杆状病毒复制和基因表达的表观遗传学调控研究

【作者】 王鑫；

【导师】 钟江；

【作者基本信息】 复旦大学 ， 微生物学， 2010， 博士

【摘要】 在过去的三十年里,表观遗传学是生命科学研究的一个热点,产生了许多重大的研究成果。但是病毒学家们似乎对此无动于衷。直到现在,我们能找到的有关病毒的表观遗传学调控的文章都很有限。在这里,比起有潜伏感染期的单纯疱疹病毒(HSV)、乙型肝炎病毒(HBV)、Epstein-Barr病毒(EBV)和人类乳头状瘤病毒(HPV),裂解性复制病毒复制周期中的表观遗传学调控更少被关注。本文重点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)在昆虫细胞中进行裂解性复制时的一些表观遗传学现象,包括病毒小染色体结构、组蛋白乙酰化修饰和DNA甲基化修饰在杆状病毒的复制和基因表达中的作用。首先,我们证明了病毒小染色体结构和组蛋白乙酰化修饰在病毒基因转录中的作用。我们证实插入在病毒polyhedrin位点下游的鸡β-珠蛋白5’-HS4绝缘子(HS4)能显著提高polyhedrin启动子控制的报告基因的表达水平。当使用增强型绿色荧光蛋白(egfp)作为报告基因时,在感染后72h和96h,带HS4的重组病毒(AcEGFP-HS4)感染的昆虫细胞比不带HS4的对照病毒(AcEGFP)感染的细胞荧光分别高3倍和2.1倍。AcEGFP-HS4感染的细胞的egfp的mRNA水平也较AcEGFP感染的细胞高。在以萤火虫荧光素酶(firefly luciferase)或分泌型碱性磷酸酶(SEAP)为报告基因时,也观察到类似的表达水平的提高。进一步的机制研究表明,polyhedrin位点插入的HS4没有显著影响到病毒复制,而HS4的作用具有方向性且对组蛋白乙酰基转移酶抑制剂敏感。从DNaseⅠ敏感实验看出,HS4的存在显著提高了附近DNA对核酸酶的敏感性,但对距离较远的DNA没有作用。这些结果表明HS4可能通过改变在病毒形成的小染色体上邻近染色体结构来影响病毒基因表达。这一研究结果为提高杆状病毒-昆虫细胞表达系统表达目标蛋白的效率提供了新的途径,也明确提示在杆状病毒裂解性复制过程中,病毒小染色体的结构及组蛋白的乙酰化修饰对病毒基因的表达起着重要的作用。为了揭示其它表观遗传学调控机制在杆状病毒复制过程中的作用,我们进一步研究了病毒复制过程中的DNA甲基化问题。我们首次证实病毒极早期基因ie0的转录受DNA甲基化调控。IE0是病毒的一个重要调控因子,在感染早期表达水平相对较高,而在感染晚期转录下调。这种表达水平的变化对病毒复制具有重要意义。我们发现：在感染后Oh (Ohp.i.) ie0启动子甲基化程度较高,8h p.i.时甲基化程度明显降低,但从18h p.i.起,这一区域甲基化程度又明显提高。体外DNA甲基化修饰实验表明DNA甲基化可以抑制ie0表达。DNA甲基转移酶抑制剂(DNMTi) DAC (50nM)的存在有助于维持ie0启动子低甲基化,并显著增加ie0的表达。这些研究结果证实启动子区域的DNA甲基化是ie0在感染晚期转录下调的原因,表明DNA甲基化在裂解性复制病毒基因表达的调控中也有重要作用。为了全局性的了解DNA甲基化调控在病毒复制和基因转录中的作用,我们还研究了DAC对病毒复制和基因转录的影响。用50nM DAC处理,可使AcMNPV感染Sf9细胞时病毒效价下降到对照的1／30,并显著地延迟病毒DNA复制。DAC处理对病毒极早期基因的表达在感染后不同阶段有不同的影响。在2h p.i., DAC可以提高病毒极早期基因iel和ie2的表达,而在感染晚期,DAC抑制iel和ie2的转录。但是在整个复制周期中,DAC都提高ie0的转录。DAC对病毒滞早期基因转录的影响不均一,但抑制晚期基因的转录。DAC对病毒极晚期启动子的活性的抑制作用尤为明显。对于DAC抑制极晚期启动子的机制我们进行了进一步的研究,发现用质粒额外表达IE1可以缓解DAC的对极晚期启动子的抑制作用。这些数据,结合前面的结果和对IE0,IE1功能已有的认识,提示在感染的后期,DAC是通过抑制ie1的转录,同时避免ie0转录的降低,改变了IEO,IE1两个蛋白的相对比例,从而抑制了极晚期启动子的激活。这一研究结果表明DNA甲基化对病毒复制的调控作用可能主要是通过影响关键调控因子的转录来实现。除了感染昆虫细胞以外,杆状病毒还能高效地侵入多种哺乳动物细胞。它作为一种新型哺乳动物转导载体正在受到越来越多的重视。但目标基因的表达沉默限制了杆状病毒这方面的应用。尽管人们早已发现组蛋白去乙酰基转移酶抑制剂可以提高杆状病毒介导的基因在哺乳动物细胞中的表达,但另一类引起表观遗传学修饰变化的药物,DNMTi却被报道没有这种效果。在本论文中,我们首次发现在病毒转导哺乳动物细胞前使用DNMTi AZA或DAC处理细胞,在多种哺乳动物细胞中杆状病毒介导的基因的表达水平均有四倍的提高；但如果添加药物的时间推迟,则其提高表达水平的作用会减弱乃至消失。同时在病毒接种期间有DNMTi的存在对其发挥作用至关重要。进一步的结果表明DNMTi可能是通过抑制细胞对病毒DNA降解起作用。这项研究为提高杆状病毒载体在哺乳动物细胞中应用的效果提供了新途径,也为揭示杆状病毒与哺乳动物细胞的作用关系,认识哺乳动物细胞对非特异性入侵物的反应及机理提供了新的数据。更多还原

