MiR-505 Inhibits Replication Of Borna Disease Virus 1 Via Regulating HMGB1-mediated Autophagy Negatively

Background: Borna disease virus 1(BoDV-1)is a highly neurotropic RNA virus that can continuously infect almost all warm-blooded animals including horses,sheep,and macaques.In recent years,BoDV-1 has been shown to cause disease in humans and may cause fatal encephalitis.Micro RNA(miRNA)are a class of small non-coding RNA molecules,containing about 22 nucleotides,involved in a wide range of biological processes.Many studies have reported that miRNA can positively or negatively regulate virus replication and affect the process of virus infection by regulating the host’s innate immune response.Our previous miRNA chip results also showed that BoDV-1 infection can cause a significant down-regulation of the expression level of miR-505 in rat brain tissue.However,the role of miR-505 in BoDV-1 infection is still unknown.Aims: Explore the possible mechanism of miR-505 in the process of BoDV-1 infection,and clarify that miR-505 can regulate HMGB1-mediated autophagy,thereby affecting BoDV-1 replication,providing a new drug target and theoretical basis for the prevention and treatment of BoDV-1 infection.Methods: BoDV-1 was used to infect primary neurons,RT-q PCR,Western Blotting and immunofluorescence were used to detect the infection of the primary neurons,and RT-q PCR was used to detect the expression of miR-505 in the primary neurons.To detect the effect of miR-505 on virus replication by regulating the expression of miR-505.The "Targetscan Human" and "miRDB" databases were used to find the downstream target gene of miR-505,and RNAhybrid2.2 software was used to locate the binding site of miR-505 and the 3’UTR of the target gene.To construct a plasmid with corresponding mutations,dual luciferase assay was used to confirm and verify the binding site of miR-505 and the 3’UTR of the target gene.In neurons infected with BoDV-1 and miR-505 mimics,the expression of autophagy-related genes and proteins was observed by RT-q PCR and Western blotting,the morphology of LC3 fluorescent protein was observed by analysis of autophagic flux,and the formation of autophagosomes was observed by transmission electron microscope.Results: RT-q PCR and WB showed that the expression of miR-505 was significantly downregulated in rat primary neurons stably infected with BoDV-1,and overexpression of miR-505 could inhibit the replication of BoDV-1 in cells.Bioinformatics analysis and dual luciferase reporter assays confirmed that during BoDV-1 infection,the high mobility group protein B1(HMGB1)that mediates autophagy is the direct target gene of miR-505.RT-q PCR and WB showed that the expression of HMGB1 was upregulated after BoDV-1 infection,and overexpression of miR-505 can inhibit the expression of HMGB1.Studies have found that many viruses can promote their own survival and replication through the activation of autophagy.In this study,autophagy-related detection found that the expression of autophagy-related ATG protein in neuronal cells infected with BoDV-1 was significantly upregulated.Autophagy-related markers LC3-I protein decreased,and LC3-II protein increased.Phagocytosis was activated.Analysis of autophagic flux showed that fluorescence plaques caused by LC3 fusion protein appeared in the cytoplasm,and autophagosomes were observed in the cytoplasm by transmission electron microscopy,which proved that BoDV-1 infection activated HMGB1-mediated autophagy.Further regulating the expression of miR-505 found that compared with the control,overexpression of miR-505 significantly downregulated the expression of ATG protein and the conversion of LC3 protein in neuronal cells,and the fluorescence plaques caused by LC3 fusion protein in autophagic flux were reduced.Transmission electron microscopy also found that the formation of autophagosomes in the cytoplasm was inhibited,indicating that overexpression of miR-505 significantly inhibited HMGB1-mediated autophagy.Conclusion: BoDV-1 infection can downregulate the expression of miR-505.Overexpression of miR-505 can inhibit the activation of autophagy mediated by HMGB1 via downregulating the expression of its target gene HMGB1,and ultimately inhibit the replication of BoDV-1.This mechanism may provide new ideas and directions for preventing and treating BoDV-1 infection in the future,and it can mediate BoDV-1infection by regulating the expression of miR-505 and HMGB1.