Preliminary Study On The Role Of Apolipoprotein L1 In The Replication Of Enterovirus A71

Background and objectiveApolipoprotein L1 is an innate immune factor in humans and is expressed only in humans and a few mammals.As the only member of APOL family that can be secreted into the blood,APOL1 is a component of high-density lipoprotein with a trypanosome action.In 2014,Taylor et al.clarified that intracellular APOL1 protein has an anti-infective effect.Intracellular APOL1 protein can inhibit the replication of HIV-1 in macrophages.Our previous study found that APOL1 protein was overexpressed in the blood of children with severe hand,foot and mouth disease caused by EV-A71 infection.Therefore,this study explores the role of APOL1 protein in intracellular replication ofEV-A71 by constructing APOL 1 knockout cell lines and overexpressing cell lines,which not only helps to understand the pathogenesis of EV-A71,but also can provides new approaches forofEV-A71's prevention and treatment.Method(1)The siRNA was designed for the Apol1 gene.Knockdown of APOL1 protein expression in HBMEC cells and the replication of EV-A71 in cellswas detected.(2)HBMEC stable cell line knocked out of Apol 1 gene was constructed by CRISPR/Cas9.(3)293T cell line stably expressing APOL1 was constructedby Cumate expression vector(4)EV-A71 infects the two cell lines constructed above,and the relationship was explored between APOL1 and EV-A71 replication.Result(1)In HBMEC,after transfecting Apol1siRNA and infecting EV-A71 for 24 h,the intracellular virus content was significantly increased relative to unknocked cells(F=170.4,p<0.05).(2)The HBMEC cells knocked out of Apol1were successfully constructed and named HBMEC-APOL1-KO,and the cells infected with the no-load virus were named HBMEC-Lenti.(3)293T cells which can induce the expression of APOL1 protein were successfully constructed and named as 293T-APOL1-GFP,and the cells infected with no load were named 293T-GFP.Real-time PCR and western blot confirmed that the amount of APOL1 expressed by 293T-APOL1-GFP cells increased with the increase of inducer concentration,and reached the maximum expression of APOLI protein at 50?g/ml and lOO?g/ml(F= 537.9,p<0.01).CCK-8 experiments showed that cell growth had no effect when the concentration of the inducer was less than 200?g/ml(F=1.082,p=0.2903).(4)The virus was infected with HBMEC-APOL1-KO and 293T-APOL1-GFP cell lines respectively,and the same result was obtained.After virus infection for 12 hours,the intracellular virus content was significantly higher than that of the control group(F=196.2,p<0.001.:F=561.5,p<0.001).ConclusionUnder normal conditions,high expression of APOL1 in HBMEC cells can inhibit the replication of EV-A71;In the absence of APOL1 expression,replication of EV-A71 was increased;In 293T overexpressing APOL1,replication of EV-A71 was also increased.In summary,this study initially determined that APOL1 played a certain role in the replication process of EV-A71.