杆状病毒DNA甲基化及其在病毒复制过程中的作用初探

发布时间：2018-03-20 21:40

本文选题：AcMNPV　切入点：DNA甲基化　出处：《复旦大学》2012年硕士论文　论文类型：学位论文

【摘要】：病毒感染宿主时,与宿主相互作用的许多方面涉及表观遗传学修饰,而这一修饰对某些病毒的感染过程和结果至关重要。已有的与病毒相关的表观调控研究集中在一些潜伏性或非裂解性复制病毒,而较少关注裂解性病毒的表观调控。本文研究了杆状病毒苜蓿银纹夜蛾核型多角体病毒(Autographa californica multicapsid nucleopolyhedrosis, AcMNPV)在昆虫细胞Sf9中进行裂解性复制时的DNA甲基化情况及其作用。首先,通过使用甲基转移酶抑制剂DAC探究了低甲基化的细胞环境对AcMNPV复制的影响。.经30nM DAC预处理的细胞中,DNA甲基转移酶DNMT1被有效降解,感染后8h的病毒DNA水平是对照组的8倍,甲基化测序表明此时ie0启动子区的甲基化水平较对照组低40%,ie1启动子区甲基化水平则降低50%。测定病毒的生长曲线发现,30nM的DAC也会促进出芽型病毒BV的产生,使得在感染后的前24h中,病毒粒子释放较对照组加快,但是这一差异随着培养时间的延长而趋于微弱,在最终的病毒效价上无明显差异。这一结果初步确定了DNA甲基化水平的降低对病毒的DNA复制和出芽病毒的产生具有促进作用为了弄清DAC是究竟通过影响病毒DNA的甲基化而直接影响病毒复制,还是通过影响细胞的基因表达状态而间接影响病毒复制,我们我们进行了在DAC存在的细胞环境下的病毒连续传代的实验。DNA甲基化测序证实,病毒的重要基因ie0, ie1启动子区的甲基化水平随病毒连续传代而持续降低。将得到的低甲基化病毒进行感染细胞,发现这种“低甲基化”病毒在前48h内DNA复制拷贝数平均为对照病毒的5倍,病毒极早期基因ie0、ie1、ie2和滞早期基因Dnapol、lef1、 lef2的转录活性也有明显提高。将‘低甲基化”的杆状病毒基因转导哺乳细胞CHO,其介导的外源基因luciferase和egfp的表达水平也3倍高于对照病毒,表现出一定的应用价值。此外,还通过体外甲基化和瞬时表达实验研究了病毒启动子甲基化对其活性的影响。构建了携带病毒关键基因启动子区的报告质粒,体外甲基化修饰后转染Sf9细胞,测定甲基化修饰对报告基因luciferase转录活性的影响,证实了病毒的一些关键基因如ie1、pe38的启动子活性受甲基化修饰抑制,出入意料的是,p35、 Dnapol、lef1、lef3、lef8的启动子活性受甲基化修饰而略有激活。综上,DNMT1抑制剂DAC可以促进病毒DNA复制和出芽病毒粒子的释放。与此相印证,低甲基化病毒感染细胞,DNA复制水平有明显提高,一些复制关键基因如ie0、ie1、ie2、Dnapol、lef1、lef2的转录水平较正常病毒也有提高。体外实验也初步显示了一些启动子活性ie1、pe38受甲基化修饰影响。这些结果为我们深入认识病毒裂解性复制过程中病毒DNA甲基化的作用提供了有益的线索。
[Abstract]:When a virus infects a host, many aspects of its interaction with the host involve epigenetic modification. This modification is critical to the infection process and results of some viruses. Previous viro-related epiregulatory studies have focused on latent or non-lytic replicating viruses. But less attention was paid to the apparent regulation of lytic virus. In this paper, we studied the DNA methylation of nucleopolyhedrosis virus Autographa californica multicapsid nucleopolyhedrosis (AcMNPV) in insect Sf9. The effect of hypomethylated cell environment on AcMNPV replication was investigated by using DAC, a methyltransferase inhibitor. The DNA methyltransferase DNMT1 was effectively degraded in the cells pretreated with 30nm DAC. The viral DNA level was 8 times higher than that in the control group at 8 h after infection. Methylation sequencing showed that the methylation level in the promoter of ie0 was 40% lower than that in the control group, and the methylation level of the promoter was 50% lower than that of the control group. The growth curve of the virus was determined. It was found that the DAC of 30 nm could also promote the production of BV of budding virus. In the first 24 hours after infection, the release of virus particles was faster than that of the control group, but the difference tended to be weak with the increase of culture time. There is no obvious difference in the final viral titer. This result preliminarily confirmed that the reduction of DNA methylation level can promote the replication of virus DNA and the production of budding virus in order to find out whether DAC is affected by the viral DNA. Methylation directly affects viral replication, Or indirectly affecting viral replication by affecting the state of gene expression in the cell, we have done a serial passage of the virus in a cellular environment where DAC exists. DNA methylation sequencing has confirmed that, The methylation level of the important gene ie0 and ie1 promoter of the virus decreases with the continuous passage of the virus. The resulting hypomethylated virus will infect the cells. It was found that the copy number of DNA replication of the hypomethylated virus was 5 times of that of the control virus in the first 48 hours. The transcriptional activity of the very early stage virus gene ie0, ie1ie2 and the hysteretic early stage gene Dnapolopsis lef1, lef2 were also significantly increased. The expression level of exogenous genes luciferase and egfp mediated by 'low methylated' baculovirus gene transduction into mammalian cells was also 3 times higher than that of the control virus. In addition, the effect of viral promoter methylation on its activity was studied by in vitro methylation and transient expression experiments. A reporter plasmid carrying the promoter region of the key gene of the virus was constructed. Sf9 cells were transfected with methylation in vitro. The effect of methylation modification on the transcription activity of reporter gene luciferase was determined. It was confirmed that the promoter activity of some key genes of the virus, such as ie1pe38, was inhibited by methylation modification. It was not expected that the promoter activity of Dnapololor lef1 and lef3lef8 was slightly activated by methylation modification. In conclusion, the DNMT1 inhibitor DAC can promote the replication of virus DNA and the release of germinated virus particles. DNA replication in cells infected with hypomethylated virus was significantly increased. The transcriptional level of some key replication genes such as ie0OZI1E1E2Dnapolopolor lef1lef2 was also higher than that of the normal virus. In vitro experiments also showed that some promoter activity ie1pe38 was affected by methylation modification. These results provide us with a better understanding of viral lytic replication. The role of viral DNA methylation in the process provides useful clues.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

【共引文献】