Study Of Effects And Mechanisms Of Pig PRDX5 In The Regulation Of AMPK Signaling Pathway During Porcine Reproductive And Respiratory Syndrome Virus Infection

Lipid,as a key cellular component and a form of energy storage,possesses important physiological functions.Viruses are obligate intracellular parasitic microorganisms that depend on the host for materials and energy,thus lipid plays an important role in the proliferation of viruses(especially enveloped viruses).Porcine reproductive and respiratory syndrome virus(PRRSV)is a positive-strand RNA virus,which causes reproductive disorders of sows and respiratory diseases of piglets,causing huge economic losses to the pig industry.It has been reported that lipids(such as fatty acid and cholesterol)are required for the optimal infection of PRRSV.The interaction between PRRSV and cholesterol has been comprehensively investigated by our lab previously,while there remains a paucity of studies investigating the interaction between PRRSV and fatty acid,especially,the specific mechanisms and biological implications underlying the regulation of fatty acid metabolism by host during PRRSV infection remains unclear.Recently,the involvement of AMPK in the regulation of fatty acid synthesis by PRDX5 has been demonstrated,and the effect of fatty acid on PRRSV infection has also been reported.Based on these,the molecular mechanism regulating AMPK signaling pathway by PRDX5 during PRRSV infection and the effect of PRDX5 on viral proliferation were investigated in this study.The results are as detailed below:1.PRRSV and PRRSV nsp4 upregulate the expression of PRDX5To explore the effect of RRRSV infection on PRDX5,the i PAMs,immortalized porcine alveolar macrophages,were infected with PRRSV,and then the protein expression level of PRDX5 was detected by Western blot assay.The results showed that the abundance of PRDX5 protein was upregulated by PRRSV.In addition,UV-inactivated PRRSV had no significant effect on the expression of PRDX5,suggesting that PRRSV nonstructural proteins possess the ability to upregulate PRDX5 expression.Then,through screening all nonstructural proteins of PRRSV,nsp4,the main protease of PRRSV,was demonstrated as the predominant viral protein inducing the upregulation of PRDX5 expression in an enzyme activity-independent manner.2.PRRSV and nsp4 inhibit fatty acid synthesis depending on the PRDX5-AMPK-ACC1 signaling pathwayPRDX5 regulates fatty acid synthesis through AMPK-ACC1 signaling pathway,and PRRSV infection influences AMPK-ACC1 signaling pathway,suggesting that PRRSV may regulate AMPK-ACC1 signaling pathway through PRDX5,thereby affecting fatty acid metabolism.To test this hypothesis,we analyzed the effects of overexpression or knockdown of PRDX5 on AMPK and ACC1 phosphorylation levels under PRRSV infection.The results showed that overexpression of PRDX5 upregulated PRRSV-induced phosphorylation levels of AMPK and ACC1 in an enzyme activity-independent manner.Knockdown of PRDX5 downregulated PRRSV-induced phosphorylation levels of AMPK and ACC1,suggesting that PRDX5 was involved in PRRSV regulation of AMPK-ACC1 signaling pathway.Furthermore,nsp4 was demonstrated to regulate AMPK-ACC1 signaling pathway through PRDX5 as well.Since AMPK-ACC1 is a key signaling pathway regulating fatty acid synthesis,we also examined the role of PRDX5 in PRRSV or nsp4 regulating fatty acid metabolism,and the results showed that knockdown of PRDX5 upregulated the contents of triglycerides and lipid droplets in the cells infected with PRRSV or transfected with nsp4,namely enhancing fatty acid synthesis.These results indicate that both PRRSV and nsp4 inhibit fatty acid synthesis through PRDX5-AMPK-ACC1 signaling pathway.3.PRRSV and nsp4 enhance the expression of PRDX5 by upregulating the production of ROSAs an antioxidant protein,PRDX5 is capable to respond to the changes of intracellular ROS content.The level of PRDX5 is upregulated with the increase of ROS content.To detect the potential role of ROS in PRRSV and nsp4 inducing PRDX5 expression,the effects of PRRSV and nsp4 on ROS were firstly analyzed.The i PAMs were infected with PRRSV or transfected with the nsp4-expressing eukaryotic expression plasmid.The results showed that both PRRSV and nsp4 upregulated the levels of ROS.Then,the protein expression level of PRDX5 was detected when the PRRSV or nsp4-induced ROS was inhibited by antioxidant NAC.The results of Western blot assay showed that NAC decreased the protein expression levels of PRDX5 induced by PRRSV and nsp4.Taken together,both PRRSV and nsp4 can induce the protein expression of PRDX5 by upregulating ROS content.4.PRDX5 antagonizes PRRSV proliferation by inhibiting fatty acid synthesisPrevious studies have demonstrated that fatty acids faciliate PRRSV infection.The above results showed that PRDX5 inhibited the synthesis of fatty acids,suggesting that PRDX5 might antagonize the proliferation of PRRSV by inhibiting fatty acid synthesis.To verify this conjecture,we first detected the effect of PRDX5 on PRRSV proliferation.The results showed that overexpression of PRDX5 inhibited PRRSV proliferation,while knockdown of PRDX5 promoted PRRSV proliferation,namely PRDX5 possessed anti-PRRSV activity.Moreover,addition of exogenous oleic acid,a kind of fatty acid,restored the inhibitory effect of PRDX5 on PRRSV proliferation.Altogether,fatty acids are involved in the inhibitory effect of PRDX5 on PRRSV proliferation.5.PRDX5 inhibits the release process in the life cycle of PRRSVTo explore the specific step(s)targeted by PRDX5 to inhibit proliferation of PRRSV,we detected the effects of PRDX5 overexpression on the adsorption,invasion and replication processes of PRRSV,and found that PRDX5 exhibited no effect on these three processes of PRRSV propagation cycle.On this basis,we further detected the effect of PRDX5 overexpression on the virus content in the supernatant.The results showed that PRDX5 decreased the content of PRRSV in supernatant.Therefore,we speculated that PRDX5 might inhibit the release process of PRRSV infection.