【作者】 周伟东；

【导师】 胡远扬；

【作者基本信息】 武汉大学 ， 微生物学， 2010， 博士

【摘要】 黑胸大蠊浓核病毒(Periplaneta fuliginosa Densovirus,简称PfDNV)是武汉大学胡远扬教授等于1990年分离鉴定。由于其独特的结构和功能,根据2008年国际病毒学分类委员会(ICTV)的第九次报告中,特别设立了黑胸大蠊浓核病毒属(Pefudensovirus),代表种为黑胸大蠊浓核病毒PfDNV。NS1蛋白是PfDNV的一个主要的非结构蛋白,也是一个多功能的调控蛋白,在病毒的复制过程中起着重要作用。已经证明它的功能有ATP结合和水解活性、位点特异的DNA识别和结合活性、位点和链特异性切割活性、解旋酶活性和启动子反式调控作用等。它对病毒DNA的复制、基因表达以及子代病毒粒子的扩增都具有调节作用。此外,NS1蛋白与宿主细胞中的调控蛋白相互作用而影响关键的细胞调控过程,并可以通过内切酶活性直接对宿主细胞的DNA进行攻击。不同浓核病毒的宿主域有宽有窄,如鹿眼蛱蝶浓核病毒(JcDNV)可引起蛱蝶和某些夜蛾的幼虫感染；而PfDNV则只能使大蠊感染,但不能感染德国小蠊。由于PfDNV的宿主特异性,目前还没有找到一个可稳定感染的细胞系。在本文中,我们将详细讨论NS1蛋白核定位信号对PfDNV感染的影响。我们根据已经发表的ns1基因的核酸序列设计特异性引物,以含有PfDNV全基因组的感染性克隆pUC-PfDNV为模板进行PCR,扩增得到编码NS1蛋白的基因。然后将其克隆至原核表达载体pET28-a,转化至大肠杆菌E.coli.BL21(DE3)菌株中,通过IPTG诱导,高效表达出了大量的NS1蛋白。表达产物经亲和层析柱分离纯化后免疫雄性新西兰大白兔,得到特异性抗NS1血清。我们利用DEAE-dextran转染技术,将含有PfDNV基因组的感染性克隆pUC-PfDNV注射入黑胸大蠊三龄幼虫体内,待蟑螂发病后,提取PfDNV病毒粒子。为了检测PfDNV对昆虫病毒的敏感性,提纯的病毒粒子感染Schneider line 2 (S2), Aedes albopictus C6/36 cells (C6/36),和蟑螂血细胞(Hemocytes)。蟑螂血细胞是我们利用原代培养的方法对蟑螂的血细胞进行维持培养。大蠊的原血细胞是一类分化较低细胞,具有极强的分裂能力,能够通过有丝分裂来保证昆虫血细胞数目的稳定。我们只有在蟑螂血细胞实验组中看到了细胞聚集、膨胀、脱落等致细胞病变效应(CPE),在昆虫S2和C6／36细胞中始终都没有看到CPE现象。蟑螂血细胞中的CPE现象大约在感染后48小时就可以看到,到72小时,CPE现象更加明显,已经有60-70%的细胞出现了病变。为了研究NS1蛋白定位,我们通过PCR的方法将nsl基因亚克隆至pAc5.1／V5-His瞬时表达载体后,利用脂质体Cellfectin转染昆虫细胞(S2,C6／36和Hemocytes)。通过荧光抗体复染的激光共聚焦显微镜检测以及Western blot检测,发现NS1蛋白在S2、C6／36细胞内能够正常表达,然而,NS1蛋白无法进入昆虫细胞的细胞核。一个在细胞质中定位的调控蛋白,无法调控PfDNV的转录和复制。NS1蛋白不但能在黑胸大蠊幼虫血细胞中表达,而且能够通过细胞核膜,进入细胞核内。单个的NS1蛋白能够表达并通过核孔进入细胞核,但在真实情况下,对整个病毒感染而言,NS1蛋白是否能够通过黑胸大蠊幼虫血细胞核孔进入细胞核还是一个疑问。于是我们用PfDNV病毒粒子感染昆虫细胞,PfDNV的启动子能够被S2、C6／36和蟑螂血细胞识别而表达出NS1蛋白,而正如含nsl基因的质粒转染的结果一样,NS1蛋白在感染中只能够进入黑胸大蠊血细胞核。NS 1蛋白能够进入黑胸大蠊血细胞核而被S2和C6／36细胞排斥在细胞核外的现象表明,NS1可能存在核定位信号。序列分析发现,NS1蛋白存在两个碱性氨基酸富集区(KAKRRGAIRKRKA (KAK region) and RRRRRR (6R region))。于是我们分别用缺失突变的方法对这两个区域进行研究,证明6R区域具有核定位信号功能,为了进一步证明,我们将6R区与流感病毒的血凝素抗原(HA)连接,在6R区的帮助下HA能够定位在细胞核。而对6R区的点突变实验证明任何一个精氨酸的缺失将导致该区域核定位功能的丧失。更多还原

