[Impacts on hepatitis B virus replication by gene engineering at apical loop region of capsid protein]

Hepatitis B virus (HBV) DNA replication takes place in the viral capsid that consists of 180 or 240 copies of HBV capsid (HBc or core) protein. The monomeric core protein contains an apical loop region that forms the spikes on the surface of viral capsid upon core dimerization and capsid assembly. To investigate the impact on HBV DNA replication through gene engineering at the spike of HBV capsid. plasmids expressing engineered HBc with linker-fused enhanced green fluorescent protein (EGFP) or shortened EGFP insertion at the spike region were constructed by Restriction Digestion and Ligation-independent Cloning (RLIC). The wildtype or mutant HBc construct was cotransfected with HBV1.1c(-), a plasmid containing 1.1 unit-length HBV genome with deficiency in HBc expression, into HEK293 cells, respectively. GFP signal was observed through a fluorescence microscope and HBV DNA replicative intermediates were assayed by Southern blotting to determine the expression and functions of different recombinants. Our results demonstrated that the RLIC method was effective to generate deletion or insertion in the apical loop region of HBc. Both HBc-EGFP recombinants with different linkers produced green fluorescence but with different subcellular distribution pattern. However, HBV DNA replication was not detected with the trans-complementation of these two HBc recombinants. In addition, other recombinants including the one only with the deletion of aa79-80 failed to support HBV replication. Taken together, our results suggest that RLIC is a robust method which can be broadly applied in gene engineering; different peptide linkers may have different influences on the functions of an engineered fusion protein; and HBc aa79-80 play a critical role for HBc to support HBV DNA replication.