乙肝病毒核心蛋白钉突部位基因工程改造对其功能的影响

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

摘要:

通过在乙肝病毒核心蛋白钉突部位插入标签蛋白EGFP及小片段多肽，研究各种改造对HBc功能的影响。采用RLIC方法，构建野生型HBc、HBc钉突部位带不同接头的EGFP融合重组体、缩短的EGFP融合重组体，并构建与HBc功能互补的质粒HBV1.1c－，将不同重组体与HBV1.1c－共转染HEK293细胞，通过观察荧光及Southern blotting检测病毒复制中间体，判断相应基因工程改造对重组蛋白中不同结构域功能的影响。RLIC方法可有效地用来进行片段缺失，且缺失片段大小及位置无明显限制。带柔性或刚性接头的重组HBc-EGFP均可产生绿色荧光，但荧光在细胞内分布形态不同，两种重组HBc-EGFP均不能支持正常的HBV复制，各种截短的插入片段以及aa79-80单独缺失体亦不能支持HBV复制。结果表明RLIC方法是一种基因工程改造的有力工具，不同类型接头对重组蛋白的结构和功能有不同影响，aa79-80对维持HBc的主要功能之一——支持HBV复制有重要作用。

Abstract:

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.