Molecular Mechanism Of Myxovirus Resistance Protein A Inhibiting HBV Replication

Objective: Chronic hepatitis B,secondary cirrhosis and hepatocellular carcinoma caused by the persistent infection of hepatitis B virus are major diseases that threaten human health.Interferon is currently a major method for the treatment of hepatitis B,but it lacks a broad-spectrum therapeutic effect in clinical treatment.MxA protein is an important antiviral protein induced by interferon,which has a good inhibitory effect on HBV,but its anti-HBV mechanism has not been fully elucidated.Previous studies have found that MxA protein inhibits HBV replication by interacting with viral core protein HBc.This study will further study the mechanism of inhibition of core particle assembly by MxA protein.Illuminating the mechanism is important for the development of new anti-HBV drugs.This research will also provide new technical means and basis for breaking through the bottleneck of today's interferon treatment of Hepatitis B.Methods: Using HepG2.2.15 cells as virus model,the Flag empty vector,Flag-MxA,mutant Flag-MxAK83 A and mutant Flag-MxAL612 K plasmid were transfected into HepG2.2.15 cells respectively.The expression of core protein was determined by western blot,and the amount of pgRNA in nucleocapsid was detected by real-time fluorescence quantitative PCR,and the shape size and aggregation of core particles were observed by transmission electron microscopy in HuH7 cells transfedcted with HBc or HBc-MxA plasmids respectively.The Myc-MxA plasmid was co-transfected with Flag-HBc or Flag-HBc-Y132 A plasmid in HuH7 cells,and the co-immunoprecipitation was used to test the interaction.The deletion mutant ?CID(central interactive domain)and the truncated CID were constructed using Myc-MxA as a template.Transfect empty-vetor,MxA,?CID and CID in HepG2.2.15 cells.Transfection efficiency was measured by immunofluorescence.Plasmid expression was detected by Western blotting.HBsAg and HBeAg in the cell supernatant were determined by enzyme-linked immunosorbent assay.The amount of HBV DNA in the supernatant was determined by fluorescence quantitative PCR to identify the anti-HBV activity of the CID segment.According to the crystal structure of CID section,9 segments of peptides such as A 1,A2 and A3 were constructed to identify the anti-hepatitis B virus activity of each segment.The regions and sites of theinteraction between MxA protein and virus were predicted by means of computational biology-molecular docking.The deletion mutant was constructed based on the screening results,and the antiviral activity of the mutant was examined.In this way,a critical segment of the MxA protein that interacts with HBc is identified.Results: There was no significant change in the HBc protein level of wild type Flag-MxA,mutant Flag-MxAK83 A and mutant Flag-MxAL612 K compared with that of control group,and the amount of pgRNA of each experimental group decreased significantly(P<0.01).Transmission electron microscope results show that compared with control group,the core particles of MxA expression group shows obvious aggregation and deformation.The results of co-immunoprecipitation show that MxA protein can also be precipitated with HBc-Y132 A,indicating HBc-Y132 A can interact with MxA protein.The sequences and expression of ?CID,CID and the 9 peptide plasmids were correct.The amounts of HBsAg,HBeAg and HBV DNA in the CID and MxA group but not the ?CID group were significantly lowered compared to that in the control group(P<0.01).Among the 9 peptides,the amount of HBsAg,HBeAg and HBV DNA in A1 decreased significantly compared with that of control group(P<0.001).The results of molecular docking shows that the interaction sites on the MxA interacting with the virus were concentrated in 384-408-bit amino acids,which was located on the A1 peptide segment.The sequence and expression of 384-408 deleted mutant plasmid were correct.There was no significant change in antiviral activity between the ?384-408 group and the blank group,and the difference was not statistically significant.Conclusion: MxA protein has no effect on the expression of HBV core protein HBc.MxA protein blocks the assembly of HBV core particles.MxA protein,mainly by interacting with the unassembled core proteins,inhibits the assembly of core particles.The MxA protein may mainly block the assembly of the nucleocapsid by the interaction of the A1 segment with the core protein HBc,thereby inhibiting viral replication.