Induction of human papillomavirus type 18 late gene expression and genomic amplification in organotypic cultures from transfected DNA templates
Induction of human papillomavirus type 18 late gene expression and genomic amplification in organotypic cultures from transfected DNA templates
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DOI：
10.1128/jvi.71.9.7068-7072.1997
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发表时间：
1997-09-01
影响因子：
5.4
通讯作者：
Laimins, LA
中科院分区：
文献类型：
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作者：
Frattini, MG;Lim, HB;Laimins, LA
人乳头瘤病毒（HPV）在植物病毒生命周期中功能的遗传分析依赖于产生维持转染病毒基因组的附加体拷贝的人角质形成细胞系的能力，我们以前已经证明，用再循环的克隆HPV-1脂质体转染正常人包皮角质形成细胞，31个基因组序列导致高频率的细胞系保持病毒基因组作为染色体外元件（M. G. Frattini，H.林和L. A. Laimins，Proc. Natl. Acad. Sci.第93章：一个女人3062-3067，1996）。在器官型（筏）培养物中生长这些细胞系后，观察到病毒晚期基因的分化依赖性表达、病毒基因组的扩增和病毒粒子生物合成。在本研究中，我证明这些方法不限于HPV-31，而是适用于其它HPV类型，包括致癌HPV-18。从细菌载体序列纯化HPV-18基因组，再连接，并与新霉素选择标记一起转染到正常人包皮角质形成细胞中。在药物选择后，扩增抗性细胞并检查病毒DNA的状态，发现所有检查的细胞系每个细胞含有约100至200个HPV-18 DNA附加体拷贝。这些细胞系在筏培养物中的生长导致E1布尔和E4和L1衣壳基因的分化依赖性表达。在对分化的筏培养物进行DNA原位杂交分析后，在基底上细胞中观察到病毒基因组扩增。这些晚期病毒功能的诱导先前已显示与分化依赖的病毒体生物合成直接相关。我们的研究表明，能够对病毒生命周期的各个阶段进行详细的遗传分析，包括使用第二致癌HPV类型控制分化依赖性晚期病毒功能。
The genetic analysis of human papillomavirus (HPV) functions during the vegetative viral life cycle is dependent upon the ability to generate human keratinocyte cell lines which maintain episomal copies of transfected viral genomes, We have previously demonstrated that lipofection of normal human foreskin keratinocytes with recircularized cloned HPV-31 genomic sequences resulted in a high frequency of cell lines which maintained viral genomes as extrachromosomal elements (M. G. Frattini, H. Lim. and L. A. Laimins, Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996), Following the growth of these cell lines in organotypic (raft) cultures, the differentiation-dependent expression of viral late genes, the amplification of viral genomes, and virion biosynthesis were observed, In the present study, me demonstrate that these methodologies are not restricted to HPV-31 but are applicable to other HPV types, including the oncogenic HPV-18, HPV-18 genomes were purified from bacterial vector sequences, religated, and transfected into normal human foreskin keratinocytes together with a neomycin-selectable marker, Following drug selection, resistant cells were expanded and examined for the state of the viral DNA, All cell lines examined were found to contain approximately 100 to 200 episomal copies of HPV-18 DNA per cell, Growth of these cell lines in raft cultures resulted in the differentiation-dependent expression of the E1 boolean AND E4 and L1 capsid genes, In addition, viral genome amplification was observed in suprabasal cells following DNA in situ hybridization analysis of differentiated raft cultures, The induction of these late viral functions has previously been shown to de directly associated with differentiation-dependent virion biosynthesis, Our studies indicate the ability to perform a detailed genetic analysis of the various phases of the viral life cycle, including control of the differentiation-dependent late viral functions, using a second oncogenic HPV type.