The Effects Of Lipid Raft And PI3K/Akt/SREBP1 Pathway On CA16 Replication

Coxsackievirus A 16(CA16)belonging to the family Picornaviriade,the geneus Enterovirus,is one of major pathogenes of Hand,foot and mouth disease in infants.In recent years,CA16 caused many epidemic outbreaks in this country,and became a serious threat to health of infants.Lipid raft is a kind of dynamic microdomain in the plasma membrane that riched in cholesterol and sphingolipid.Lipid raft can increase quantities of particular proteins,include the viral receptors and signaling molecules.And it allow lipid raft to take part in the process of viral infection,and induce signal transduction,such as the activation of Akt.So this study focused on CA16 and explained the roles of lipid raft and PI3K/Akt pathway in CA16 infection.Cholesterol is a necessary compent of stabilization and function of lipid rafts.So this study used M?CD to disturb the structure of lipid raft.Through MTT assay,10 m M was choosed for the max work concentration of M?CD.After M?CD treatment,RT-PCR,Western blot and indirect immunofluorescence were used to detect the effect of M?CD on CA16 infection.To confirm M?CD inhibit which stage of infection,M?CD was amplied in the different stage of CA16 infection,then RT-PCR and Western blot were used to dectect CA16 infection.In order to testify whether M?CD inhibit CA16 infection through cholesterol detetion,exogenous cholesterol was added to cells after M?CD treatment.Western blot was used to detect the phosphorylation of Akt at the different timings in the early stage of CA16 infection and the effect of M?CD treatment on PI3K/Akt pathway.The change of PI3K/Akt pathway after inactivated CA16 infection was also detected by Western blot.The inhibitor of PI3 K,wortmannin was used to block PI3K/Akt pathway and Western blot was used to detect the replication of CA16.Western blot was used to detect the phosphorylation of Akt in the middle and advanced stage of CA16 infeciton.To further study the function of PI3K/Akt pathway activated in the middle and advanced stage of CA16 infeciton,Western blot was used to observe the expression of SREBP1 and INSIG-1,indirect immunofluorescence was used to confirm the effect of CA16 infection on the subcellular localization of SREBP-1a and SREBP-1c,and Western blot was used to dectect the change of SREBP1 after wortmannin treatment.Indirect immunofluorescence was used to observe the subcellular localization of SREBP-1a and SREBP-1c.At last,SREBP was overexpressed and CA16 VP1 was detected by Western blot.The result showed that M?CD can inhibit CA16 infection.Inhibition of M?CD treatment was most obvious before CA16 infection.And exogenous cholesterol restore CA16 infection.The phosphorylation of Akt increased distinctly in the early stage of CA16 infection,but it wasn't change after M?CD treatment.The phosphorylation of Akt also increased after inactivated CA16 infect.The replication of CA16 was suppressed when PI3K/Akt pathway was unactivated.Phosphorylation of Akt increased at 12 h and 24 h after CA16 infection,but the phosphorylation of Akt at 12 h was inhibited after M?CD treatment.SREBP1 was cleaved and the expression of INSIG-1 decreased after CA16 infection.SREBP-1a and SREBP-1c were transfered from cytoplasma to cell nucleus.But SREBP1 wasn't cleaved after wortmannin treatment.Overexpression of SREBP inhibits the replication of CA16.These results reveal that lipid raft is necessary for CA16 to inavde host cell and the activation of PI3K/Akt pathway in the early stage and middle stage of CA16 infecion,PI3K/Akt pathway regulate CA16 replication in the early stage of infection and induce the cleavage and nuclear translocation of SREBP1 in the middle and advanced stage of infection.