The Role Of 3'flap Endonuclease MUS81 In The CccDNA Formation Of Hepatitis B Virus CccDNA

Objective: To investigate the role of 3'flap endonuclease MUS81 in the formation of cccDNA of Hepatitis B virus.Methods: In vitro purification of mus81-eme1 wild-type and mutant enzyme complexes was carried out to evaluate the activity of mus81-eme1 enzyme complexes to different HBV flap fragments.We used CRISPR-Cas9 system to knock out the genes of mus81 in HepAD38 and HepG2-NTCP cell lines as well as evaluate the effect of inactivation of MUS81 on the production of cccDNA in HBV stable cell line and HBV infection cell model.Results: The wild-type and mutant MUS81-EME1 protein complexes were obtained,which had cleavage activity to the HBV 3'flap structure.The enzymolysis activity increased with the increment of enzyme complex,and MUS81's mutant nuclease domain did not have this cleavage activity.The MUS81 knocked out stable cell line of HepAD38 and HepG2-NTCP were successfully established,which confirmed that the inactivation of MUS81 could significantly reduce the level of cccDNA.Conclusion: MUS81 plays an important role in the formation of HBV cccDNA,which may be manifested in the removal of rcDNA 3 'redundant sequence of Hepatitis B virus and the cyclization of rcDNA.