Interaction Between Japanese Encephalitis Virus Replication And Cellular Autophagy

Autophagy is a eukaryotic cellular degradation pathway that involves the delivery of cytoplasmic cargo to the lysosome. Autophagy is essential for intracellular homeostasis in eukaryocytes and it participates in innate immunity. It has been shown that autophagy plays an important role in stress response, including fighting against pathogens. On the other hand, some microbes have also evolved mechanisms to evade host autophagy and in some cases microbes can even exploit host autophagy to benefit themselves. Some viruses can inhibit host autophagy by their self-encoded proteins. Certain plus-stranded RNA viruses, including dengue virus, which up-regulate autophagy activity of host cells, are able to use autophagy-related membrane structures as viral replication sites. Though the modulation of autophagy on virus infection is of great importance, little is known about the regulatory role of autophagy in Japanese encephalitis virus (JEV) replication. It is a fascinating question that whether JEV infection induces or inhibits autophagy and whether autophagy facilitates or suppresses JEV replication. The answer to this question will enrich the content of JEV and host interactions, help to understand the pathogenic mechanism of JEV infection, and provide new strategies for therapeutics to viral diseases.In this study, to discuss interactions between autophagy and JEV infection, autophagy of hepatoma cells infected with JEV was monitored by western blotting and transmission electron microscope. Furthermore, autophagy of JEV-infected cells was pharmacologically or genetically induced/ inhibited, then viral replication under those conditions was assessed.1. JEV infection induces host autophagyIn order to investigate the effect of JEV infection on autophagy, we observed autophagosome formation in JEV-infected cells by transmission electron microscope. Compared with mock cells, in which autophagic vacuoles were rarely seen, accrual of 0.5-1μm double-membrane vacuoles in both rapamycin-treated and virus-infected cells were observed. The amount of LC3-II or the ratio of LC3-II/ LC3-I had been used to represent the level of autophagy, thus we investigated the expression profile of LC3 protein upon JEV infection. It was shown that the ratio of LC3-II/ LC3-I evidently increased in both rapamycin-treated and JEV-infected cells compared to mock-treated cells. To further validate these results, BHK-21 and HepG2 cells were transfected with a plasmid coding GFP-LC3 fusion protein (pEGFPC1-LC3), an autophagy marker, followed by JEV infection. GFP-LC3 protein was able to aggregate to form puncta in autophagy-induced cells and it was found that more punctas appeared in virus-infected cells than in mock cells. These findings indicate that JEV infection induces autophagy of host cells.2. Host autophagy suppresses JEV replicationTo study the regulatory role of autophagy in JEV replication, the conditions of pharmacological induction/ inhibition of autophagy were optimized firstly. It is demonstrated that autophagy in HepG2 was activated efficiently by rapamycin incubation at 0.1μM for 6 hours, and was significantly suppressed by wortmannin (0.5μM) treatment for 1 hour. Then autophagy of HepG2 cells was induced or inhibited pharmacologically under those above conditions, followed by JEV infection. The yields of virus in culture supernatants were determined by plaque assay. When cells were treated with autophagy inhibitor, virus titers of wild and vaccine strain were increased by about 50% and 2 times, respectively. The titer of wild strain was dosage dependent in certain extent.. These results indicated that autophagy in virus-infected cells could suppress virus replication. However, induction of autophagy did not affect the yield of wild strain, but moderately raised the vaccine strain output in supernatants. This might due to immunosuppression effect of rapamycin.3. Autophagy-related genes involve in suppression of JEV replicationTo further discuss the suppression mechanism of JEV replication by autophagy, we analyzed some of ATGs involved in suppression of JEV replication. LC3 is an essential factor in autophagosome formation, and Beclin1's regulation on autophagy is also critical. LC3 or Beclin1 gene was knocked down using RNA interference followed by JEV infection, and the yield of virus was assessed by plaque assay. Compared to mock cells, the amount of LC3 and Beclin1 proteins was significantly decreased in HepG2 cells transfected with corresponding siRNA. Silencing of LC3 gene markedly boosted the replication of both two strains, while viral replication had no significant difference between mock and Beclin1-silenced cells. These results illustrated expression of LC3 was essential for host autophagy to restrain JEV replication and Beclin1 was not required in this process, it was also indicated that not all ATGs took effect in autophagy-dependent inhibition of JEV replication. To conclude, in this study we showed that JEV infection could activate host autophagy and caused autophagosome formation, which decreased JEV replication in a LC3 dependent manner. These results will contribute to further investigation of JEV-host interactions and will be helpful for the new strategies for the control and treatment of JEV infection.