The Mechanisms Study Of Cellular Autophagy And ER Stress Induced By PRRSV

Autophagy is a lysosomal-dependent degradation pathway which plays an important role in the maintenance of intracellular homeostasis. Cellular autophagy function to limit viral replication and pathogenesis. However, some viruses can counteract and/or subvert the host autophagy machinery in order to enhance their own growth or survival. In addition, autophagy plays specific roles in immune.The endoplasmic reticulum is an important place for protein synthesis, assembly and maturation. Virus infection often leads to a large number of unfolded or misfolded protein accumulation and leading ER stress response (unfolded protein response UPR). The UPR also regulates the replication of the virus. Moreover, the UPR can further activate the cellular autophagy.Porcine reproductive respiratory syndrome (PRRS) characterized by severe reproductive failure in sows, respiratory distress high mortality in piglets and growing pigs often leads a higher mortality rate. There have been many researches about PRRSV, but little was known about themechanism of immunosuppression during PRRSV infection.Hence, in the study we investigated whether the cellular autophagy and ER stress response were activated during PRRSV infection. The main contents include:1. PRRSV infection induced the formation of autophagyTo determine whether PRRSV infection triggers cellular autophagy, we first examined the expression of LC3-II. The results showed that PRRSV infection induce the conversion of LC3-I to LC3-II, suggested that PRRSV infection may induce cellular autophagy. Next, laser confocal microscopy was performed to investigated the punctate accumulation of EGFP-LC3, the results showed that cells with both Nsp2-positive and EGFP-positive showed punctate accumulation of LC3, this further indicated that PRRSV infection induce the cellular autophagy. Lastly, the cells ultrastructure was observed by TEM during PRRSV infection, directly demonstrate autophayg was induced during PRRSV infection.2. PRRSV infection facilitates the degradation of the autophagosomeTwo pathways can lead to the accumulation of autophagosome, induced the formation or inhibited the degradation of autophagosome. To investigated the mechanism of autophagosome accumulation during PRRSV infection. degradation pathway. First, lysosome was stained by Lyso-Tracker and the distribution of lysosome and EGFP-LC3were observed. We founded the clearly co-localization of lysosome and EGFP-LC3in PRRSV-infected cells. Furthermore, the turnover of LC3and the expression of p62were detected by Western Blot. Taken together, the results suggested that PRRSV infection facilitate the degradation of the autophagosome.3. Induction of autophagy enhances PRRSV replicationCells treated by3-Methyladenine (3-MA) or rapamycin (Rapa) to inhibit/induce the cellular autophagy, then, plaque assay was performed to analysis the effects of PRRSV replication. The results suggested that induction of autophagy enhance the PRRSV replication. Another, we performed RNA interference experiments to knockdown the expression of two critical genes of the autophagy pathways:ATG7and Beclin-1. We founded the siRNA can inhibit the autophagy induced by PRRSV and leading to the repression of PRRSV replication.4. PRRSV encodes protein which involved in the induction of autophagyTo screens the protein which participate in the PRRSV-induced autophagy, we detected whether the UV-inactivated virus can activate the autophagy. We founded that the PRRSV inactivated by UV cannot induce the autophagy. We concluded that PRRSV encoded non-structural proteins play a major role in autophagy induction. Furthermore, overexpression Nsps of PRRSV demonstrated that several PRRSV-encoded proteins were involved in the autophagy induction.5. Knockdown of autophagy enhances the innate immune response in PRRSV-infected MARC-145cellsPRRS is a strong immunosuppressive diseases. It has been demonstrated that PRRSV infection inhibits the expression of IFN which induced by poly(I:C). Several researches reported that activate the autophagy result of decreased IFN production. Hence, the luc reporter was used to investigate whether PRRSV-induced cellular autophagy was involved in the immunosuppressive. The result indicated that PRRSV-induced autophagy impairs innate immune response, inhibit of autophagy enhances the innate immune response in PRRSV-infected cells.6. PRRSV infection elicits ER dilation and over expression of the ER chaperone, GRP79and GRP94To observed cellular changes that may accompany response to PRRSV infection, ultrastructure analysis using electron microscopy was performed. As shown in the result, compared to the mock infection, the most prominent change observed is the expansion of the ER in PRRSV infection cells. The result suggested that the ER stress would be induced during