The Inhibitory Mechanism Of Cholesterol 25-Hydroxylase On Porcine Deltacoronavirus Proliferation

Cholesterol 25 hydroxylase(CH25H)is a hydroxylase located in the endoplasmic reticulum membrane.Its main function is to catalyze excessive cholesterol in vivo to produce soluble 25-hydroxycholesterol(25HC).25 HC can control cholesterol biosynthesis by regulating nuclear receptors and sterol reaction element binding protein(SREBP),thus reducing the accumulation of cholesterol in vivo.In recent years,it has also been found that CH25 H acts as a broad antiviral host restriction factor to catalyze synthesis of 25 HC,which prevents the membrane fusion between viruses and cells,leading to the inhibition of virus invasion.For example,25 HC can significantly inhibit the proliferation of human immunodeficiency virus(HIV),vesicular stomatitis virus(VSV),human herpes virus(HSV)and other viruses.Porcine deltacoronavirus(PDCoV)is an emerging swine enteropathogenic coronavirus that mainly causes vomiting,diarrhea,dehydration and even death in newborn piglets,causing great economic losses to the swine industry.It was reported that treatment with 25 HC reduced PRRSV titer in the lungs of pigs and showed good protective effect.Based on this,the search concentrates on the influence and mechanism of CH25 H on the proliferation of PDCoV.In this study,the expression of CH25 H in cells after PDCoV infection and the influence of CH25 H and its enzyme active mutant on the proliferation of PDCoV were analyzed.It was confirmed that PDCoV infection induced CH25 H expression,and CH25 H inhibited the proliferation of PDCoV but did not completely dependent on its enzyme activity.The main research contents are as follows:1.PDCoV infection upregulates the expression level of CH25 H in vitroPorcine intestinal epithelial cells(IPI-FX)were infected with different doses of PDCoV(0.25 MOI,0.5 MOI,1 MOI).q RT-PCR and Western blot analysis showed that m RNA and protein levels of CH25 H were significantly up-regulated at 12 h post-infection(hpi)in a dose-dependent manner.At the same time,IPI-FX cells were infected with PDCoV(MOI of 1),m RNA levels and protein levels of CH25 H at different time(6 hpi,12 hpi,18 hpi)were analyzed.It was found that there was no significant change in m RNA and protein levels of CH25 H at 6 h post-infection,while both m RNA and protein levels of CH25 H at 12 h and 18 h post-infection were significantly up-regulated.2.CH25 H inhibits PDCoV proliferationIn order to determine the effect of CH25 H and its enzyme-activity mutant(CH25HM)on PDCoV proliferation,IPI-FX cells were transfected with expression constructs encoding CH25 H or CH25H-M and then were inoculated with PDCoV(MOI of 0.5).The proliferation levels of PDCoV at 12 h and 18 h post-infection were detected by q RT-PCR,plaque assay,and indirect immunofluorescence assay.It was found that overexpression of both CH25 H and CH25H-M significantly inhibited the proliferation of PDCoV,but the inhibitory effect of CH25H-M was weaker than that of CH25 H,suggesting that CH25 H inhibits PDCoV proliferation and does not completely depend on its enzyme activity.To further determine whether CH25 H is a host restriction factor against PDCoV infection,IPIFX cells were transfected with specific si RNA to interfere the expression of CH25 H,and then were inoculated with PDCoV(MOI of 0.5).The proliferation level of PDCoV at 18 h post-infection was detected by q RT-PCR and plaque assay.It was found that knockdown of CH25 H in cells significantly promoted PDCoV proliferation,indicating that CH25 H is a host restriction factor against PDCoV infection.3.25 HC inhibits PDCoV proliferationTo further analyze the effect of 25 HC generated by CH25 H on PDCoV proliferation,IPI-FX cells were pretreated with 25HC(12.5 ?mol/L)for 8 h and then were inoculated with PDCoV(MOI of 0.5).The proliferation levels of PDCoV at 12 h and 18 h postinfection were detected by q RT-PCR,plaque assay and indirect immunofluorescence assay.It was found that 25 HC treatment significantly inhibited the proliferation of PDCoV,further confirming that CH25 H inhibits PDCoV infection by exerting its enzyme activity.4.25 HC inhibits PDCoV invasionIn order to explore which phase of PDCoV replication cycle 25 HC targets,the effects of 25 HC on the adsorption,invasion,replication and release of PDCoV were analyzed.It was found that 25 HC inhibited the invasion of PDCoV,but it had no effect on the adsorption,replication and release stages.Moreover,25 HC could not directly inactivate PDCoV in vitro.These results further suggest that 25 HC may inhibit PDCoV infection by modifying cell membrane.