The Mechanism Of HMGB1 In Porcine Epidemic Diarrhea Virus Infection

Porcine epidemic diarrhea (PED) is an acute,highly contagious viral enteric disease of swine caused by porcine epidemic diarrhea virus (PEDV),a severe PED characterized by high mortality rates. PEDV affects pigs of all ages in China, resulting in enormous economic losses. Although many efforts have been made to explore the pathogenesis of PEDV, the factors that affect PEDV infection in host species/cells are still poorly understood. We explore the role of HMGB1 in PEDV infection. The main contents of this thesis are the following:1. Glycyrrhizin as antiviral agent against PEDVGlycyrrhizin (GLY),a natural product isolated from licorice root,has been used as the traditional Chinese medicinal herb for many years. In our studies, we found GLY had antiviral activity against PEDV infection as suggested by western blot analysis, qRT-PCR and plaque formation assay. In addition, we explored which step of the infection process was affected. We revealed that GLY had no affect on cells and PEDV particle. GLY did not inhibit assembly or release of PEDV. GLY inhibited the entry and replication of PEDV.2. HMGB1 has an important role on the antiviral effect of glycyrrhizinHigh-mobility group box 1 (HMGB1) is an abundant nuclear protein, which is released into extracellular space to serve as an important inflammatory mediator. We explored the antiviral mechanism of GLY against PEDV. We found that GLY reduced the mRNA levels of proinflammatory cytokines (1L-1β, IL-6, IL-8, TNF-α) during PEDV infection. By HMGB1 antibody and siHMGB1 treatment, we elucidated that HMGB1 had an important role on the replication of PEDV and the mRNA levels of proinflammatory cytokines.HMGB1 was contributed to the replication of PEDV and increase of proinflammatory cytokines via TLR4 and RAGE. HMGB1 mutant plasmids inhibited the replication of PEDV and the increase of the proinflammatory cytokines during the infection. Furthermore,we revealed that HMGB1 activated the P38 MAPK through TLR4 or RAGE to increase the replication of PEDV and the proinflammatory cytokines. Our research provided insights to the development of new therapeutic drugs of PEDV.3. PEDV uses cell surface heparan sulfate as attachment factorIt is well known that many viruses use heparin sulfate as the initial attachment factor.In the present study, we determined whether porcine epidemic diarrhea virus (PEDV), an emerging veterinary virus, infects Vero cells by attaching to heparan sulfate. Western blot analysis, real-time PCR, and plaque formation assay revealed that PEDV infection was inhibited when the virus was pretreated with heparin (an analogue of heparan sulfate).There was no inhibitory effect when the cells were pre-incubated with heparin. We next demonstrated that enzymatic removal of the highly sulfated domain of heparan sulfate by heparinase I treatment inhibited PEDV infection. We also confirmed that sodium chlorate,which interfered with heparan sulfate biosynthesis, also inhibited PEDV infection.Furthermore, we examined the effect of two heparin derivatives with different types of sulfation on PEDV infection. The data suggested de-N-sulfated heparin, but not N-acetyl-de-O-sulfated heparin, inhibited PEDV infection. In addition, heparin inhibited the replication of PEDV and the mRNA of HMGB1. In summary, our studies revealed that heparan sulfate acted as the attachment factor of PEDV in Vero cells.4. PEDV nucleoprotein contributes HMGB1 transcription, acetylation and release by interaction with C/EBP-βWe showed that PEDV infection resulted in the acetylation and the release of High mobility group box 1 (HMGB1) in Vero cells, which is an important proinflammatory mediator and contributes to the pathogenesis of various inflammatory diseases. PEDV infection and PEDV nucleoprotein regulated the acetylation of HMGB1 through SIRT1,histone acetyltransferase or NF-κB. Overexpression of full length PEDV nucleoprotein promoted HMGB1 acetylation and release. Our CHIP experiment suggested that HMGB1 promotor DNA could be immunoprecipitated by PEDV-N. Dual-luciferase reporter gene assay showed that PEDV-N mediated HMGB1 transcription was associated with transcription factor C/EBP binding motif in HMGB1 promotor region. Our co-immunoprecipitation experiment further suggested that viral nucleoprotein interacted with C/EBP-β to promote the up-regulation of HMGB1 gene expression. Collectively,our data demonstrated that PEDV infection caused the release and acetylation of HMGB1,PEDV-N is actively involved in this process. Our results provided new insights on the pathogenesis of PEDV infection and a molecular basis to develop therapeutic methods and drugs targeted HMGB1.5. PEDV induces the release of HMGB1 through DNA damageDNA damage response activated by DNA viruses has been extensively studied.However, Few studies on DNA damage response caused by RNA viruses have been reported. Thus, we explored DNA damage response associated with PEDV infection. In our study, we revealed that the phosphorylation of H2AX protein at serine 139 (yH2AX) was emerged rapidly in PEDV infection through western blot and immunofluorescence. An inducer of DNA damage, HY, increased the expression of PEDV N protein. Furthermore,DNA-PK inhibitor (NU7441) and ATM kinase inhibitor (KU-55933) decreased the release of HMGB1 and PEDV infection, especially DNA -PK inhibitor, whereas ATR inhibitor(VE-821) has no effect on PEDV. In addition, PEDV increased the activity of PARP1. The inhibitor of PARP1, 3-AB, inhibited the release of HMGB1 and decreased PEDV N protein expression or the virus titer. DNA-PK inhibitor (NU7441) and ATM kinase inhibitor(KU-55933) decreased the activity of PARP1, especially NU7441. These results demonstrated that PEDV caused the DNA damage mainly depending on DNA-PK to elevate the activity of PARP1 for the PEDV proliferation, partly depending on ATM. In conclusion, PEDV induced the release of HMGB1 to favor itself replication through DNA damage/PARP 1 /ROS.