Untargeted Metabolomics Analysis Of IPAM Cells Infected With Porcine Reproductive And Respiratory Syndrome Virus

Viruses are specialized intracellular parasitic organisms that rely exclusively on cellular metabolic substances and energy to synthesize the components of viruses.There are both commonalities and differences in the regulation of host cell metabolic reprogramming by different viruses.Porcine reproductive and respiratory syndrome virus(PRRSV),a single-stranded positive-stranded RNA virus with capsule,has caused enormous economic losses to the world pig industry.Currently,few studies have been reported on the interactions between PRRSV and host cell metabolism.In this thesis,we conducted a non-targeted metabolomic study of PRRSV-infected IPAM cells(immortalized porcine alveolar macrophages),and resolved the inter-regulation of glucose metabolism,amino acid metabolism,nucleotide metabolism and lipid metabolism with PRRSV to provide theoretical references for exploring the drug targets of PRRSV and elucidating the pathogenic mechanism of PRRSV.The main research contents are as follows:1.Preparation of non-targeted metabolomics samples and preliminary analysis of histological dataBased on the results of multi-step growth curves of PRRSV(WUH3 strain)infected IPAM cells obtained by in our laboratory in the previous periodly,we examined the infection of PRRSV(0.5 MOI)in IPAM cells at different time points(6 h,12 h and 24 h)and found that the progeny virus could be detected at 6 h post-infection and the virus infection rate reached more than 90% at 24 h post-infection.Therefore,samples of experimental and control groups were collected at 6 h,12 h and 24 h post-infection,then cellular metabolites were detected by the untargeted metabolomics based on liquid chromatography-tandem mass spectrometry.Subsequently,the multivariate statistical analysis models(principal component analysis and orthogonal partial least squares-discriminant analysis)were established to screen for differential metabolites by the combination with unidimensional statistical analysis(t-test).The results showed that PRRSV infection of IPAM cells caused significant alterations in 540,612 and 514 metabolites at 6 h,12 h and 24 h,respectively,covering glucose metabolism,amino acid metabolism,nucleotide metabolism and lipid metabolism.2.Clustering analysis and validation of metabolomics dataCluster analysis of the differential metabolites revealed that(i)for glucose metabolism,glucose content decreased and glucose 6-phosphate content increased after PRRSV infection,and most intermediates of the tricarboxylic acid cycle were down-regulated and intermediates of the pentose phosphate pathway were up-regulated.It is speculated that glucose preferentially enters the pentose phosphate pathway after PRRSV infection,which may provide the carbon skeletons for nucleotide and amino acid synthesis;(ii)in terms of amino acid metabolism,most free amino acid contents were down-regulated after PRRSV infection,such as glutamine,serine and histidine;(iii)in terms of nucleotide metabolism,AICAR(key substance of purine nucleotide de novo synthesis),AMP and GMP were upregulated after PRRSV infection,but the catabolic products of purine nucleotides were not upregulated;the intermediate product of pyrimidine nucleotide de novo synthesis,UDP,and the catabolic products were downregulated,presumably PRRSV may prefer to hijack cellular purine synthesis rather than pyrimidine synthesis;(iv)regarding lipid metabolism,carnitine involved in fatty acid transport were down-regulated after PRRSV infection,while the PC and PE that are involved in biofilms constitution were up-regulated.It is speculated that PRRSV may inhibit fatty acid β-oxidation and convert fatty acids to membrane components such as glycerophospholipids to facilitate viral particle package.To determine the accuracy of the metabolomic data,glucose metabolism was examined by glucose labeling transport assay and enzyme activity kit after PRRSV infection with IPAM.It was found that glucose uptake by cells increased after PRRSV infection and the activity of 6-phosphoglucose dehydrogenase(a key metabolic enzyme of the pentose phosphate pathway)was enhanced,indicating that glucose catabolism into pentose phosphate after PRRSV infection pathway was increased.Further assays using the glutamine content assay kit revealed that PRRSV-infected IPAM cells significantly downregulated intracellular glutamine content.All of these results were consistent with the metabolomics data,indicating that the metabolomics data are plausible.3.PRRSV induces glutamine catabolism involved in purine nucleotide synthesisPRRSV infection significantly down-regulated intracellular glutamine content,but its effect on PRRSV proliferation is unclear.Detection by RT-q PCR and TCID50 assay revealed that the proliferation titer of PRRSV in IPAM was significantly reduced when cultured with glutamine-free medium,while the addition of α-ketoglutarate,non-essential amino acids and nucleosides partially rescued PRRSV replication from glutamine depletion,with the effect of nucleosides strongest.Then we determined the type of nucleoside that regulates PRRSV proliferation through exogenous addition of purine nucleotides(adenosine,guanosine or hypoxanthine)or pyrimidine nucleotides(uridine)to the glutamine-free medium,the results showed that purine nucleotides partially rescued PRRSV proliferation,and pyrimidine nucleotides did not rescue PRRSV proliferation.This suggests that PRRSV infection-induced glutamine catabolism may be involved in the synthesis of purine nucleotides,which in turn promotes its own proliferation.4.Purine nucleotide synthesis facilitates PRRSV proliferationThe effect of purine nucleotide synthesis on PRRSV proliferation was further examined,and it was found that AVN-944,an inhibitor of purine nucleotide de novo synthesis,significantly antagonized PRRSV proliferation,and that exogenous addition of guanosine reversed the inhibitory effect of AVN-944 on PRRSV proliferation,but inhibition of pyrimidine nucleotide de novo synthesis did not affect PRRSV proliferation,suggesting that purine nucleotide de novo synthesis facilitates PRRSV proliferation.The effect of one-carbon units on PRRSV proliferation was examined,and it was found that the one-carbon metabolism inhibitor SHIN1 and the folate metabolism inhibitor MTX both inhibited PRRSV proliferation,while the addition of one-carbon group donor formate or hypoxanthine completely rescued the inhibitory effect of SHIN1 or MTX on PRRSV proliferation,respectively.The results showed that one-carbon units promoted PRRSV proliferation,further confirming that de novo synthesis of purine nucleotides promoted PRRSV proliferation.In addition,OA,an inhibitor of the purine nucleotide synthesis salvage pathway,was also found to inhibit PRRSV proliferation,suggesting that PRRSV can hijack both two purine nucleotide synthesis pathways to establish optimal infection.