本文選題：AcMNPV　切入點(diǎn)：DNA甲基化　出處：《復(fù)旦大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：病毒感染宿主時(shí),與宿主相互作用的許多方面涉及表觀遺傳學(xué)修飾,而這一修飾對(duì)某些病毒的感染過(guò)程和結(jié)果至關(guān)重要。已有的與病毒相關(guān)的表觀調(diào)控研究集中在一些潛伏性或非裂解性復(fù)制病毒,而較少關(guān)注裂解性病毒的表觀調(diào)控。本文研究了桿狀病毒苜蓿銀紋夜蛾核型多角體病毒(Autographa californica multicapsid nucleopolyhedrosis, AcMNPV)在昆蟲(chóng)細(xì)胞Sf9中進(jìn)行裂解性復(fù)制時(shí)的DNA甲基化情況及其作用。首先,通過(guò)使用甲基轉(zhuǎn)移酶抑制劑DAC探究了低甲基化的細(xì)胞環(huán)境對(duì)AcMNPV復(fù)制的影響。.經(jīng)30nM DAC預(yù)處理的細(xì)胞中,DNA甲基轉(zhuǎn)移酶DNMT1被有效降解,感染后8h的病毒DNA水平是對(duì)照組的8倍,甲基化測(cè)序表明此時(shí)ie0啟動(dòng)子區(qū)的甲基化水平較對(duì)照組低40%,ie1啟動(dòng)子區(qū)甲基化水平則降低50%。測(cè)定病毒的生長(zhǎng)曲線(xiàn)發(fā)現(xiàn),30nM的DAC也會(huì)促進(jìn)出芽型病毒BV的產(chǎn)生,使得在感染后的前24h中,病毒粒子釋放較對(duì)照組加快,但是這一差異隨著培養(yǎng)時(shí)間的延長(zhǎng)而趨于微弱,在最終的病毒效價(jià)上無(wú)明顯差異。這一結(jié)果初步確定了DNA甲基化水平的降低對(duì)病毒的DNA復(fù)制和出芽病毒的產(chǎn)生具有促進(jìn)作用為了弄清DAC是究竟通過(guò)影響病毒DNA的甲基化而直接影響病毒復(fù)制,還是通過(guò)影響細(xì)胞的基因表達(dá)狀態(tài)而間接影響病毒復(fù)制,我們我們進(jìn)行了在DAC存在的細(xì)胞環(huán)境下的病毒連續(xù)傳代的實(shí)驗(yàn)。DNA甲基化測(cè)序證實(shí),病毒的重要基因ie0, ie1啟動(dòng)子區(qū)的甲基化水平隨病毒連續(xù)傳代而持續(xù)降低。將得到的低甲基化病毒進(jìn)行感染細(xì)胞,發(fā)現(xiàn)這種“低甲基化”病毒在前48h內(nèi)DNA復(fù)制拷貝數(shù)平均為對(duì)照病毒的5倍,病毒極早期基因ie0、ie1、ie2和滯早期基因Dnapol、lef1、 lef2的轉(zhuǎn)錄活性也有明顯提高。將‘低甲基化”的桿狀病毒基因轉(zhuǎn)導(dǎo)哺乳細(xì)胞CHO,其介導(dǎo)的外源基因luciferase和egfp的表達(dá)水平也3倍高于對(duì)照病毒,表現(xiàn)出一定的應(yīng)用價(jià)值。此外,還通過(guò)體外甲基化和瞬時(shí)表達(dá)實(shí)驗(yàn)研究了病毒啟動(dòng)子甲基化對(duì)其活性的影響。構(gòu)建了攜帶病毒關(guān)鍵基因啟動(dòng)子區(qū)的報(bào)告質(zhì)粒,體外甲基化修飾后轉(zhuǎn)染Sf9細(xì)胞,測(cè)定甲基化修飾對(duì)報(bào)告基因luciferase轉(zhuǎn)錄活性的影響,證實(shí)了病毒的一些關(guān)鍵基因如ie1、pe38的啟動(dòng)子活性受甲基化修飾抑制,出入意料的是,p35、 Dnapol、lef1、lef3、lef8的啟動(dòng)子活性受甲基化修飾而略有激活。綜上,DNMT1抑制劑DAC可以促進(jìn)病毒DNA復(fù)制和出芽病毒粒子的釋放。與此相印證,低甲基化病毒感染細(xì)胞,DNA復(fù)制水平有明顯提高,一些復(fù)制關(guān)鍵基因如ie0、ie1、ie2、Dnapol、lef1、lef2的轉(zhuǎn)錄水平較正常病毒也有提高。體外實(shí)驗(yàn)也初步顯示了一些啟動(dòng)子活性ie1、pe38受甲基化修飾影響。這些結(jié)果為我們深入認(rèn)識(shí)病毒裂解性復(fù)制過(guò)程中病毒DNA甲基化的作用提供了有益的線(xiàn)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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R373

【共引文獻(xiàn)】

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本文編號(hào)：1640898