HBc二聚体组装核衣壳相关功能域突变对HBV复制的影响

目的 探讨乙型肝炎病毒核心蛋白(hepatitis B virus core protein,HBc)二聚体组装相关功能域突变对核心颗粒组装及HBV复制的影响.方法 基于HBc空间结构,PCR定点诱变二聚体组装核衣壳相关功能域重要氨基酸位点,以pcDNA3.1为载体构建4个突变体表达质粒pHBc14-18M、pHBc120-135M、pHBc23-39M和pHBc122-139M.将突变质粒与HBc缺失的含1.2拷贝HBV基因质粒pHBV1.2-core-共转染HepG2细胞,通过Northern blot检测HBV前基因组(pgRNA),Southern blot检测复制中间体,非变性琼脂糖凝胶电泳(native agarose gel electrophoresis)及Western blot检测细胞核心颗粒观察突变质粒自身形成核衣壳情况.将突变质粒与含有1.2拷贝HBV基因质粒pHBV1.2共转染HepG2细胞观测突变质粒对野生型HBc组装及HBV复制的影响.结果 突变质粒与pHBV1.2-core-质粒共转染HepG2细胞,pHBc14-18M、pHBc120-135M和pHBc122-139M突变能够组成核衣壳样结构.pHBc23-39M不能形成核衣壳样结构.Northern blot结果显示所有突变质粒组均未见pgRNA条带,Southern blot检测复制中间体也未见条带.将突变质粒与pHBV1.2共转染HepG2细胞,pHBc14-18M、pHBc120-135M和pHBc122-139M组HBV复制中间体及上清中病毒颗粒明显减少,而pHBc23-39M组无减少.结论 HBc23-39位氨基酸突变可阻止二聚体多聚化,不能形成核衣壳样结构,且不能与野生HBc二聚体相互作用.HBc14-18、120-135、122-139区域的氨基酸突变,能够形成核衣壳样结构但不支持HBV DNA复制,并且能与野生HBc二聚体相互作用,形成杂合体,干扰野生型HBV DNA的复制.

The effect of mutation of hepatitis B virus core protein dimer interface domain related to nucleocapsid formation on HBV replication

Objective To investigate the effect of hepatitis B virus core protein (HBc) dimer interfaces amino acids mutation on nucleocapsid assembly and HBV DNA replication. Methods Based on HBc three dimension structure, four HBc dimer interfaces domain mutation plasmids, pHBc14-18M,pHBc120-135M,pHBc23-39M and pHBc122-139M were constructed in pcDNA3.1 vector by PCR site-directed mutagenesis, there was a flag-tag at the C-terminal of all mutants for easy detection. Wild type core protein plasmid 1-183flag was also constructed as a positive control. The 4 mutants were cotransfected HepG2 cells with pHBV1.2 core negative plasmid (pHBV1.2-core-) ,which contained 1.2 copies of HBV whole genome but the core protein would not express due to a stop codon. The capsid formation, HBV pregenome(pgRNA) and HBV DNA replication mediate were analyzed by native agarose gel electrophoresis and Western blot, Northern blot and Southern blot , respectively. The 4 mutants were also cotransfected HepG2 cells with HBV wild type plasmid pHBV1.2 and examined by Southern blot. Virions in the medium were determined by native agarose gel electrophoresis and Western blot. Results Cotransfecting HepG2 cells with pHBV1.2-core- plasmid, pHBc14-18M,pHBc120-135M and pHBc122-139M mutant groups formed nucleocapsid-like structure but pHBc23-39M could not, Northern and Southern blot displayed no signal in all mutants except 1-183flag conrol group. In pHBV1.2 cotransfection experiment, HBV DNA replication was blocked in pHBc14-18M, pHBc120-135M and pHBc122-139M mutant groups, sharply decreased in pHBc120-135M and pHBc122-139M groups, correspondingly virons production in medium were also inhibited. pHBc23-39M mutant exerted no influence on HBV replication. Conclusion pHBc23-39M mutant can neither form nucleocapsid-like structure nor interact with wild type HBc dimmer to interfere HBV replication.On the contrast, pHBc14-18M, pHBc120-135M and pHBc122-139M mutants can form nucleocapsid-like structure by themselves, but this structure does not support HBV DNA synthesis. Besides, they can effectively inhibit wild type HBV DNA replication by contacting with wild HBc dimmers resulting in nucleocapsid dysfunction.