Probing The Function Of Cellular Prion Protein During PRRSV Infection Of Marc-145 Cells

Porcine reproductive and respiratory syndrome virus(PRRSV)is one of the most influential pathogens which affect the healthy development of the pig industries worldwide.Cellular prion protein(PrPc)is a membrane glycoprotein universally expressed on the surfaces of mammalian cells.PrPc possesses lots of physiological functions and involves in many cellular events.Our previous study has primarily shown that PrPc plays a role in PRRSV infection of the host cells,but the mechanism is not clear.This paper further explored the role of PrPc during PRRSV infection process.This study analyzed the influence of PrPc on the multiplication of type 2 PRRSV strain QH-08 when it infected African green monkey kidney cell line Marc-145 which had been treated to upregulate PrPc exrpession or inhibit PrPc function.The results are as follows:(1)Firstly,PrPc changes were studied when PRRSV QH-08 infected the wild-type Marc-145 cells via q PCR and confocal methods.The results demonstrated that the expression profile of PrPc greatly changed after virus QH-08 incubation with Marc-145 cells over time.mRNA level of PrPc reached the climax at 24 hour after PRRSV infection.However,CD163,one of PRRSV receptors,achieved the peak at 48 hour after PRRSV inoculation.Laser confocal further confirmed that the localization phenomenon happened between PRRSV and PrPc when Marc-145 cells were incubated with PRRSV at 24 hour while PRRSV and CD163 overlapped at 48 hour.CD163 involves the process of PRRSV uncoating and releasing the genomes at the late stage of virus replication.Comparison with CD163,the results above indicated that PrPc should play a role in the early stage of PRRSV infection of Marc-145 cells.(2)PcDNA3.1/Myc-His(-)B was used as the expression vector to construct the eukaryotic recombinant expression plasmid containing full-length PrPc encoding gene.The corresponding recombinant plasmids were transfected into Marc-145 cells to enhance PrPc expression level.The changes of PRRSV replication were measured by qPCR at 24 hour after virus strain QH-08 infected the Marc-145 cells with PrPc overexpression.The results showed that mRNA level of PRRSV significantly increased when PrPc expression was enhanced in Marc-145 cells.(3)Monoclonal antibody SAF-32 against PrPc was applied to block the function of PrPc of Marc-145 cells and the changes of PRRSV propagation was analyzed after infection of Marc-145 cells with lower PrPc function.Experimental data showed that the levels of PRRSV mRNA were significantly reduced after Marc-145 cells were incubated with SAF-32 for 1 hour.Furthermore,SAF-32 dose-dependent pattern was shown.This study preliminarily proved that PrPc plays a role in the early process of PRRSV infection of Marc-145 host cells,however,the specific mechanism remains to be further studied.The results have laid a solid foundation for further exploring the role of PrPc in PRRSV infection.