In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression
In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression
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DOI：
10.1073/pnas.93.7.3062
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发表时间：
1996-04-02
影响因子：
11.1
通讯作者：
Laimins, LA
中科院分区：
文献类型：
--
作者：
Frattini, MG;Lim, HB;Laimins, LA
人乳头瘤病毒（HPV）16、18、31和51型是包括宫颈癌在内的许多肛门生殖器癌的病原体，这些“高危”HPV特异性靶向生殖器鳞状上皮，并且它们的裂解生命周期与上皮分化密切相关。我们已经开发了一种在病理发生过程中HPV功能的遗传测定，使用再环化的克隆HPV 31基因组，其仅与耐药标记物一起转染到正常人包皮角质形成细胞（天然宿主细胞）的单层培养物中。在药物选择后，分离稳定维持HPV 31 DNA作为附加体的细胞系，并且当在器官型筏培养物中生长时经历终末分化，在分化的筏中，在基底上细胞中检测到晚期病毒基因的表达、病毒DNA的扩增和病毒颗粒的产生，这证明了从转染的DNA模板合成HPV 31病毒体的能力，并允许检查HPV在植物病毒生命周期中的功能，然后，我们使用该系统来研究附加型基因组是否是诱导晚期病毒基因表达所必需的。当在El开放阅读框中含有错义突变的HPV 31基因组（31 E1 *）转染到正常人角质形成细胞中时，发现突变病毒序列整合到宿主细胞染色体DNA中，早期和晚期区域都是完整的。虽然观察到高水平的早期病毒基因转录，但在含有突变病毒基因组的细胞系的筏中没有检测到晚期基因表达，尽管有终末分化的证据。因此，晚期病毒基因表达的诱导需要病毒基因组保持为染色体外元件，仅终末分化是不够的。这些研究为详细检查HPV在病毒发病过程中的功能提供了基础。
Human papillomavirus (HPV) types 16, 18, 31, and 51 are the etiologic agents of many anogenital cancers including those of the cervix, These ''high risk'' HPVs specifically target genital squamous epithelia, and their lytic life cycle is closely linked to epithelial differentiation. We have developed a genetic assay for HPV functions during patho genesis using recircularized cloned HPV 31 genomes that mere transfected together with a drug resistance marker into monolayer cultures of normal human foreskin keratinocytes, the natural host cell, After drug selection, cell lines were isolated that stably maintained HPV 31 DNA as episomes and underwent terminal differentiation when grown in organotypic raft cultures, In differentiated rafts, the expression of late viral genes, amplification of viral DNA, and production of viral particles were detected in suprabasal cells, This demonstrated the ability to synthesize HPV 31 virions from transfected DNA templates and allowed an examination of HPV functions during the vegetative viral life cycle, We then used this system to investigate whether an episomal genome was required for the induction of late viral gene expression, When an HPV 31 genome (31E1*) containing a missense mutation in the El open reading frame was transfected into normal human keratinocytes, the mutant viral sequences were found to integrate into the host cell chromosomal DNA with both early and late regions intact, While high levels of early viral gene transcription were observed, no late gene expression was detected in rafts of cell lines containing the mutant viral genome despite evidence of terminal differentiation. Therefore, the induction of late viral gene expression required that the viral genomes be maintained as extrachromosomal elements, and terminal differentiation alone was not sufficient, These studies provide the basis for a detailed examination of HPV functions during viral pathogenesis.