Effects Of Nucleic Acid G-quadruplex Structure On The Expression Of EPO Gene And The Replication Of Pseudorabies Virus

G-rich DNA or RNA can form a special secondary structure of nucleic acid,named G-quadruplex structure,which widely distributes in the genome of eukaryotes,prokaryotes and viral.The G-quadruplexes not only take part in important biological processes,such as DNA replication,transcription,and translation,but also affect some physiological and pathological processes.Pseudorabies(Aujeszky's disease)is a kind of acute infectious disease which is caused by pseudorabies virus(PRV).This virus cause high mortality rate in piglets,abortion in pregnant sows,and neurological disorder symptoms.The GC content of PRV genome is as high as 70-80%,we speculated that the G-rich sequences of PRV have ability to form G-quadruplex.EPO,as an early protein of PRV,plays an important role in regulating other viral genes expression.However,little is known about the regulation mechanism of EPO gene expression.Using bioinformative analysis,we found that there existed a guanine-rich sequence in the promoter region of the EPO gene.This sequence also contained three overlapping SP1 binding sites.Circular Dichroism and gel electrophoresis proved that the sequence can form G-quadruplex structure.The dimethyl sulfate footprinting assay and the Taq polymerase stop assay indicated that the G-quadruplexes folded in the EPO promoter were polymorphic.Furthermore,using fluorescence energy resonance transfer,gel electrophoresis,and Taq polymerase stop assay,we verified that the small molecule ligand PDS can stabilize the formed G-quadruplex structure.Finally,the luciferase reporter assay indicated that G-quadruplex structure diplayed a negative regulatory effect on the activity of EPO promoter.The mutation of SP1 binding site in EPO promoter region leads to the decrease of promoter activity.Next,we tested the ability of Sp1 to recognize and bind DNA containing G quadruplex structure and DNA without G quadruplex structure to explore the mechanism.The truncated Sp1 containing three zinc-finger-motif was tagged with six soluble tags on N-domain,and the fusion Sp1 protein was expressed by using prokaryotic expression system.As a result,a high purity fusion protein MBP-Sp1 was obtained.Using electrophoretic mobility shift assay(EMSA),it was concluded that:compared to double-stranded DNA and single-stranded DNA without G-quadruplex,the Sp1 selectively bound to double-stranded DNA and single-stranded DNA having G-quadruplex In cells with Spl were overexpressed or knocked down,the luciferase reporter assay showed that Sp1 exhibited important regulation on EPO promoter activity.Finally,we tested the antiviral ability of small molecule ligands against PRV.Using flow cytometry analysis,we found that PDS and BRACO19 mainly inhibited the replication phase in the life cycle of PRV.Fluorescence quantitative PCR and western blotting results showed that PDS inhibited the expression of immediate early protein IE 180,early gene EPO,TK and virulence protein gB of PRV HN-1201 strain.Moreover,addition of PDS led to a significant reduction of the titer of PRV HN-1201,and displayed an anti-viral effect.In summary,The G-rich sequence of EPO promoter can form G quadruplex structure,which not only inhibited the promoter activity,but also changed the recognition and binding of cell transcription factor Spl to EPO promoter.The small molecule ligand stabilized the structure of the G quadruplex,and also inhibited virus's replication.To the best of our knowledge,this study is the first to explain the molecular mechanism by which EPO gene expression was regulated based on nucleic acid structure,and also will provide a theoretical basis to find novel anti-PRV targets.