Meyers Craig, Bromberg-White Jennifer L, Zhang Jiaping, Kaupas Michelle E, Bryan Janine T, Lowe Robert S, Jansen Kathrin U
Department of Microbiology and Immunology, College of Medicine, The Pennsylvania State University, Hershey, Pennsylvania 17033, USA.
J Virol. 2002 May;76(10):4723-33. doi: 10.1128/jvi.76.10.4723-4733.2002.
The organotypic raft culture system has allowed the study of the differentiation-dependent aspects of the human papillomavirus (HPV) life cycle. However, genetic strategies to more completely understand the HPV life cycle are limited. The generation of chimeric viruses has been a useful tool in other virus systems to analyze infection and replication. To investigate the specificity of the interaction of nonstructural genes of one HPV type with the structural genes of another HPV type, we have replaced the L2 and L1 open reading frames (ORFs) of HPV type 18 (HPV18) with the L2 and L1 ORFs of HPV type 16 (HPV16). The resulting HPV18/16 chimeric construct was introduced into primary keratinocytes, where it was stably maintained episomally at a range of 50 to 100 copies of HPV genomic DNA, similar to that typically found in HPV-infected cells in vivo. The integrity of the HPV18/16 genomic DNA chimera was demonstrated. Upon differentiation in raft cultures, late viral functions, including viral DNA amplification, capsid gene expression, and virion morphogenesis, occurred. Chimeric HPV18/16 virions were purified from the raft cultures and were capable of infecting keratinocytes in vitro. Additionally, infection was specifically neutralized with human HPV16 virus-like particle (VLP)-specific antiserum and not with human HPV18 VLP-specific antiserum. Our data demonstrate that the nonstructural genes of HPV18 functionally interact with the structural genes of HPV16, allowing the complete HPV life cycle to occur. We believe that this is the first report of the propagation of chimeric HPV by normal life cycle pathways.
器官型筏式培养系统有助于研究人乳头瘤病毒（HPV）生命周期中与分化相关的方面。然而，用于更全面理解HPV生命周期的遗传策略有限。嵌合病毒的产生在其他病毒系统中是分析感染和复制的有用工具。为了研究一种HPV类型的非结构基因与另一种HPV类型的结构基因相互作用的特异性，我们用HPV16型（HPV16）的L2和L1开放阅读框（ORF）替换了HPV18型（HPV18）的L2和L1开放阅读框。将所得的HPV18/16嵌合构建体导入原代角质形成细胞，在其中它以50至100拷贝的HPV基因组DNA的范围稳定地维持在游离状态，类似于在体内HPV感染细胞中通常发现的情况。证明了HPV18/16基因组DNA嵌合体的完整性。在筏式培养中分化后，发生了晚期病毒功能，包括病毒DNA扩增、衣壳基因表达和病毒体形态发生。从筏式培养物中纯化出嵌合HPV18/16病毒体，其能够在体外感染角质形成细胞。此外，感染被人HPV16病毒样颗粒（VLP）特异性抗血清特异性中和，而不被人HPV18 VLP特异性抗血清中和。我们的数据表明，HPV18的非结构基因与HPV16的结构基因在功能上相互作用，从而使完整的HPV生命周期得以发生。我们认为这是关于嵌合HPV通过正常生命周期途径传播的首次报道。
