Research On Mechanism Of Host CircRNA?0050463 Promoting IAV Replication

Influenza A virus(IAV)is a single stranded negative strand RNA virus,belonging to Orthomyxoviridae.As zoonotic pathogen,IAV causes yearly outbreaks in human and animals with not only rapid transmission and variation but also high rate of morbidity and fatality.Circular RNA(circ RNA)is a kind of noncoding RNA.It has been shown that circ RNA is involved in regulating interaction between viruses and hosts as an important epigenetic regulator.Based on our preliminary transcriptome profiles of A549 cells responding to IAV infection,we selected one of the most significantly up-regulated host circ RNAs,circ?0050463,to validate its expression,explore its roles in IAV replication and illustrate molecular mechanisim underlying its regulating effect on IAV replication.Firstly,we applied PCR with divergent/convergent primers to identify the cyclic structure of circ?0050463,which verified that circ?0050463 is a real circ RNA.To verify the relationship between IAV and circ?0050463,we infected A549 cells with IAV different strains,different time courses,different MOI,and determined expression of circ?0050463 using RT-q PCR.These results demonstrated that the increment of circ?0050463 level is associated with IAV infection.To determine whether the v RNA is the key factor to induce circ?0050463,we treated cells with Poly I:C,LPS,cisplatin and serum withdrawl,respectively,and detected the expression of circ?0050463 using RT-q PCR.These datas showed that circ?0050463 was strongly induced by double-strand RNA poly I:C in A549 cells,and the expression of circ?0050463 is the result of the cells responsing to IAV infection.To investigate whether IAV-induced expression of circ?0050463 is dependent on NF-?B,we treated cells with NF-?B inhibitor,and monitored circ?0050463 expression with RT-q PCR.These findings indicated that the circ?0050463induction is regulated by NF-?B activation.In order to study the effect of circ?0050463 on IAV replication,we applied si RNA to knockdown endogenous expression of circ?0050463 and infected cells with IAV.Then we determined IAV replication using RT-q PCR,Western Blot and TCID₅₀assay.The results showed that knockdown of circ?0050463 attenuated IAV replication in A549 cells.We want to figure out the molecular mechanisim,using separation of nuclear and cytoplasmic fractions and RT-q PCR,we investigated the subcellular localization of circ?0050463,which revealed that circ?0050463 was predominantly localized to the cytoplasm.Next,we used Arraystar software to predict possible micro RNAs binding with circ?0050463,TOP 5:mi R-629-3p,mi R-92a-1-5p,mi R-511-5p,mi R-150-5p and mi R-33b-5p.The highly conserved binding site between mi R-33b-5p and circ?0050463 was observed.Subsequently,RNA pull down assay and dual luciferase reporter gene assay confirmed that circ?0050463 can bind to mi R-33b-5p.To investigate whether circ?0050463 affected the expression of EEF1A1 by binding to mi R-33b-5p,after co-transfected A549 cells with mi R-33b-5p inhibitor and circ?0050463 si RNA and infected cells with IAV,we examined the expression of EEF1A1 using Western Blot and RT-q PCR.The results showed that circ?0050463 promotes the expression of EEF1A1 through binding with mi R-33b-5p.The same method was used to detect the effect of mi R-33b-5p expression on IAV replication.The data demonstrated that mi R-33b-5p expression inhibited IAV replication.To explore the effect of circ?0050463/mi R-33b-5p/EEF1A1 axis on IAV replication,we transfected cells with si-EEF1A1 to knockdown the expression of EEF1A1 and examined the expression of IAV M1 protein using Western Blot.We finally proved that knockdown of EEF1A1 resulted in a significant decrease of IAV replication,suggesting that circ?0050463/mi R-33b-5p/EEF1A1 axis regulates IAV replication.Taken together,we identify a new function of circ?0050463 that facilitate IAV replication by circ?0050463/mi R-33b-5p/EEF1A1 axis.