HPV16主要衣壳蛋白L1与hnRNP H1的相互作用及其功能

发布时间：2018-04-16 02:10

本文选题：人乳头瘤病毒 + 核糖核蛋白　；参考：《厦门大学》2008年硕士论文

【摘要】： 流行病学和相关研究证实人乳头瘤病毒(HPV)感染与尖锐湿疣、[鄙掀ち鲅錾凸卑┑姆⑸叵得芮小PV感染机体后各种病毒基因的表达是受严格调控的,尤其是晚期基因,在未分化的细胞中几乎不表达,随着细胞分裂,病毒基因组随子细胞上移到表皮上层后,晚期蛋白的表达才被启动。这种分化依赖的晚期蛋白表达调控机制尚不明确。在以往的研究中发现,晚期蛋白在转录后水平受多种负调节元件的控制,其中早期多聚腺苷酸化信号(pAE)在晚期基因的转录后调控中起到核心的作用,它能使mRNA的转录提前终止,在HPV16 L2的N端有一个GU含量丰富的序列(GU-rich sequence,GRS),是hnRNP H1蛋白结合位点,能够增强pAE的活性,进一步抑制晚期蛋白的表达。hnRNPH1在细胞中的分布水平有随着细胞的分化而降低的特点,晚期蛋白表达的组织特异性很可能与hnRNP H1的表达有关。我们用免疫共沉淀技术发现hnRNP H1与HPV16 L1蛋白有相互作用,并证实这种相互作用是天然状态下存在的。我们构建了一个模拟HPV转录调控的质粒模型(pGPG),用两个GFP分别代替早期基因和晚期基因,中间加以pAE和下游的GRS序列。通过检测下游的GFP(G2)的RNA以及蛋白质的表达,来反映hnRNP H1蛋白及L1对晚期蛋白转录翻译的影响。在293F了细胞中转染pGPG质粒,几乎检测不到G2的转录产物,蛋白表达量也很低,沉默hnRNP H1基因后,G2的转录和表达量大大提高,印证了hnRNP H1对晚期蛋白的转录抑制效应起重要作用。我们首次发现:HPV16 L1的表达能够有效地提高G2的转录和蛋白表达水平,在HeLa和Huh7等细胞上也发现相同的现象,将hnRNP H1沉默后再转染HPV16 L1质粒,发现在没有H1的情况下,L1对G2的表达没有额外的刺激效应。提示HPV16 L1对晚期蛋白表达的提高可能是通过L1与hnRNPH1结合,抑制了hnRNP H1的功能来实现的。在病毒的感染过程中,L1蛋白很可能参与了晚期基因的转录后调控,对自身的表达有正协同的效应。L1与hnRNPH1相互阻遏,hnRNP H1表达过高时L1的表达被抑制,在分化的细胞中hnRNPH1的表达量降低,pAE活性不高,晚期基因可以通读、表达,随着L1蛋白表达量增多并结合到hnRNP H1上,hnRNP H1失活,最终L1蛋白得以大量表达,启动病毒成熟的后续过程。本文章为更进一步揭示HPV晚期蛋白的表达和病毒生活周期的调控规律提供了一些思路,为HPV引发的多种疾病的致病机理提供理论支持,并为相关治疗药物的研发提供有利的启示。
[Abstract]:Human papillomavirus (HPV) infection and condyloma acuminatum are confirmed by epidemiology and related studies.Wahoo?Chisel?Convex?Perem 5?The expression of various viral genes is strictly regulated, especially the late genes, which are hardly expressed in undifferentiated cells. As the cells divide, the virus genome moves up to the upper epidermis with the daughter cells.Late protein expression was only activated.The regulatory mechanism of this differentiation dependent late protein expression is unclear.In previous studies, it has been found that advanced proteins are controlled by many negative regulatory elements at the post-transcriptional level, among which the early polyadenylation signal (PAE) plays a central role in the post-transcriptional regulation of advanced genes, which can lead to early termination of mRNA transcription.In the N terminal of HPV16 L2, there is a GU-rich sequence of GU-rich sequence GRSs, which is a binding site of hnRNP H1 protein, which can enhance the activity of pAE and further inhibit the expression of late protein .hnRNPH1.The tissue specificity of late protein expression may be related to the expression of hnRNP H 1.The interaction between hnRNP H1 and HPV16 L1 protein was found by immunoprecipitation technique, and it was confirmed that this interaction exists in natural state.We constructed a plasmid model to mimic the transcriptional regulation of HPV. Two GFP were used to replace the early gene and the late gene, respectively, with pAE and downstream GRS sequences.The effect of hnRNP H1 and L1 on the transcriptional translation of advanced proteins was studied by detecting the expression of RNA and protein in the downstream GFP G2).After transfection of pGPG plasmid into 293F cells, the transcription product of G2 was hardly detected, and the protein expression was very low. After silencing hnRNP H1 gene, the transcription and expression of hnRNP H1 was greatly increased, which confirmed that hnRNP H1 played an important role in the transcriptional inhibition of late protein.We found for the first time that the expression of 1: HPV16 L1 could effectively improve the transcription and protein expression of G2. The same phenomenon was found in HeLa and Huh7 cells. HnRNP H1 was silenced and then transfected into HPV16 L1 plasmid.It was found that the expression of G _ 2 was not stimulated by L _ 1 without H _ 1.The results suggest that the increase of HPV16 L1 protein expression may be due to the binding of L1 to hnRNPH1, which inhibits the function of hnRNP H1.In the process of virus infection, it is possible that the L1 protein is involved in the post-transcriptional regulation of late gene, and the positive synergistic effect of the expression of L1 on itself. L1 interacts with hnRNPH1 to suppress the expression of hnRNP H1 when the expression of H1 is too high, and the expression of L1 is inhibited when it is overexpressed.The expression of hnRNPH1 in differentiated cells was not high and the late stage genes could be read and expressed. With the increase of L1 protein expression and the inactivation of hnRNP H1, L1 protein could be expressed in large quantities.Initiate the subsequent process of virus maturation.This article provides some ideas for further revealing the expression of late HPV protein and the regulation of virus life cycle, and provides theoretical support for the pathogenesis of many diseases caused by HPV.And provide beneficial enlightenment for the research and development of related therapeutic drugs.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373

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