人细小病毒B19类染色质结构调控基因组复制及RNA加工

发布时间：2018-01-17 17:19

本文关键词：人细小病毒B19类染色质结构调控基因组复制及RNA加工　出处：《中国科学院武汉病毒研究所》2017年博士论文　论文类型：学位论文

【摘要】：人类细小病毒B19(B19V)属于细小病毒科的嗜红细胞属,基因组为单链DNA,全长5.6kb。B19V和人博卡病毒是现今可以感染人类的仅有的两种细小病毒科的病毒。B19V感染主要引起传染性红斑、急性再生障碍性贫血、关节炎等,感染孕妇后可导致胎儿水肿,流产或者死胎。B19V基因组内有两个polyA信号,位于3'端的称为(pA)d,位于第二个内含子内的称为(pA)p。(pA)p直接抑制病毒转录出全长pre-mRNA,在转录后水平与第二个内含子的剪切相互竞争抑制病毒结构蛋白mRNA的生成,而病毒的复制利于启动子通读过(pA)p位点,使用(pA)d位点。病毒基因组的复制和第二个内含子的剪切都影响(pA)p的使用,但是复制如何影响(pA)p的使用还不清楚。细小病毒MVM和腺相关病毒AAV感染敏感细胞后,可以和组蛋白结合,形成类染色质结构,但是类染色质结构有哪些功能还没有相关报道。因此,本研究主要目的是探讨B19V基因组是否可以在细胞内形成类染色质结构,研究B19V基因组甲基化修饰以及类染色质结构对基因复制和RNA加工的影响,进一步揭示基因组复制影响(pA)p使用的分子机制。为了研究B19V基因组DNA是否形成类染色质结构,转染B19V DNA到HEK293T细胞内,分别在12、24、36和48小时提取细胞核,微球菌核酸酶消化后用苯酚-氯仿抽提,同时在这四个时间段提取Hirt DNA,Southern blot检测。结果证实B19V基因组DNA可以在细胞内形成类染色质结构,且类染色质结构的形成早于复制。为了研究病毒染色质的修饰是否影响病毒的复制、转录或转录后加工,我们应用甲基转移酶抑制剂5-Aza-2’-deoxycytidine(DAC)处理转染B19V基因组DNA后的HEK293T细胞,48小时后提取小分子量DNA(Hirt DNA),利用DpnI消化,用甲基化检测试剂盒处理后进行PCR扩增反应,通过测序我们发现,DAC处理后基因组上特定位点的甲基化修饰被抑制,表明在B19V基因组内存在甲基化修饰位点。用终浓度为20μM的DAC处理转染后的293T细胞,48h后分别提取细胞核、Hirt DNA和总RNA,并用Southern blot和RNA酶保护实验进行检测。结果发现,与DMSO处理组和未处理组比较,DAC明显抑制B19V类染色质结构的形成及基因组的复制。RPA实验证实DAC抑制第二个内含子的剪切,促进(pA)p的使用。为了研究B19V基因组复制影响(pA)p使用的具体机制,分别利用IRES2-eGFP和pBluescript KS II载体构建了基因组5’和3’端一半基因的复制型和非复制型克隆,并在此基础上获得了敲除D1和D2剪切位点的克隆,转染HEK293T细胞,48小时后提取总RNA,RNA酶保护实验进行检测。结果发现B19V基因组的复制主要通过影响第二个内含子的剪切影响(pA)p的使用。本研究证实B19V基因组上存在甲基化修饰,DAC可以使基因组去甲基化,抑制类染色质结构的形成和复制,导致基因组第二个内含子的剪切被抑制,进而影响(pA)p的使用。本研究首次报道了B19V基因组甲基化修饰和类染色质结构影响基因组的复制和RNA转录后加工,揭示了B19V基因组中央poly位点选择性使用的分子调控机制,为B19V的防治提供了理论依据。
[Abstract]:Human parvovirus B19 (B19V) belongs to the Parvoviridae of eosinophilic cells, single stranded DNA genome, full-length 5.6kb.B19V and Boka virus can infect humans is now only two minute virus of.B19V infection mainly caused by infectious erythema, acute aplastic anemia, arthritis, after infection of pregnant women lead to fetal edema, abortion or stillbirth within the.B19V genome with two polyA signal in 3'terminal called D (pA), located in the second intron called (pA) P. (pA) P directly inhibit viral transcription of full-length pre-mRNA, competing suppression of virus structural protein mRNA in shear transcription after the level and second introns, and the replication of the virus to read through the promoter (pA) P locus, using (pA) d site. The shear replication of the virus genome and second introns are affected (pA) the use of P, but how to copy. Ring (pA) the use of P is not clear. Parvovirus MVM and adeno-associated virus AAV infection sensitive cells, and can be combined with histones, chromatin structure formation, but the type of chromatin structure which function has not been reported yet. Therefore, the main purpose of this study is to investigate whether B19V gene group can be formed the class structure of chromatin in the cells, the study of B19V genome methylation and influence of chromatin structure on gene duplication and RNA processing, to further reveal the influence of genome replication (pA) molecular mechanism used by P. In order to study the B19V genomic DNA formation of chromatin structure, transfection of B19V DNA into HEK293T cells were extracted in 48 hours and 12,24,36 nuclei, micrococcal nuclease digestion by phenol chloroform extraction, DNA extraction and Hirt in the four time, Southern blot detection. The results demonstrated that B19V genome DNA can be in the cell In the formation of chromatin structure, formation and chromatin structure in early replication. In order to study whether the virus modified chromatin influence viral replication, transcription or post transcription processing, we used the methyltransferase inhibitor 5-Aza-2 -deoxycytidine (DAC) transfected B19V gene group DNA after the HEK293T cells after 48 hours the extraction of low molecular weight DNA (Hirt DNA), using DpnI digestion treatment test kit with methyl PCR after amplification by sequencing, we found that after the treatment of DAC methylation specific sites on the genome was inhibited, showed that in the memory in the B19V genome methylation sites. With a final concentration of 20 M DAC after transfection of 293T cells after 48h nuclei were extracted, Hirt DNA and RNA, and were detected by Southern blot and RNA enzyme protection experiments. The results showed that DMSO treatment group and untreated group, DAC significantly Copy the.RPA experimental inhibition of B19V dyeing formation and genome structure of the matter confirmed that the shear DAC inhibition of the second introns (pA) to promote the use of P. In order to study the influence of B19V genome replication (pA) specific mechanism used by P, IRES2-eGFP and pBluescript respectively by KS II carrier to construct genomic 5 'and 3' at the end of half gene replicating and non replicating clones, and based on the cloning of knock except D1 and D2 splice sites, were transfected into HEK293T cells, 48 hours after the total RNA was extracted from experiment RNA enzyme protection were detected. The results showed that B19V genome replication mainly through the influence of shear second introns (pA the use of P). This study confirmed the presence of methylation of the B19V genome, DAC can make the genome demethylation and inhibition of chromatin structure formation and replication, leading to shear genomic second introns were inhibited, and Effect of P (pA). This is the first report of B19V genome methylation and chromatin structure of genome replication and RNA transcription, revealing the molecular mechanism of B19V genome poly central site selective use, provides a theoretical basis for the prevention and treatment of B19V.

【学位授予单位】：中国科学院武汉病毒研究所
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q78;R373

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