Effect Of Polyamines On The Proliferation Of Porcine Reproductive And Respiratory Syndrome Virus

Polyamines are small,abundant,aliphatic molecules consisting of two or more amino groups that is positively charged at neutral pH.It exists in animals,plants,algae and microorganisms.Mammalian cells mainly include putrescine,spermidine and spermine.Studies have shown that polyamines play an important role in viral infections such as middle east respiratory syndrome virus?MERS?,poliovirus?PV?,and zika virus?ZIKV?.Porcine reproductive and respiratory syndrome virus?PRRSV?is a virus that seriously harms the pig industry.It mainly causes reproductive failure in sows and a large number of deaths in newborn piglets,causing huge losses to the pig industry.At present,the role of polyamines in PRRSV infection and replication has not been reported.In this study,polyamines were used as an entry point to explore the relationship between polyamine anabolic pathways and porcine reproductive and respiratory syndrome viruses,and the mechanism of polyamines regulating PRRSV proliferation was revealed,which laid a foundation for elucidating the infection mechanism of PRRSV.The main research contents and results are as follows:1.The levels of intracellular polyamine were reduced in PRRSV inflection.To explore the levels of cellular poyamine levels after PRRSV inflection,PK-15^CD163cells?a PK-15 cell expressing PRRSVreceptor CD163?were infected with PRRSV,then detecting polyamines st different times after PRRSV infection by Thin-layer Chromatography.It is was found that the levels of spermidine and spermine was reduced but the level of putrescine was increased in a time-dependent manner.This indicates that PRRSV infection can cause the changes of intracellular polyamine levels.2.Polyamines promote PRRSV proliferationDFMO is an inhibitor that specifically inhibits the activity of ornithine decarboxylase?ODC?in the polyamine metabolic pathway.PK-15^CD163D163 cells were treated with DFMO,and it was confirmed by thin layer chromatography that DFMO treatment can significantly reduce the polyamine levels of cells in a time-dependent manner.The cells were pretreated with DFMO,then inoculated with PRRSV,and the copy number and viral titer of the viral genome were detected by fluorescence quantification and plaque assay,respectively.It was found that the inhibition of polyamine metabolic pathway significantly reduced PRRSV replication and virus titer,indicating the decrease of intracellular polyamines levels can inhibit the proliferation of PRRSV.At the same time,DFMO was used to down-regulate the level of polyamines in the cells,then the exogenous polyamines were added.It was found that the virus titer could be recovered,and was dose-dependent with the concentration of exogenous polyamines added,further demonstrating that polyamines promote the proliferation of PRRSV.3.Spermidine and spermine promote PPRRSV proliferationIn order to further investigate which specific or polyamine molecules are involved in the regulation of PRRSV proliferation.DFMO was used to down-regulate intracellular polyamines,and then putrescine,spermidine and spermine were added respectively.Real-time quantitative PCR showed that spermidine and spermine can improve the PRRSV genome copy number,while putrescine had no significant effect on PRRSV replication.Western Blot showed the expression level of PRRSV N protein was similar.It further showed that spermidine and spermine promote PRRSV proliferation,and putrescine had no significant effect on the proliferation of PRRSV.4.SAT1 inhibits PRRSV proliferationSAT1?spermidine/spermine decarboxylase?is the first rate-limiting enzyme in polyamine metabolism,which reduces spermine and spermidine levels.To further investigate the relationship between polyamines and PRRSV proliferation,real-time fluorescence quantitative PCR assay was used to detect the effect of PRRSV infection on SAT1 expression.It was found that PRRSV infection significantly up-regulated SAT1mRNA expression in a dose-dependent manner,suggesting that SAT1 may be involved in the regulation of PRRSV proliferation.Then,the expression of SAT1 was further up-regulated by SAT1 activator DENSpm,and its effect on PRRSV proliferation was examined by plaque assay and Western Blot.The results indicate that DENSpm inhibits viral proliferation and protein expression,further indicating that spermidine and spermine can promote PRRSV proliferation.