PRRSV Infection Regulates Host Cell Central Carbon Metabolism And The Screening Of Drug Targets

Given the relatively simple structure and the lack of a complete enzyme system required for viral proliferation,viruses are completely dependent on the metabolic network of the host cell to provide energy and biomolecules.Therefore,it is important to study the metabolic remodelling of host cells after virus infection to unravel virus infection mechanisms and drug targets.Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)is one of the most important pathogens affecting the global pig industry.The highly variable nature of PRRSV genome has made it difficult for existing vaccines to provide effective immune protection and there is an urgent need to develop alternative control technologies and products.Central carbon metabolism(CCM),which includes glycolysis,the tricarboxylic acid(TCA)cycle and the pentose phosphate pathway,is the main source of energy required by the organism and provides metabolite precursors for other metabolic pathways.Previous studies have found that many viruses modulate the CCM to promote viral proliferation.Given the important physiological functions of CCM in cells and its role in viral infections,making it an important antiviral target.However,it is currently unknown how PRRSV infection regulates the CCM.This study focuses on the interaction between PRRSV and CCM,with a view to screening for promising anti-PRRSV drug targets with clinical applications.The main study components and results are as follows:1.PRRSV hijacks host cell CCM to promote viral proliferationGlucose and glutamine are the main carbon sources for cells,providing energy and carbon skeleton for life activities.In this study,we found that PRRSV infection increased cellular uptake of glucose and glutamine;meanwhile,deprivation of glucose and/or glutamine significantly inhibited PRRSV proliferation,suggesting that PRRSV infection promotes glucose and glutamine uptake to optimise viral proliferation.Glucose and glutamine are mainly catabolised through the CCM pathway,and CCM inhibitor assays showed that restricting glucose and glutamine entry into the CCM significantly limited PRRSV proliferation,suggesting that unobstructed CCM facilitates PRRSV proliferation.Further studies showed that PRRSV infection activates glycolysis(increases glucose uptake and lactate secretion,upregulates lactate dehydrogenase activity)and maintains TCA cycle flux(maintains acetyl coenzyme A abundance);also,inhibitors of either glycolysis or the TCA cycle decreased PRRSV proliferation,suggesting that glycolysis and the TCA cycle are metabolic pathways required for optimal PRRSV proliferation.In addition,the addition of an exogenous TCA cycle intermediate metabolite,α-ketoglutarate(α-KG),partially restored PRRSV proliferation inhibited by glutamine deficiency.These findings illustrate that PRRSV infection drives cells to take up more glucose and glutamine into the glycolysis and TCA cycle to increase viral proliferation,suggesting that glycolysis and the TCA cycle are potential drug targets for the control of PRRSV infection.2.PRRSV nsp1β promotes glycolysis by stabilizing HIF-1α expressionGiven that PRRSV infection activates glycolysis to promote viral proliferation.To explore potential anti-PRRSV drug targets in the glycolytic pathway,how PRRSV activates the glycolytic pathway was further analyzed.By re-analyzing the transcriptomic data of PRRSV-infected IPAM cells(immortalized porcine alveolar macrophages),which had been conducted in our laboratory,we found that several metabolic pathways were significantly enriched after PRRSV infection,including the glycolytic pathway and the hypoxia-inducible factor 1α(HIF-1α)pathway.HIF-1α is an important transcription factor regulating glycolysis,but its involvement in PRRSV-induced glycolysis is still not known.The transcriptomic data was first validated and found that PRRSV infection upregulated HIF-1α expression.Overexpression of HIF-1α up-regulated PRRSV-induced glycolytic flux;interference with HIF-1α down-regulated PRRSV-induced glycolytic flux,suggesting that HIF-1α is involved in PRRSV-induced glycolysis.Subsequently,screening of PRRSV-encoded proteins involved in the regulation of HIF-1α expression revealed that PRRSV non-structural protein 1β(nsp1β)significantly increased HIF-1αexpression.Nsp1β promoted HIF-1α transcription through its N-terminal enzymatic activity and inhibited the ubiquitinated degradation of HIF-1α through its C-terminal enzymatic