colony growth was observed on the standard deviations (SDs)/−Trp/X-α-Gal/AbA plates, indicating that there was no self-activation phenomenon for the reporter genes (Figure 1D). These results indicate that the constructed pGBKT7-S1 bait vector could be used the yeast two-hybrid system for screening host proteins that interact with the TGEV-S1 protein
Summary
Transmissible gastroenteritis virus (TGEV) is a pathogenic agent of porcine transmissible gastroenteritis (TGE), which causes vomiting, diarrhea, and high mortality in suckling piglets, resulting in heavy losses to the pig breeding industry [1]. The genome contains nine open reading frames (ORFs) encoding 16 kinds of nonstructural proteins and four structural proteins, spike (S), envelope (E), membrane (M), and nucleoprotein (N), which are arranged in the order of 5′-replicase-(1a/1b)-S-3a-3b-E-M-N-7-3′ [2]. In the early stage of TGEV infection, TGEV-S1 is mainly responsible for binding to cellular receptors, and the S2 domain is associated with membrane fusion between the virus and host cells. Compared with the S2 domain, the S1 domain has many significant functions similar to those of the entire S protein, including enteric tropism [6], sialic acidbinding activity [7], neutralizing antibody induction, and host receptor binding. Determinants for enteric tropism and sialic acid recognition are located in a 224 aa region at the N terminus of the TGEV S1 domain. The immunogenicity of TGEV S1 is stronger than that of the whole S protein and is the major inducer of TGEV-neutralizing antibodies [9]
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