Functional interaction between gene expression and DNA replication of HPV lifecyle

人乳头瘤病毒（HPV）的基因表达和基因组扩增受宿主细胞分化状态的严格调控。在基底层细胞中，可检测到病毒早期基因的弱表达和病毒基因组的低拷贝数。在角质形成细胞分化的过程中，病毒基因表达和DNA扩增的水平上调，并且仅在完全分化的细胞中可以检测到病毒颗粒的产生。因此，有必要在反映角质形成细胞分化程序的系统中分析HPV基因的功能和复制机制，发展角质形成细胞的器官型培养以重建皮肤结构，其中HPV DNA可以表达病毒基因并以分化特异性方式复制。我们采用了由Kuroki博士等人开发的器官型培养系统用于HPV分析。采用脂质体法将HPV 18基因组DNA导入人原代角质形成细胞，并将其用于体外培养。 ...更多信息 或器官型培养。在基底层细胞中，HPV DNA难以通过原位杂交检测，尽管其可通过Southern印迹分析检测到。在分化的细胞中发现DNA扩增，表达分化标志物之一的转氨酶。mRNA表达水平也随着细胞分化而增强。结果表明，器官型培养体系适合于HPV分化特异性功能的分析，在器官型培养体系中可以进行HPV基因功能的分析。两种主要的病毒癌蛋白，E6和E7，在角质形成细胞中表达，并分析对细胞增殖和分化的影响。正常情况下，细胞分裂仅限于基底层，但E6/E7表达细胞即使在部分分化层也能增殖。然而，即使在E6/E7表达的情况下，分化程序也保持完整。我们现在正在扩大对其他病毒基因和低风险型E6/E7基因的分析。我们观察到器官型培养中基因表达和DNA复制之间的联系。这两种功能主要受HPV基因组LCR的调节，其中病毒E2蛋白参与其中。我们获得的证据表明，E2蛋白作为两个活动之间的调解人。我们正在研究E2蛋白在分化特异性HPV复制机制中的作用。少

Gene expression and genome amplification of human papillomavirus (HPV) are tightly regulated by the status of host cell differentiation. In basal layer cells, weak expression of viral early genes and low copy number of viral genome are detectable. In the course of keratinocyte differentiation, the levels of viral gene expression and DNA amplification are upregulated, and the viral particle production can be detected only in fully differentiated cells. It is necessary, therefore, to analyze the mechanisms of HPV gene function and replication in the system that reflects the keratinocyte differentiation program.The organotypic culture of keratinocytes is developed to reconstitute the skin structure, in which HPV DNA can express viral genes and replicate in differentiation specific manner. We adapted an organotypic culture system developed by Dr. Kuroki et al. for HPV analysis. HPV 18 genomic DNA was introduced into human primary keratinocytes by lipofection, and then the cells were used f … More or organotypic culture. In the basal layer cells, HPV DNA was difficult to detect by in situ hybridization, although it was detectable by Southern blot analysis. The DNA amplification could be found in the differentiated cells, in which transglutaminase, one of the differentiation markers, was expressing. mRNA expression level was also enhanced through cell differentiation. The result indicated that the organotypic culture system was suitable for the analysis of differentiation specific HPV function.The gene function of HPV was analyzed in the organotypic culture system. Two major viral oncoproteins, E6 and E7, were expressed in keratinocytes and the effects on cell proliferation and differentiation were analyzed. In normal condition, cell division was restricted only in the basal layer, but E6/E7 expressing cells could proliferated even in partially differentiated layer. However, differentiation program was maintained intact even with E6/E7 expression. We are now expanding the analysis for other viral genes and low-risk type E6/E7 genes.We observed a linkage between gene expression and DNA replication in the organotypic culture. Both functions are regulated mainly by LCR of HPV genome, in which viral E2 protein is involved. We obtained evidence that the E2 protein functioned as a mediator between both activities. We are now investigating the involvement of E2 protein in differentiation specific HPV replication mechanism. Less