Strenuous expression of Porcine epidemic diarrhea virus ORF3 protein suggests role in pathogenesis

Abstract

Abstract Background and objective The swine enteric disease etiology, porcine epidemic diarrhea virus (PEDV) is attenuated upon adaptation to cell culture. Genomic analyses between the wild type and the cell adapted (ca) strains have demonstrated nucleotide deletions exclusively on ORF3 gene of ca PEDV. Curiously, we sought to express the ORF3 protein of the virus in both mammalian and bacteria cells as a prerequisite for investigation of the protein’s putative role in pathogenesis. Materials and methods For prokaryotic expression, two vector systems, pET28-a(+) and pGEX-4T-1 were constructed and expressed in Escherichia coli cells. For eukaryotic analyses, ORF3/pEGFP-C1 vector constructs were expressed in human embryonic (HEK-293T) cells, green monkey kidney (Vero) cell and mouse fibrous cells (NIH3T3) cells. Intriguingly, there was minimal expression of the ORF3 gene. Following a hint from Shmitz (1998), that truncated ORF3 revealed higher expression, ORF3 gene was truncated following the Simple Modular Architecture Research Tool (SMART). The analysis predicted two transmembrane domains, one at position amino acid (aa) 41-63 and aa 76-98. Consequently, we generated two fragments; ORF-N (aa 1-98) which included the transmembrane domains and ORF3-C (aa 99-224). These truncated sequences were constructed in similar fashion as the whole gene here referred to as ORF3 wild type (wt). Results Coomassie blue stained gels revealed bands of ORF3-C expressed as a fusion protein of 17.5 and 39 kDa in pET28-a(+) and pGEX-4T-1 vectors, respectively. In contrast, there were no apparent bands visualized for ORF3-N. In addition, ORF3-N induction decreased the total levels of cellular proteins suggesting inhibition of protein synthesis or metabolism in the transformed cells. Further, solubility tests carried out at 30oC, 25oC and 18oC showed that ORF3 formed inclusion bodies. Similar expression patterns were observed in mammalian cells. Conclusion The difficult expression of ORF3 protein and mutants may have resulted from host cell immune system. This would be true especially if the protein is critical in virus pathogenesis. Noteworthy, morphological distortions appeared exclusively in mammalian cells expressing ORF3 protein or truncated mutants suggesting that the protein may be significant in pathogenesis.