Study On The Effects Of Lipid Raft On The Infection Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS),caused by porcine reproductive and respiratory syndrome virus,is a kind of infective diseases.It is characterized by severe reproductive failure in sows,and respiratory distress in piglets and growing pigs.Since first reported in the United States in 1987,PRRS had spreaded in world-wide and it has been one of the most economically important diseases of the global swine industry.In recent years,an outbreak of so called "Ardent Fever Disease" in China was identified as the result of PRRSV.Accumulated evidences indicate that cellular membrane lipid bilayer is inhomogeneous,there are lots of microdomains which contain abundant cholesterol and sphingolipid in the membrane.These microdomains,which are characterized by special structure and biological function,are called lipid microdomain.Due to lipid microdomains are floated in the cellular membrane as rafts,they are also called lipid rafts. Lipid raft participate in numerous biological processes,such as signal transduction, protein sorting,T cell activation,entry of bacterium and virus,assembly and budding of virus and so on.The abundant cholesterol in lipid raft plays an important role in the structure and function of lipid raft.It was reported that the entry of enveloped virus depend on lipid raft and cholesterol;moreover,most virus utilize lipid raft in assembly and budding.But the effect of lipid raft on PRRSV infection is unclear,so we studied on the infection of PRRSV in this research,determined the effect of lipid raft,cholesterol and caveolin-1 on PRRSV infection.The main contents are as follow:1.The effect of Methyl-β-cyclodextrin(MβCD) on MARC-145 cellMethyl-β3-cyclodextrin(Mβ3CD) is cholesterol extraction agent,which can remove cellular membrane cholesterol while has little toxicity on cells.In the research, MARC-145 cells were dealed with various concentration of MβCD,and the results indicated that the effect of MβCD on cholesterol depletion was dose-dependent beween 0mM to 10mM,and 75%cellular cholesterol was depleted by 10mM MβCD,but the effect was not significantly enhanced when the concentration was raised more than 10 mM.Moreover,exogenous cholesterol can replenish cholesterol by about 84%.Based on the results,10mM MβCD was applied in later research.2.The effects of cholesterol on PRRSV infectionThe effects of cellular membrane cholesterol on different stages of PRRSV infection were studies by cholesterol depletion and replenishment assay.And the results indicated that cellular membrane cholesterol played an important role in many stages of PRRSV infection.Depletion of cellular membrane cholesterol inhibited PRRSV proliferation by about 99.71%;the ability of virus entry decreased while the amount of cholesterol decreased by MβCD,and this correlation was mainly displayed on virus attachment,the result of flow cytometry indicated that virus attached to control cells was 3.8 times more than that on cholesterol depletion cells;additionally,cholesterol depletion facilitated virus releasing from MARC-145 cell by 1.06 to 4.92 times,but decreased the infectivity of released virus particles by 90%.And all the impacts mentioned above could be partially neutralized by cholesterol replenishment.3.The effects of lipid raft and its specific protein on the endocytosis of PRRSVIn this research,the entry process of PRRSV into MARC-145 cell was studied.At first,Nystatin,a lipid raft-dependent endocytosis disruptor was applied,the result indicated that PRRSV proliferation was decreased to 0.18%compared with control cells while lipid raft-dependent endocytosis was disrupted.Meanwhile,eukaryotic expression plasmid of pEGFP-C₂-cav-1 and pEGFP-C₂-cav-1-△1-81 were constructed.And the effect of caveolin-1 on PRRSV infection was detected by transfection and Real-Time PCR.The result indicated that the depletion of the function of caveolin-1 had little effect on PRRSV infection.Later,a clathrin-mediated endocytosis disruptor(CPZ) was applied to detect whether clathrin was involved in the endocytosis of PRRSV,and it was discovered that CPZ notably inhibited PRRSV infection by 99.83%compared with control cells.So,the endocytosis of PRRSV relied on lipid raft and clathrin.