activity.Further studies revealed that nsp1β interacted among HIF-1α and the von Hippel-Lindau tumour suppressor protein(p VHL)and forms a ternary complex.Interestingly,nsp1β stabilized own expression by hijacking the E3 ubiquitin ligase activity of p VHL,meanwhile,inhibited the ubiquitinated degradation of HIF-1α by p VHL.In addition,nsp1β promoted lactate dehydrogenase activity,suggesting that nsp1βupregulates glycolytic flux.The above findings suggest that nsp1β activates glycolysis by stabilizing HIF-1α expression,suggesting that HIF-1α is a potential drug target for controlling PRRSV infection.3.Glycolytic end product lactate is utilized by PRRSV to promote viral proliferationBecause PRRSV infection produces large amounts of lactate,recent studies have found that lactate is not a "metabolic waste" but a substrate for histone lactylation that are involvement in the regulation of various biological processes,including interferon(IFN)response,inflammation,and tumour formation.However,the role of elevated lactate in PRRSV infections remains unknown.Here,we found that lactate was required for optimal PRRSV proliferation,and PRRSV infection increased histone lactylation in a dose-dependent manner.By using the Cleavage Under Targets and Tagmentation(CUT&Tag)combined with RNA sequencing(RNA-seq)to screen the downstream genes regulated by lactylation in PRRSV-infected cells,we found that PRRSV-induced lactylation activated the expression of heat shock 70 k Da protein 6(HSPA6).Follow-up experiments showed that HSPA6 is important for PRRSV proliferation by negatively modulating interferon(IFN)-β induction.Mechanistically,HSPA6 impeded the interaction between TNF-receptor-associated factor 3(TRAF3)and inhibitor of nuclear factor kappa-B kinase subunit epsilon(IKKε),thereby hindering the production of IFN-β.These results suggest that activated lactate-lactylation-HSPA6 axis promotes viral growth by impairing IFN-β induction,providing new therapeutic targets for the prevention and control of PRRSV infection.4.TCA cycle metabolite itaconate significantly inhibits PRRSV proliferationPrevious findings have confirmed that PRRSV infection maintains TCA cycle fluxe to optimize viral proliferation.To explore potential anti-PRRSV metabolites in the TCA cycle,non-targeted metabolomic data from PRRSV-infected IPAM cells already performed in our laborotory were re-analysed and revealed that in terms of the TCA cycle,PRRSV infection only up-regulated the expression of the TCA cycle main-chain metabolite cis-aconitine.However,exogenous addition of cis-aconitate did not affect PRRSV proliferation.Further analysis of the reason for PRRSV infection increasing the abundance of cis-aconitate but not affecting viral proliferation revealed that PRRSV infection downregulated the expression of the cis-aconitate metabolizing enzyme immune response gene 1(IRG1)and the abundance of the IRG1 metabolite itaconate,suggesting that PRRSV infection reduced cis-aconitate consumpution through downregulation of IRG1 expression.The effect of itaconate on PRRSV proliferation was further analysed using the itaconate derivative 4-octyl itaconate(4-OI),and it was found that 4-OI treatment significantly inhibited PRRSV proliferation,mainly by suppressing the adsorption,replication and release stages of viral life cycle.It was also found that 4-OI attenuated PRRSV-induced inflammatory factors expression by upregulating the expression of nuclear factor 2-related factor 2(Nrf2)inhibited by PRRSV.Since 4-OI inhibited both PRRSV proliferation and PRRSV-induced inflammatory factors expression,suggesting that 4-OI may be a promising anti-PRRSV drug.In summary,by analyzing the effects of PRRSV infection on CCM,this study found that PRRSV infection activated cellular glycolysis and maintained TCA cycle flux,both of which were metabolic pathways required for optimal PRRSV proliferation,suggesting that glycolysis and TCA cycle could be potential drug targets for controlling PRRSV infection;it was demonstrated that PRRSV promoted glycolytic flux by stabilizing HIF-1α expression,suggesting that HIF-1α may be a drug target for the control of PRRSV infection;lactate,a glycolytic end product,was found to optimize PRRSV proliferation,indicating that lactate inhibitors are potential PRRSV antagonists;it was determined that itaconate,a metabolite of TCA cycle,significantly inhibited PRRSV proliferation and PRRSV-induced inflammatory factors expression,suggesting that it may be a candidate for clinical control of PRRSV infection.