【Abstract】 Periplaneta fuliginosa Densovirus (PfDNV) isolated from cockroaches in Wuhan, PRC, has been systematically studied for about 20 years. In 2004, International Committee on Taxonomy of Viruses (ICTV) created a new genus (Pefudensovirus) in the subfamily Densovirinae with Periplaneta fuliginosa densovirus as type species owing to its divergences in the genome structure and the gene expression strategy between PfDNV and other DNVs.The regulatory protein NS1 is a key molecule in viral replication.It is a multi-functional protein that has several catalytic activities, including ATP binding and hydrolysis, oligomerization, sitespecific DNA binding to a cognate recognition motif, site-and strand-specific nicking, helicase activity, and promoter trans-regulation. These activities allow this pleiotropic protein to execute the different functions necessary for progeny virion production, including regulation of viral DNA amplification and gene expression. Additionally, some of the latter functions may also act upon the host cell genome and may contribute to the cytotoxic activities of NS1. Moreover, the ability of NS1 physically interacts with a variety of host cell proteins, including components of the DNA replication machinery and transcriptional regulators may represent another "scavenging"mechanism by which NS1 negatively interferes with essential cellular processes.The host ranges of different densoviruses are variable. JcDNV can infect butterfly and some nymph of nocturnal moth; while PfDNV just infects Periplaneta fuliginosa cochroch. Lack of a suitable cell line that permits virus replication to high titers has been a long-standing problem in densovirus research. In this study, infected primary hemocyte cells produced a typical cytopathic effect characterized by cell clumping and syncytia. So the hemocyte cells of P. fuliginosa nymphs, which were permissive for both infection with the purified PfDNV virions and transfection by plasmids, were used to study the nuclear localization signal of the NS1 protein and PfDNV infection.Firstly, the cDNA clone encoding NS1 of PfDNV was prepared from the total mRNA of PfDNV-infected crockroach by reverse transcription-PCR. For expression in E. coli, the DNA fragment was cloned in pET28a plasmid and the protein with his-tagged was expressed and purified by Ni-NTA affinity chromatography for generating polyclonal rabbit antibodies. Affinity and specificity of the anti-NS1 serum were tested by Western blot analysis.The infectious plasmid pUC119-PfDNV was mixed with DEAE-dextran and injected in 3 star nymph of Periplaneta fuliginosa. The PfDNV virion was purified from the infected cochroch. To understand clearly the sensitivity of insect cells, we infected the insect cells of hemocyte, S2 and C6/36 by PfDNV strains. The characteristic cytopathic effect typical for PfDNV, in the form of clumping and ballooning of cells and syncytia formation, was observed in hemocyte cells at around 48 h post-infection. Hemocyte showed an extensive CPE (60-70%) at 72 h post-infection. And the cells were split seriously after 72 h post-infection, and no difference was found between the mock-cells and the insect cell lines, S2 and C6/36.To understand clearly the localization of NS1, we constructed a plasmid, pAcNS1, by PCR the nsl sequence from pUC-PfDNV. Immunofluorescence was then used to determine the cellular distribution of NS1 in the transfected cells. After the pAcNS1 was transfected into the insect cells (hemocytes, S2 and C6/36), NS1 protein was detected at 48 h post-transfection.We found that NS1 was localized to both cytoplasm and nucleus of hemocytes, while it was localized to only the cytoplasm of S2 and C6/36. Transfection efficiency of the plasmid pAcNS1 of hemocyte, S2 and C6/36 cells were 32%,41%and 38%, respectively. In order to confirm these results, cytoplasmic and nuclear extracts were isolated from NS1-transfected cells and analyzed by Western blot. NS1 protein (approximately 71 kDa, with tag) was present in the cytoplasm of the insect cell lines, while NS1 was detected in the nuclear extracts of hemocytes.We next asked whether the NS1 protein was expressed and localized to the nucleus in these insect cells following PfDNV infection. Infection of insect cells using virions of PfDNVwas carried out. Because of the cytopathic effect, immunofluorescence diagnosis directed against NS1 using polyclonal antibodies were performed at 36 h post-infection. NS1 could be observed in insect cells, and was localized to the nucleus exclusively in the hemocyte cells. Infection efficiencies of hemocyte, S2 and C6/36 cells, which accorded to the number of cells expressing NS1, were 16%,31%and 25%, respectively. Western blotting of the fractionated lysates confirmed that NS1 was in the nucleus of hemocyte.更多还原