Mitophagy In Transmissible Gastroenteritis Virus Infection In Intestinal Epithelial Cells

Coronaviruses (CoVs), a genus in the coronaviridae family, are pleomorphic,enveloped viruses, and include alpha-, beta-, and gammacoronavirus, as well as a tentative new genus, deltacoronavirus. Transmissible gastroenteritis virus (TGEV), a coronavirus in the alphacoronavirus genus, causes clinical watery diarrhea, dehydration and vomiting in piglets less than 2 weeks old, and persists in lung or intestine up to 104 days, but does not cause obvious apoptosis in intestinal epithelial cells. Although intestinal epithelial cells are the targets for TGEV, little is known about TGEV infection or the mechanisms underlying intestinal injury, diarrhea, dehydration and long periods carry of virus.Intestinal epithelial cells play an important role in the nutrition absorption and immune response against pathogens. The absorption and barrier functions are heavily dependent on mitochondrial function. There has a little reports between mitochondria and viral infection.Although TGEV infection induce severe enteritis and diarrhea, and also cause cytopathic effect (CPE) in some of cell lines, but does not induce obvious cell apoptosis in intestinal epithelial cells. Mitochondria play an important role in the regulation of innate immunity and metabolism, and it will play an important role in the pathogenesis of the virus. The study of viral infection and mitochondrial danymic could help us to understand the interaction between the virus and host, and to understand the pathogenesis of the virus.Therefore, in order to explore the role and significance of mitochondrial autophagy in TGEV infection, we conducted the following studies:1. The study of mitochondrial injury and mitophagy induced by TGEV infectionTGEV infected IPEC-J2 cells had no obvious cytopathic effect (CPE). In order to explore the reasons, we first observed the ultrastructure of IPEC-J2 cells infected with TGEV by transmission electron microscopy. It was found that the morphology of mitochondria was changed after TGEV infection. We also found mitophagosome-like vesicles was formed after TGEV infection. Next, through Western blot, flow cytometry and confocal microscopy observation, we demonstrated TGEV infection induces mitochondrial damage and mitophagy. We found that, LC3-II/LC3-I was more abundant in TGEV-infected IPEC-J2 cells; BECN1 was also over-expressed in TGEV infected cells. We also found that the tendency of total mitochondrial mass is similar with that of AΨ after TGEV infection and the ratio of dysfunctional mitochondria does not change significantly. However, the expected apoptosis did not occur after TGEV infection. Through confocal microscopy, we found that TGEV infection could induce mTFP-LC3 aggregation, and co-location with mitochondria. To determine whether a complete autophagic response is triggered by TGEV infection, we used western blot analysis to measure the degradation of SQSTM1 (p62).TGEV-infected cells have lower levels of SQSTM1 protein post-infection. We also examined autophagic flux by using fluorescence microscopy with pLVX-mRFP-mWasabi-LC3 and pLVX-mRFP-mWasabi-Bcl-xL lentiviral transfected cells.In TGEV-infected cells, mRFP puncta have accumulated in the cytoplasm at 12 h to 48 h post-infection, whereas mWasabi puncta are weak. Together, these data show that a complete autophagic response is induced in IPEC-J2 cells following TGEV infection.2. The study of TGEV replication and apoptosis by mitophagyThe proliferation of virus is highly dependent on cell status. Mitophagy has obvious effect on cell survival. Therefore mitophagy may affect the replication of TGEV. To explore the role of autophagy in TGEV infection, IPEC-J2 cells were treated with rapamycin or CCCP, and then infected with TGEV. The expression of N was monitored as a proxy for viral infection. An increase in N expression was found in both rapamycin- and CCCP-treated cells. While, 3-MA treatment reduced the expression of LC3-Ⅱ and N at 48 h post-infection and decreased virus titer. To examine the relationship between autophagy and virus infection further, shRNA knockdown experiments were performed to specifically deplete endogenous ATG5 protein. The result indicated suppression of ATG5 expression reduced the expression of N and the viral progeny yield in TGEV infected IPEC-J2 cells compared with the control shRNA transfection. The apoptosis of IPEC-J2 cells treated with CCCP was similar to that observed in untreated cells. In contrast, 3-MA treatment and ATG5 knockdown both increased apoptosis in infected cells. Together, these experiments demonstrate that mitophagy promote the infection of TGEV partly by attenuating cell apoptosis.3. The study of oxidative stress in mitophagy induced by TGEV infectionMitochondria is the main organelle produced ROS. To explore the relationship between ROS and mitophagy induced by TGEV, we detect the ROS and mtROS level after TGEV infection. We also detected the expression of some antioxidant genes expression after TGEV infection. The results indicated a moderate oxidative stress was induced after 12 h TGEV infection. Since oxidative stress plays important roles in mitophagy, we then used GSH to suppress oxidative stress, and detected the change of autophagy and mitochondrial injury. The results indicated GSH could suppress LC3-Ⅰ convert to LC3-Ⅱ,and inhibit mitochondrial degradation. Then, we knockdown DJ-1 expression and detected the viral replication, conversion of LC3-Ⅰ to LC3-Ⅱ, apoptosis and abnormal mitochondrial ratio after TGEV infection. The results indicated DJ-1 knockdown suppressed TGEV replication, induced more severs mitochondrial injury and inhibited autophagy. These results suggest that DJ-1 play an important role in suppression oxidative stress and induction mitophagy during TGEV infection.4. The study of the role of TGEV nucleocapsid protein in mitophagyIn order to further explore the causes of mitochondrial abnormalities and mitophagy induced by TGEV, we observed the localization of some viral proteins in cells by confocal microscopy. We found the nucleocapsid protein could located on mitochondria. Through flow cytometry analysis, we found N protein could induce ROS and mtROS increase, and the number of mitochondria decrease, but no affect Ay and apoptosis. During TGEV infection, we also observed the co-localization of N protein and mitochondria. To confirm the location of autophagic proteins and N, mitochondrial and cytoplasmic proteins were separated and subjected to western blot analysis. Result displayed that protein N is highly enriched in the mitochondrial fraction. Together, these data indicate that the nucleocapsid protein of TGEV may contribute to mitochondrial dysfunction and induces mitophagy in IPEC-J2 cells.In conclusion, this study provides evidence for the likely involvement of TGEV-induced mitophagy in facilitating the infection of infection and the pathogenesis of gastrointestinal disease associated with infection in vitro. A closer look at the relationship between the mitochondrial location of nucleocapsid protein and the elimination of dysfunction mitochondria may provide new strategies for the control of viral infection.