Peptides that Lead Nuclear Entry of Nucleocapsid of Hepatitis B Virus in HepG2.2.15 Cells X.B. Pan 1,2 , L. Wei 1,2 , J.C. Han 1,2 , K. Deng 1,2 1 Peking University Hepatology Institute, 2 Peking University People's Hospital Background: The nuclear entry of nucleocapsid is a key step for the HBV life cycle and the formation of covalently closed circled DNA (cccDNA). It has been supposed that the carboxyl-terminal arginine-rich domain of the core protein contains a signal for nuclear localization (NLS). Whereas HBcAg was primarily distributed in cytoplasm and no marked cccDNA was detected in HepG2.2.15 cells. Methods: We designed peptides containing a cell-penetrating sequence (RRRRRRR) and a nucleocapsid binding sequence (GSLLGRMKGA) with/without a classic nuclear localization sequence (PKKKRKV) and these sequences were linked by a soft linker Acp. HepG2.2.15 cells were treated with the peptides at levels of 0 M, 25 M and 50 M for 4 days. Results: Compared with that of control cells, the results showed HBV DNA levels in culture medium decreased at least 2 log10 both in 50 M of peptide RRRRRRRAcpGSLLGRMKGA treatment group and RRRRRRRAcpGSLLGRMKGAAcpPKKKRKV treatment group; whereas HBsAg and HBeAg increased at 1.34+0.21 folds and 2.15+0.37 folds respectively. The signal strength of cytoplasmic HBcAg increased at about 2.5-fold in both groups. In RRRRRRRAcpGSLLGRMKGAAcpPKKKRKV treatment group, nuclear HBcAg increased about 3.2-fold and obvious cccDNA signal was detected by southern blot. Conclusion: Our results implied that the NLS of core protein likely does not expose to surface of nucleocapsid in HepG2.2.15 cells, the artificial peptide containning NLS binds to the nucleocapsid and leads nuclear entry of nucleocapsids and then facilitates the formation of cccDNA. Our study presents a tool for study on cccDNA formation and nuclear entry of nucleocapsid.