家蚕核型多角体病毒(BmNPV)lef-11基因在病毒感染宿主细胞中的表达特征

Expression Characterization of Bombyx mori Nucleopolyhedrovirus lef-11 Gene in the Virus-infected Host Cells

杆状病毒晚期表达因子(late expression factor,LEF)是参与病毒基因组DNA复制和病毒基因转录调控的一个重要家族,该家族成员LEF-11为13 k D的小分子蛋白质。通过比对目前已经测序的63种杆状病毒基因组序列,发现LEF-11的编码基因lef-11在除宿主为双翅目昆虫的Cuni NPV外的所有杆状病毒基因组中都存在,暗示lef-11基因可能在包括家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,Bm NPV)在内的杆状病毒复制过程中行使重要功能。通过RT-PCR检测和Western blotting分析lef-11基因及其编码蛋白质在Bm NPV侵染的家蚕卵巢培养细胞Bm N中的转录与表达时相,其转录本从侵染后6h开始持续表达,蛋白质从侵染后12 h开始持续表达。当利用DNA聚合酶特异抑制剂阿非迪霉素(aphidicolin)阻断Bm NPV病毒基因组DNA复制之后检测LEF-11的表达受到抑制,暗示LEF-11的表达起始于病毒DNA复制之后。以上结果表明:BmNPV lef-11基因在杆状病毒中非常保守,是一个晚期表达基因,可能不直接参与病毒的起始复制,而是参与病毒的大规模复制过程。

Baculovirus late expression factor（ LEF） are involved in viral DNA replication and viral gene transcriptional regulation. Baculovirus lef-11 gene encodes a putative protein with predicted molecular weight of 13 k D. Sequence-based alignment showed that Bombyx mori nucleopolyhedrovirus（ Bm NPV） lef-11 gene is conserved in 63 sequenced baculovirus genomes except dipteran-specific nucleopolyhedrovirus Cuni NPV,which suggests that lef-11 may play an important role in viral replication cycle. After RT-PCR and Western blotting assay were employed to characterize the temporal transcription pattern of lef-11 and the expression pattern of LEF-11 in Bm N cells,it was found that lef-11 gene and LEF-11 protein were continuously expressed after 6 h and 12 h posterior infection respectively. While aphidicolin,a specific inhibitor against DNA polymerase,was utilized to block viral DNA replication, the expression of LEF-11 was also inhibited,suggesting that LEF-11 protein expression is initiated after DNA replication. These results indicate that Bm NPV lef-11 gene is highly conserved in baculoviruses and is a late expression gene. This gene may not be directly involved in the initiation of virus replication but functions in large-scale replication of virus at late stage.