中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2010年
8期
730-736
,共7页
邓凯%蒋栋%韩进超%潘孝本%王豪%魏来
鄧凱%蔣棟%韓進超%潘孝本%王豪%魏來
산개%장동%한진초%반효본%왕호%위래
乙型肝炎病毒%核心蛋白%核心颗粒组装%突变体
乙型肝炎病毒%覈心蛋白%覈心顆粒組裝%突變體
을형간염병독%핵심단백%핵심과립조장%돌변체
hepatitis B virus%HBc%Core particle assembly%Mutants
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.
目的 探討乙型肝炎病毒覈心蛋白(hepatitis B virus core protein,HBc)二聚體組裝相關功能域突變對覈心顆粒組裝及HBV複製的影響.方法 基于HBc空間結構,PCR定點誘變二聚體組裝覈衣殼相關功能域重要氨基痠位點,以pcDNA3.1為載體構建4箇突變體錶達質粒pHBc14-18M、pHBc120-135M、pHBc23-39M和pHBc122-139M.將突變質粒與HBc缺失的含1.2拷貝HBV基因質粒pHBV1.2-core-共轉染HepG2細胞,通過Northern blot檢測HBV前基因組(pgRNA),Southern blot檢測複製中間體,非變性瓊脂糖凝膠電泳(native agarose gel electrophoresis)及Western blot檢測細胞覈心顆粒觀察突變質粒自身形成覈衣殼情況.將突變質粒與含有1.2拷貝HBV基因質粒pHBV1.2共轉染HepG2細胞觀測突變質粒對野生型HBc組裝及HBV複製的影響.結果 突變質粒與pHBV1.2-core-質粒共轉染HepG2細胞,pHBc14-18M、pHBc120-135M和pHBc122-139M突變能夠組成覈衣殼樣結構.pHBc23-39M不能形成覈衣殼樣結構.Northern blot結果顯示所有突變質粒組均未見pgRNA條帶,Southern blot檢測複製中間體也未見條帶.將突變質粒與pHBV1.2共轉染HepG2細胞,pHBc14-18M、pHBc120-135M和pHBc122-139M組HBV複製中間體及上清中病毒顆粒明顯減少,而pHBc23-39M組無減少.結論 HBc23-39位氨基痠突變可阻止二聚體多聚化,不能形成覈衣殼樣結構,且不能與野生HBc二聚體相互作用.HBc14-18、120-135、122-139區域的氨基痠突變,能夠形成覈衣殼樣結構但不支持HBV DNA複製,併且能與野生HBc二聚體相互作用,形成雜閤體,榦擾野生型HBV DNA的複製.
목적 탐토을형간염병독핵심단백(hepatitis B virus core protein,HBc)이취체조장상관공능역돌변대핵심과립조장급HBV복제적영향.방법 기우HBc공간결구,PCR정점유변이취체조장핵의각상관공능역중요안기산위점,이pcDNA3.1위재체구건4개돌변체표체질립pHBc14-18M、pHBc120-135M、pHBc23-39M화pHBc122-139M.장돌변질립여HBc결실적함1.2고패HBV기인질립pHBV1.2-core-공전염HepG2세포,통과Northern blot검측HBV전기인조(pgRNA),Southern blot검측복제중간체,비변성경지당응효전영(native agarose gel electrophoresis)급Western blot검측세포핵심과립관찰돌변질립자신형성핵의각정황.장돌변질립여함유1.2고패HBV기인질립pHBV1.2공전염HepG2세포관측돌변질립대야생형HBc조장급HBV복제적영향.결과 돌변질립여pHBV1.2-core-질립공전염HepG2세포,pHBc14-18M、pHBc120-135M화pHBc122-139M돌변능구조성핵의각양결구.pHBc23-39M불능형성핵의각양결구.Northern blot결과현시소유돌변질립조균미견pgRNA조대,Southern blot검측복제중간체야미견조대.장돌변질립여pHBV1.2공전염HepG2세포,pHBc14-18M、pHBc120-135M화pHBc122-139M조HBV복제중간체급상청중병독과립명현감소,이pHBc23-39M조무감소.결론 HBc23-39위안기산돌변가조지이취체다취화,불능형성핵의각양결구,차불능여야생HBc이취체상호작용.HBc14-18、120-135、122-139구역적안기산돌변,능구형성핵의각양결구단불지지HBV DNA복제,병차능여야생HBc이취체상호작용,형성잡합체,간우야생형HBV DNA적복제.
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.
