Molecular Mechanism Of EGCG Inhibiting PRRSV Proliferation

Porcine Reproductive and respiratory syndrome virus(PRRSV)belongs to the family arteritis virus,which is a single-stranded plus STRAND RNA virus with capsule.PRRSV infection leads to abortion in late pregnancy in sows,decreased semen quality in boars,and respiratory diseases and death in piglets.PRRSV is one of the most important pathogens that harm the global pig industry and bring huge economic losses to the global pig industry.Epigallocatechin gallate(EGCG)is a polyphenolic compound in green tea.EGCG is the most active substance in catechin and has antiviral activity against a variety of viruses.In this study,the molecular mechanism of EGCG inhibiting PRRSV proliferation was studied in depth,providing a new strategy for the prevention and control of PRRSV.The main results are as follows:1.EGCG restricts PRRSV replication and assemblyIn this study,the effects of EGCG on cell viability,cell cycle and apoptosis were detected by CCK-8 assay.The results showed that EGCG(0-10 μM)had no significant effect on cell viability,cell cycle progression and apoptosis of porcine alveolar macrophages and MARC-145 cells.The results of virus titer assay showed that EGCG inhibited the replication of both high and low pathogenicity PRRSV in porcine alveolar macrophages and MARC-145 cells.To determine how EGCG inhibits PRRSV replication,the effects of EGCG on PRRSV life cycle were examined.The results showed that EGCG inhibits PRRSV replication and assembly,but does not affect virus attachment,entry and release.2.EGCG inhibits PRRSV-induced lipid droplet formation and lipid synthesisThis study then examined whether EGCG affected pr RSV-induced lipid droplet formation and lipid synthesis.Oil red O and BODIPY staining showed that PRRSV infection significantly increased the number of lipid droplets in porcine alveolar macrophages and MARC-145 cells,while EGCG inhibited this phenomenon.Qrt-pcr and Western blotting showed that EGCG inhibited the mRNA and protein expression of pr RSV-induced genes related to lipid synthesis(SREBF1,FASN,ACACA,ACACB,SCD,SREBF2,HMGCR).PRRSV increased cholesterol, triglycerides,and free fatty acids,while EGCG restored cholesterol,triglycerides,and free fatty acids to control cell levels.These results suggest that EGCG inhibits prRSV-induced lipid droplet formation and lipid synthesis.3.EGCG inhibits PRRSV-induced lipophagyFinally,this study examined whether EGCG affected pr RSV-induced lipophagy.Western blotting and immunofluorescence showed that EGCG inhibited PRRSV-induced LC3-Ⅱ expression,P62 degradation and autophagy flow.After EGCG treatment and exogenous oleic acid supplementation,PRRSV replication and assembly could be restored,and then PRRSV proliferation could be restored.These results suggest that EGCG inhibits PRRSV proliferation by inhibiting intracellular lipid metabolism.This study found that EGCG treatment could make the accumulation of lipid droplets caused by PRRSV disappear.Subsequently,this study clarified the molecular mechanism of EGCG inhibiting PRRSV proliferation through the changes of lipid synthesis and autophagy level after EGCG treatment in infected cells,providing a new idea for clinical virus prevention and control.