Studies On The Molecular Mechanism That PRRSV Infection Activates The Endoplasmic Reticulum Stress

Endoplasmic reticulum is a place for viral protein folding, assembly and maturation, the virus infection often lead to unfolded protein characterized by accumulation of endoplasmic reticulum stress. After the occurrence of stress, cells activate the unfolded protein response (UPR) in order to maintain the steady-state and functions of the endoplasmic reticulum, by inhibiting viral protein synthesis, degradation of virus protein which resides in the endoplasmic reticulum or inducing the expression of folding chaperone, etc., in response to endoplasmic reticulum stress caused by infection.With the purpose of achieving effective replication and infection, virus eliminate the negative effects for proliferation through refined regulation to UPR, and create micro-environment beneficial to its reproduction and proliferation.At the same time, the virus also inhibit expression of other natural anti-viral molecules by regulating UPR interferon (IFN), thereby evading the immune inhibition, and ultimately achieving the purpose of infection.PRRSV is infectious virus characterized by causing pregnant sow's serious reproductive disorders and piglet's respiratory diseases and high mortality, and it has caused tremendous economic losses to pig industry.Although people have conducted extensive research on PRRSV and got a series of significant results in the past 20 years, it is difficult to reveal this "mystery disease", especially about the mechanism of virus infection and immune which is little known.View of important role of UPR in virus infection and immune evasion, molecular mechanisms of endoplasmic reticulum stress response induced by PRRSV infection was approached in this study in order to further reveal the pathogenic mechanism of PRRSV. The main contents are as follows:1. PRRSV infection induced endoplasmic reticulum stress responseObserved by electron microscopy, the endoplasmic reticulum after PRRSV infection in Marc-145 cells were obviously swelling;Marc-145 cells intracellular GRP78 and GRP94 levels at different time after PRRSV infection were detected by SYBR Real-time PCR, discovering that PRRSV infection could up-regulate the level of GRP78 and GRP94 mRNA significantly, and it was time-dependent. This phenomenon illustrated that PRRSV infection could induce endoplasmic reticulum stress response.2. PRRSV infection activated PERK pathwayPhosphorylation of eIF2 which was the direct substrate of PERK was detected by Western blotting, phosphorylating eIF2a could be detected after PRRSV infection at 6h, reached the highest level at 36h, and decreased at 48h, while the total eIF2a protein levels did not change obviously.It illustrated that PRRSV infection activated PERK pathway by phosphorylating eIF2a inhibiting protein translation at early stage.While cells could not overcome the UPR stress after PRRSV persistent infection, the phosphorylating eIF2a induced expression up-regulation of downstream CHOP and GADD34 by activating transcription factors ATF4. CHOP gene mediated endoplasmic reticulum-related apoptosis, and GADD34 gene conducted negative feedback regulation of UPR at the same time.3. PRRSV infection activated IRE1/XBP1 pathwayXBP1 was the only substrate role of IRE1. Observed by RT-PCR and restriction enzyme detection for Marc-145 cells after PRRSV infection, XBP1 expression increased which mostly were the splicing forms XBP1s. Using SYBR Real-time RT-PCR and dual-luciferase reporter gene assay, XBPls target genes such as endoplasmic reticulum-related degrading protein EDEM, UPR negative feedback regulatory protein p58IPK and UPRE element depending on XBP1s identification in Marc-145 cells after PRRSV infection all increased. It illustrated that PRRSV infection activated IRE1/XBP1 pathway.4. PRRSV infection could not activate IRE1/XBP1 pathwayUsing luciferase reporter gene assay on the expression of transcription factors ATF6 at different times Marc-145 cells after PRRSV infection, we found ATF6 was not activated. From further analysis of its target genes, we found ERSE element and some important endoplasmic reticulum chaperones PDI, ERp57, calnexin, calreticulin induced by ATF6 ATF6 did not change significantly in Marc-145 cells after PRRSV infection. It illustrated that PRRSV infection could not activate IRE 1/XBP1 pathway.5. Proteins encoded by PRRSV ORF2a, ORF2b, ORF3, ORF4, ORF5 involved in UPR regulationEukaryotic expression plasmid and luciferase reporter plasmid XBP1-Fluc of PRRSV structural proteins were transfected Hela cells. By detecting luciferase activity for XBP1s, we found proteins encoded by PRRSV ORF2a, ORF2b, ORF3, ORF4, ORF5 could induced expression of XBP1s, indicating that several proteins involved in the regulation of PRRSV-induced UPR. After analysis, these proteins altogether contained glycosylation sites, and glycosylation of proteins could inhibit the activation of endoplasmic reticulum stress. So we speculated that it was easier for the proteins containing glycosylation sites to induce endoplasmic reticulum stress response.6. PRRSV induced apoptosis through CHOP geneCHOP was the key cytokine of the endoplasmic reticulum pathway.The mechanism of apoptosis induced by PRRSV infection was studied further after we confirmed PRRSV infection could induce CHOP gene expression. We found PRRSV infection could up-regulate the expression of apoptosis marker genes caspase3,9, and when the expression of CHOP was inhibited by siRNA, caspase3,9 expression was also significantly decreased, indicating PRRSV infection induced apoptosis through CHOP gene. By detecting PRRSV proliferation in cells, when the CHOP gene was inhibited, we found PRRSV proliferation significantly enhanced, indicating CHOP could promote apoptosis through CHOP to inhibite PRRSV replication and proliferation in host body, which was a spontaneous host protection response.