| Rotavirus(RV) is one of the major pathogens that cause the severe gastroenteritis in young animals and children, especially in developing countries. Rotaviruses have been shown to be responsible for an estimated 450,000 deaths each year occuring in children aged below five 5 years. Porcine rotavirus(PoRV) is the leading cause of viral diarrhea in piglets, which is marked by dehydration due to diarrhea, causes serious damage in the development of animal husbandry. Nonstructural proteins 4(NSP4), a 175-amino acid-long protein, has been identified as the viral enterotoxin. NSP4 domain(amino acids [aa] 47 to 91) has structural characteristics of viroporin, NSP4 and viroporin play an important role in viral pathogenesis. Study showed that NSP4(47-91 aa) mutants form a transmembrane puncta, which disrupts calcium homeostasis and increases the levels of free cytoplasmic Ca2+. NSP4 and viroporin may be closely related to RV-induced endoplasmic reticulum stress(ERS) and apoptosis. The further research on NSP4 and viroporin has a significance in public health and deep study with the mechanism of rotavirus. In this study, we used the plasmid with PoRV NSP4 and viroporin gene to transfect cells and detected cell apoptosis the relationship between ERS and cell apoptosis induced by NSP4 and viroporin. The results were as follows:(1)The NSP4 gene and NSP4(47-91 aa) gene segment were cloned by RT-PCR from PoRV, and Flag tag were added to the C-terminal by primer design. Then they were inserted into pc DNA3.1(+) vector to construct eukaryotic expression recombinant plasmid pcDNA3.1-NSP4-Flag and pcDNA3.1-Viroporin-Flag. The double enzyme identification and sequencing results were consistent with the expected results, suggesting that eukaryotic expression plasmid of NSP4 and NSP4(47-91 aa) were constructed successfully.(2)The swine intestinal epithelial cells(SIEC02) were transfected with recombinant plasmid NSP4-GFP and viroporin-GFP, subsequently they were analyzed by fluorescent inverted microscope and Western blot, the results showed that NSP4 and NSP4(47-91 aa) were successfully expressed in SIEC02 cells. DNA ladder is one of the apoptosis index, it is DNA fragmentation. The total DNA was extracted from cells transfected with recombinant plasmids and detected by 1% agarose gel electrophoresis, the result showed that NSP4 and NSP4(47-91 aa) induced cell apoptosis. The expression of Bax and Bcl-2 were analyzed by real-time quantitative PCR. The results elucidated that Bax was upregulated and Bcl-2 was down-regulated; Western blot analysis showed that caspase-3, caspase-8 and caspase-9 were cleaved, suggesting that NSP4 and NSP4(47-91 aa) induced apoptosis were dependent on the activation of caspase and Bax.(3)Cell apoptosis is closely related to endoplasmic reticulum stress(ERS). To determinewhether ERS was involved in NSP4 and NSP4(47-91 aa) induced apoptosis, we detected the expression of ERS factors such as GRP78, CHOP, caspase-12, ATF6, ATF4 and IERα by real-time quantitative PCR and Western blot analysis. The result showed that NSP4 and NSP4(47-91 aa) promote the expression of caspase-12, ATF6, ATF4 and IERα. These results indicated that NSP4 and NSP4(47-91 aa) induced ERS-mediated apoptosis.This study constructed the eukaryotic expression plasmids of NSP4 and viroporin with Flag tag successfully; the effect of NSP4 and viroporin on cell apoptosis and the relationship between NSP4 and NSP4(47-91 aa) induced ERS and apoptosis were explored initially.Our results suggested that ERS was also one of signaling pathways to trigger apoptosis by NSP4, and viroporin might be the important domain. This study provided a new insight for the pathogenesis of RV-induced diarrhea. |
