Studies On The Anti-HBV Effect Of Neddylation Inhibitor MLN4924 And Its Mechanism Investigation

Background and aimsHepatitis B virus(HBV)is a hepatotropic virus that can establish persistent and chronic infections in the human body.Currently,about 3.5% of the global population is chronically infected with hepatitis B virus.If HBV infection is not diagnosed or treated in time,life-threatening complications such as liver cirrhosis and hepatocellular carcinoma(HCC)will occur.It is estimated that only about 10% of all HBV infections in the world are diagnosed,and the proportion receiving formal antiviral treatment is even smaller.The current application of preventive vaccines has significantly reduced the number of children under 5 years of age who are infected with HBV.However,for patients already suffering from chronic hepatitis B(CHB),the current treatments are still only antiviral drugs that directly act on virus replication and immunomodulators that act on the body’s immunity.These treatment options have drug resistance and Problems with greater side effects.Therefore,there is an urgent need to develop new anti-HBV drugs.Neddylation modification is an important protein post-translational modification process,and it is an important modification link used by the body to adapt to changes in the external environment to maintain internal homeostasis.Neddylation is essentially completed by binding of NEDD8 to the substrate protein to alter protein function.It has a very close relationship with developmental diseases,tumors and viral infections.This study will use in vivo and in vitro HBV expression models to verify the antiviral effects of Neddylation inhibitors MLN4924 and explore its anti-HBV mechanism.It aims to clarify the possibility and effectiveness of the Neddylation inhibitor MLN4924 as a treatment plan for HBV infection,and to provide new methods for the diagnosis and treatment of clinical HBV infection.MethodsPart 1: HBV stable expression model Hep G2.2.15 cell and transient expression cell model(constructed by Huh7,Hep G2 transfected with p HBV1.37 plasmids)to verify the antiviral effect of MLN4924 in vitro;use high-pressure hydrodynamic method to inject p AAV-1.2HBV into the tail vein(10μg per mouse),a mouse model of HBV replication was constructed to verify the antiviral effect of MLN4924 in vivo.Collect mouse sera to determine glutamate pyruvate aminotransferase(ALT)and aspartate aminotransferase(AST).HBV DNA in the cell supernatant and blood were detected by RT-q PCR;the HBV antigens including HBs Ag and HBe Ag in cell supernatant and blood were detected by enzyme-linked immunosorbent assay(ELISA).Use immunofluorescence technology to detect intracellular virus antigens,and use immunohistochemistry technology to detect mouse liver virus antigens and related proteins.Detect the level of HBV 3.5k RNA and ccc DNA by fluorescence quantitative PCR;detect HBV ccc DNA by Southern Blot.Part 2: Western Blot was used to detect the NEDD8 levels in the stable-expressing HBV Hep G2.2.15 cell line,Hep AD38 cell line as well as HBV transient expression cell model.(constructed by Huh7,Hep G2 transfected with p HBV1.37 plasmid).Using dual fluorescent reporter gene detection technology to detect the expression activity of HBV promoter.Fluorescence quantitative PCR was used to detect the RNA expression of liver-enriched transcription factors.Use Western Blot to detect the expression of related proteins.The cell thermal transition analysis experiment(CETSA)method was used to study the affinity of MLN4924 with the target protein in the cell.RT-q PCR was used to detect HBV DNA and virus antigens HBs Ag and HBe Ag in cell supernatant were detected by ELISA.ResultsPart 1: HBV can promote intracellular neddylation modification in vitro and vivo.In vitro,Neddylation inhibitor MLN4924 can inhibit virus replication in HBV stable and transient expression models,and reduce HBV DNA and viral antigen titer;in vivo,MLN4924 can reduce the HBV plasmid constructed by high-pressure hydrodynamic tail vein injection HBV expresses the viral load in mice.More importantly,MLN4924 can reduce the content of HBV 3.5k RNA and ccc DNA,the most important transcription template of HBV.Part 2: MLN4924 can inhibit the activity of four HBV promoters(X,pre S1,pre S2,C)as well as the levels of liver-rich transcription factors HNF1α HNF1α,C/EBPa and HNF4α.The MAPK pathway activated by MLN4924 lead to the de-regulation of four transcription factors mentioned above,thereby playing an anti-HBV effect at the transcriptional level.Conclusions(1)Neddylation inhibitor MLN4924 can inhibit HBV replication in vitro and in vivo.(2)Neddylation inhibitor MLN4924 can inhibit the important transcription template of HBV(HBV ccc DNA).(3)The ERK/HNF1α/C/EBPa/HNF4α axis plays an important role in the anti-HBV effect of the Neddylation inhibitor MLN4924.