【Abstract】 In the last three decades, epigentics has been one of the fast developing areas in life science. Many great results were reported. However, virologists seem to be spectators in this field. Until recently, only a few reports about viral epigenetic regulation could be found. Compared with viruses with latent infection phase, such as herpes simplex virus (HSV), hepatitis B virus (HBV), Epstein-Barr virus (EBV) and human papillomavirus (HPV), less concern was given to the epigenetic regulation in the replication of lytic viruses. In this dissertation, we focused on epigentics regulation of baculovirus AcMNPV(Autographa californica multicapsid nucleopolyhedrovirus) during its lytic infection in insect cells. We studied the roles of viral minichromosome structure, histone acetylation and DNA methylation in viral gene expression and virus replication.We first studied the roles of viral minichromosome structure and histone acetylation in viral gene expression. We found that chickenβ-globin 5’-HS4 insulator (HS4), which was placed downstream of the polyhedrin promoter-directed foreign gene expression cassette in AcMNPV, markedly increased the expression of reporter genes. When enhanced green fluorescence protein gene (egfp) was used as the reporter gene, cells infected by the recombinant virus with HS4 (AcEGFP-HS4) showed 3.0 and 2.1-fold stronger fluorescence than that by the control virus without HS4 (AcEGFP) at 72 and 96 h post infection, respectively. The level of egfp mRNA was also much higher in cells infected by AcEGFP-HS4 than that by AcEGFP. An increase in gene expression was also seen when firefly luciferase gene or secreted alkaline phosphatase gene was used as a reporter. The insertion of HS4 in the polyhedrin locus has no significant effect on virus replication. The effect of HS4 was orientation-dependent, and sensitive to inhibitors of histone acetyltransferase. In DNase I sensitivity assay, HS4 significantly increased the sensitivity of neighbouring DNA to nuclease, but had little effect on DNA of a distal locus. These results suggested that HS4 insulator might affect baculovirus gene expression by modifying the structure of neighbouring chromatin in the virus minichromosome. These results provided a new method to improve baculovirus-insect cell expression system, and clearly suggested viral minichromosome and histone acetylation play an important role in virus replication and viral gene expression.To analyse other epigenetic regulation mechanisms in baculovirus replication, we then investigated DNA methylation during virus replication. We for the first time showed that the expression of viral immediate-early gene ieO was affected by the DNA methylation in the promoter area. IEO was an important viral regulator that was expressed at relatively higher level at the early phase of infection, and then down-regulated at late stage. The down-regulation of ieO was known to be imporant for normal virus replication. We found that ieO promoter was hypermethylated Oh p.i., hypomethylated 8h p.i., and back to hypermethylated state again after 18h p.i. In vitro DNA methylation test showed that DNA methylation inhibited ieO expression. Treatment with 50nM DAC, a kind of DNA methyltransferase inhibitor (DNMTi) maintained the hypomethylation of ieO promoter and increased ieO expression. These results indicated that DNA meythlation in the ieO promoter contributed to the down-regulation in ieO expression at the late phase of viral infection, and showed that DNA methylation played a role in the regulation of gene expression in lytic virus replication.To understand the general relationship between DNA methylation and virus replication and viral gene expression, we also studied the effect of DNA methylation inhibitor, DAC on the virus replication and viral gene expression. Presence of 50nM DAC decreased viral titer to only 1/30 and delayed viral DNA replication significantly. Treatment with DAC has different effect on immediate early gene expression in different phases of infection. DAC increased the expression of immediate early gene iel and ie2 2h p.i., and inhibited their expression later. Meanwhile, DAC increased the expression of another immediate early gene, ie0, both early and late in the infection. The effect of DAC on the expression of viral delayed early genes was not uniformed, but it inhibited the expression of all late genes tested. DAC had more significant effect on the expression of viral very late genes, resulted in almost complete elimination of the protein production. The mechanism of DAC to inhibit very late gene expression was further studied. It was found that additional expression of IE1 by plasmid relieved the inhibitory effect of DAC on very late promoter. These data, in combination with results above, and known function of IEO and IE1 proteins, implied that DAC inhibited very late promoter by decreasing the expression iel, while while preventing the down-regulation of ieO expression. This will resulted in the improper ratio of IEO and IE1 in infected cells, which was known to be essential for the activation of viral very late promoter late in infection.These results suggested that viral DNA methylation might affect virus replication and viral gene expression by modifying the expression of a few important viral regulatory genes.Apart from infecting insect cells, baculovirus could also enter many mammalian cells, too. In recent years, baculovirus has attracted more and more attentions as a gene delivery vector for mammalian cells. However, the silencing of target gene mediated by baculovirus limited its application. Although histone deacetylation inhibitors were known to improve baculovirus-mediated gene expression in mammalian cells for long time, another group of chemicals that induces epigenetic changes, DNMTi, was reported to have no such effect. In this dissertation, we for the first time found that DNMTi AZA or DAC improved baculovirus-mediated gene expression by fourfold or more in all four mammalian cell lines tested when they were added prior to virus inoculation. This improvement of gene expression was less significant or even abolished when drugs were added after virus inoculation. The further results indicated that DNMTi improved baculovirus-midated gene expression by inhibiting viral DNA degradation in transduced mammalian cells. This research provided a new method to improve the application of baculovirus vector in mammalian cells, and offered new insights to understand the relationship between baculovirus vector and mammalian cells, and the response to non-specific invasion by mammalian cells.更多还原