PRRSV infection. Then, glucose-regulated protein78and94(GRP78and GRP94) expression were both enhanced at various time-points after PPRSV infection Taken together, the results indicate that PRRSV infection induce ER stress.7. PRRSV infection activates the PERK/eIF2a/ATF4signaling pathwaywe tested the expression of eIF2a which can be phosphorylated by active PERK, and found that the expression of phosphorylated form of eIF2a was enhanced at12h post-infection and remained elevated throughout the infection time course.In addition, qRT-PCR were performed to measure the levels ofActivating transcription factor4(ATF4) and GADD34mRNA which can be induced by eIF2a. The results suggested that, there were a great increase in the level of ATF4and GADD34mRNA after PRRSV infection or treated with TG. Taken together, the results implied that PRRSV infection can activate the PERK signal pathway and may resulting in a translational attenuation.8. PRRSV infection activates the IRE1/Xbpl branch of UPRWe tested the splicing of the Xbpl mRNA by RT-PCR using primers which overlapping the26-bp intron. The results showed that, the448-bp fragment amplified from Xbp1s mRNA was detected starting at24h after PRRSV infection, and the level of this mRNA continued to increase through48hpi. Dual-luciferase reporter assay was performed to evaluate the level of Xbpl splicing and the similar results were found.This further demonstrated that PRRSV infection induce the splicing of Xbpl mRNA. In addition, we found both ERSE and UPRE response elements were activated significantly during PRRSV infection, and the ER degradation-enhancing a-mannosidase-like protein (EDEM), which transcription is solely dependent on Xbp was induced. These results indicated that PRRSV infection activate the IRE1pathway leading to the splicing of Xbpl RNA and resulting in activation of downstream target genes.9. PRRSV infection cannot activate the ATF6pathwayIn response to ER stress, the90-kDa precursor of ATF6was cleaved into a50-kDa protein which functions as a transcription factor. To examine the effects of PRRSV on the ATF6pathway, Western Blot was performed to assess the status of ATF6,both90-or50-kDa ATF6protein were detected in MARC-145cells which treated with TG for4h. However, corresponding to the mock-infection cells, the50-kDa cleavage products was undetectable at any time during PRRSV infection. In addition, the expression of calnexin, calreticulin, ERp57and PDI were no obvious change during PRRSV infection. These results demonstrated that PRRSV infection induce the ER stress but cannot activate the ATF6pathway.10. ER stress modulates the PRRSV replicationPlaque assay were performed to determine the effect of4-PBA on PRRSV replication, the dates showed that4-PBA reduced PRRSV growth significantly at micromolar concentrations. To extend these studies, we performed RNA interference (RNAi) experiments to knockdown the PERK and IRE1which can be activated during PRRSV infection. The result showed that knockdown of PERK or IRE1significantly decrease PRRSV yield. Based on these results, we concluded that the UPR induced by PRRSV plays a positive role to its replication.11. PRRSV infection induces the CHOP expression leading ER stress-mediated apoptosisTranscription factor CHOP/GADD153can be activated in cells suffering from ER stress, and CHOP activation appears to contribute to subsequent cell growth arrest and apoptosis. To investigate the activation of CHOP during PRRSV infection, MARC-145cells were co-transfeced with CHOP-luc and pRL-TK. The results suggest that, he CHOP promoter was activated during PRRSV infection. Besides, to demonstrate induction of CHOP at mRNA level, we performed qRT-PCR. The results showed that treated by TG induce the transcription of CHOP, and CHOP mRNA induced by PRRSV was observed at24h and sustained to48h. The results indicated that PRRSV infection activate the expression of CHOP. Caspase3is the effector caspase activated by proteolytic cleavage and ER stress-mediated apoptotic signals ultimately converge on it. Here, we demonstrate that, caspase3was activated during PRRSV infection. In addition, this activation can be blocked by siRNA of CHOP,Taken together, our results indicated that the persistent activation of UPR pathways by PRRSV leading to the induction of apoptosis.12. Induction of complete autophagic response by PRRSV via the unfolded protein responseUPR, the major ER stress pathway, is a potent stimulus of autophagy. We have demonstrated that PRRSV could induce ER stress to activate PERK and IRE1arms of the UPR. To test whether PRRSV indeed induced the accumulation of autophagy via the induction of ER stress, we treated cells with siRNA and4-PBA, we then examined whether the suppression of UPR would affect the lipidation of LC3by PRRSV. All the results indicated that ER stress was important for the lipidation of LC3induced by PRRSV.