Characterization Of The Amino Acids Essential For HBC Dimerization

Purpose: Homodimeric core protein(HBC)is the basic subunit in hepatitis B virus nucleocapsid assembly.Lacking proper method to detect the HBC dimerization,the primary amino acid sequences,that are essential for HBC dimerization,are not identified thoroughly.A new method,which reflects the HBC dimerization in a convenient way,was used to screen the primary amino acid sequences essential for HBC dimerization.Method: Split Renilla Luciferase complementation system was used to detect HBC homodimerization.Renilla luciferase N terminal(1-229aa)and C terminal(229-331aa)were fused separately to the N terminal of HBC(2-183)through a long G4 S linker,named RlucN-HBC and RlucC-HBC.When a heterodimer of the fusion protein formed(RlucN-HBC +RlucC-HBC),the N 、 C terminal of Rluc closed each other mediated by HBC dimer,thus recover partly the luciferase activity.Series of deletion mutants of RlucC-HBC were constructed and screened using this reporter system,aiming at identifying the primary amino acid sequences essential for HBC dimerization.Based on 3xFlag-HBC,the formation of capsid of several deletion mutants were assayed.Result:1.Plasmids using in this article were constructed by golden gate clone method,including RlucN-HBC and RlucC-HBC as positive control,RlucN-d HBc and Rluc C-d HBc as negative control,while RlucN-HBc127 Q,RlucC-HBc127 Q,Rluc N-HBc132 A,RlucC-HBc132 A,RlucN-HBc42 A,RlucC-HBc42 A,RlucN-HBc23 A and Rluc C-HBc23 A each bearing a assembly deficient mutation.2.Co-transfection of RlucN-HBC and RlucC-HBC gain more than 25 times luciferase activity of the negative group,namely RlucC-Dhbc + RlucN-Dhbc,RlucC-HBC+RlucN-Dhbc,Rluc C-d HBC+Rluc N-HBC.3.Capsidation deficient mutants RlucN-HBc127 Q,RlucC-HBc127 Q,RlucN-HBc132 A,Rluc C-HBc132 A,RlucN-HBc42 A,RlucC-HBc42 A,RlucN-HBc23 A and RlucC-HBc23 A gained equivalent signal with RlucN-HBC + RlucC-HBC.4.30 deletion mutants spanning HBC N terminal 、C terminal and α-helixes regions were constructed(based on RlucC-HBC).5.Co-transfected with RlucN-HBC,deletion mutants were separated into 3 groups according to luciferase activity.D1-5 、d6-17、d30-41、d75-81、d86-90、d122-183 retained 80% of WT-HBC,d26-29、d61-65、d71-75 、d17-120 retained 40% while d18-25、d50-60、d66-70、d81-85、d91-116 retained less than20%.6.3xFlag-HBCd81-85、d86-90 form capsid while 3xFlag-HBCd38-41 d50-55、d91-95、d106-110 not。Conclusion:1.The split luciferase reporter system reflected HBV core protein(HBC)dimerization.2.Sequence50-55、91-95、106-110 are essential for HBC dimerization.3.Sequence 18-25、50-60、66-70、91-116 may important for HBC dimerization.