The Role Of Lipid Metabolism In Porcine Reproduction And Respiratory Syndrome Virus Infection

Porcine reproductive and respiratory syndrome virus?PRRSV?,is an enveloped,positive-stranded RNA virus,which belongs to order Nidovirale,family Arteriviridae.Typical clinical symptom of PRRS are reproductive failure in pregnant sows,the decreasing boar semen quality and severe respiratory diseases in infected newborn and growing pigs.Since its emergence in the late 1980s,PRRS has been a continuous threat and a huge loss to the global swine industry.Lipids are important components of all kinds of cells and lipid metabolism is an important cellular bioprocess.Lipids and its metabolites are playing an important role in the viral replication cycle since virus is absolutely intracellularly parasitic.Lipids could be the receptors and form a"scaffolding"for viral replication complexes.Also,Some viruses can select cholesterol-enriched membrane domains on the cell surface for budding and secretion.Moreover,virus would target lipid signaling and rearrange lipids to form an optimal environment for their replication and to escape the natural immune response.Previous studies have demonstrated that the intracellular cholesterol levels can influence PRRSV replication.However,it still remains unclear whether PRRSV could alter intracellular lipid metabolism.This study aims to investigate the metabolic changes of cholesterol and fatty acids and its regulatory mechanism caused by PRRSV infection,which could not only clarify the replication mechanism of PRRSV,but also provide a theoretical basis for antiviral therapeutic approaches and prevention of PRRS.1.PRRSV infection upregulates cellular cholesterol levels by activating HMGCRTo observe the changes of intracellular cholesterol level in PRRSV infected cells,the total cholesterol content were determined in PK-15^CD163 cells infected with PRRSV at different times.Results showed a rising cholesterol content in PRRSV infected cells.3-hydroxy 3-methylglutaryl-coenzyme A reductase?HMGCR?is the rate-limiting enzyme in the cholesterol synthesis pathway.In order to investigate the changes of HMGCR activity in PRRSV-infected cells,the level of HMGCR total and HMGCR phosphorylated were further analyzed.Results show that the total HMGCR level was not remarkably changed,but its phosphorylation level was significantly down-regulated?activated state?in a dose-dependent manner,confirming that PRRSV infection can modulate intracellular cholesterol synthetic pathway.PK-15^CD163 cells were pretreated with HMGCR inhibitor Lovastatin prior to PRRSV infection and samples were harvested for indirect immunofluorescence and western-blot assay.Results demonstrates that inhibition of HMGCR activity can restrict viral proliferation and viral protein expression,which is in agreement with the previous reports.2.PPRSV-nsp4 activates HMGCR significantly and PP2A is mainly responsible for this process.In order to further explore which viral proteins are involved in HMGCR activation caused by PRRSV,PK-15^CD163 cells were transfected with plasmids expressing PRRSV-proteins.Western-blot results showed that PRRSV-nsp4 could significantly activate HMGCR in a dose-dependent manner.At the same time,indirect immunofluorescence assay showed that nsp4 and HMGCR protein were co-localized during PRRSV infection.These results indicate that nsp4 plays an important role in the activation of HMGCR.HMGCR activity is regulated by two kinases:AMPK?Adenosine 5'-monophosphate?AMP?-activated protein kinase?and PP2A?Protein phosphatase 2?.PP2A dephosphorylates HMGCR and boosts its activity,whereas activated AMPK decreases HMGCR activity.Due to the lack of PP2A antibodies,PK-15^CD163 cells were infected with PRRSV,followed by incubation of Okadaic acid?PP2A inhibitor?,western-blot assay was performed to detect HMGCR activity.Results showed that PRRSV infection inhibited HMGCR phosphorylation levels,whereas Okadaic Acid treatment lead to a significant increase in HMGCR phosphorylation levels?inactivation?.Based on this,it is speculated that PRRSV infection activates HMGCR mainly via PP2A,which ultimately leads to an increase of cholesterol synthesis.Besides,the phosphorylation level of AMPK was significantly up-regulated after PRRSV infection.Typically,activation of AMPK inhibits HMGCR activity,thereby inhibiting cholesterol synthesis,which is contrary to the results before.In this case,the HMGCR activity increased by PRRSV was not modulated by AMPK,or this trend was counteracted by other pathways.3.PRRSV infection down-regulates cellular free fatty acids through AMPK activation.To investigate the significance of AMPK activation induced by PRRSV,the effect of AMPK activation on PRRSV proliferation was firstly analyzed.Treatment with AMPK activator A769662 would restrict PRRSV infection in a dose-dependent manner,while knockdown of AMPK by siRNA promoted PRRSV replication,suggesting that AMPK acts as a host restriction factor to limit PPRSV infection.Apart from HMGCR,AMPK also regulates the ACC1?acetyl-CoA carboxylase?activity,which is a rate-limiting enzyme for fatty acid synthesis.Western blot analysis confirmed that ACC1 phosphorylation was up-regulated in PRRSV infected PK-15^CD163cells?inactivated form?,and ACC1 phosphorylation level was attenuated due to knockdown of AMPK by siRNA,confirming the regulation of ACC1 activity was mediated by AMPK during PRRSV infection.Furthermore,the effect of free fatty acid level on PRRSV infection was analyzed.PK-15^CD163 cells were pretreated with the fatty acid synthesis inhibitor C75 and then inoculated with PRRSV.Results showed that inhibition of fatty acid synthesis significantly inhibited the proliferation of PRRSV,which was consistent with assay conducted by AMPK activator,demonstrating that AMPK restricts PRRSV infection through the inhibition of ACC1 activity,the first rate-limiting enzyme in fatty acid synthesis.